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Universidade de So Paulo

Biblioteca Digital da Produo Intelectual - BDPI


Departamento de Fsica e Cincias Materiais - IFSC/FCM

Artigos e Materiais de Revistas Cientficas - IFSC/FCM

2011-12

Polypyrrole/phytase amperometric biosensors


for the determination of phytic acid in standard
solutions
Sensors and Actuators B, Lausanne : Elsevier, v. 160, n. 1, p. 222-226, Dec. 2011
http://www.producao.usp.br/handle/BDPI/50047
Downloaded from: Biblioteca Digital da Produo Intelectual - BDPI, Universidade de So Paulo

Sensors and Actuators B 160 (2011) 222226

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical


journal homepage: www.elsevier.com/locate/snb

Polypyrrole/phytase amperometric biosensors for the determination of phytic


acid in standard solutions
Valquria Cruz Rodrigues a , Marli Leite de Moraes b , Andr Brisolari a , Juliana Coatrini Soares a ,
Marystela Ferreira b , Dbora Goncalves a,
a
b

Instituto de Fsica de So Carlos, Universidade de So Paulo, CP 369, 13560-970, So Carlos, SP, Brazil
Universidade Federal de So Carlos, Campus Sorocaba, 18052-780, Sorocaba, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 2 March 2011
Received in revised form 6 July 2011
Accepted 21 July 2011
Available online 27 July 2011
Keywords:
Biosensors
Phytase
Polypyrrole
Phytic acid detection

a b s t r a c t
Amperometric biosensors based on the physical immobilization of phytase (PhyA) into polypyrrole (PPy)
lms were prepared in aqueous medium. The PPy/PhyA lms were characterized by cyclic voltammetry,
and surface and structural characterization techniques, SEM and FTIR. Both voltammetric and amperometric transduction methods were used in order to detect phytic acid in acetate buffer at pH 5.5 at room
temperature. The biosensors exhibited a detection limit of 0.15 mmol L1 and a linear range of phytic acid
content from 0.5 to 2.0 mmol L1 , which are adequate values for typical analyses of phytic acids in most
seeds, grains, and vegetables.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Phytase (myo-inositol hexaphosphate phosphohydrolase)
(PhyA) catalyzes the hydrolysis of phytic acid (myo-inositol
hexaphosphate or phytate) into phosphorous compounds, which
is an essential nutrient for animals [1] according to the reaction
shown below.
phytase

phytic acid + H2 O inositol + PO4 3


Phytic acid is the main storage form of phosphate in grain seeds,
pollen, vegetables, and oilseeds [2,3], but it is also considered an
antinutrient due to its chelation ability, binding dietary minerals
such as magnesium, zinc, and calcium [2]. Monogastric animals,
such as pig and poultry, and also humans, are not able to digest
phytic acid because they lack sufcient amounts of the digestive enzyme, PhyA [4,5]. In this case, unabsorbed phosphorus
compounds pass into the feces and can be degraded by aquatic
microorganisms, being one of the causes of eutrophication of water
bodies downstream the rural areas [6]. In order to alleviate adverse
effects of high levels of phytic acid, PhyA has been used as supplementary feeding in swine and poultry rations [79].
The presence of phytic acid in food and feedstuffs can be
determinate by many methods; a well-known procedure involves

Corresponding author. Tel.: +55 16 3373 9825x216; fax: +55 16 3371 5365.
E-mail address: gdebora@if.sc.usp.br (D. Goncalves).
0925-4005/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2011.07.038

the precipitation of iron (III)PhyA complex, and further analysis of the endpoint of titration [10]. In addition, techniques such
as nuclear magnetic resonance spectroscopy, high-performance
liquid chromatography, and SDSpolyacrylamide gel electrophoresis [11,12] have been also used for the detection of phytic
acid in different analytes. The use of biosensors based on the
immobilization of PhyA into polymeric matrices appoints as an
alternative for the detection of phytic acid. Previous studies have
reported the preparation of poly(carbamoylsulphonate) hydrogel
co-immobilized with PhyA and pyruvate oxidase onto Pt electrodes
[13]; the authors obtained for these amperometric biosensors a
detection limit of 2.0 103 mmol L1 and a linear response ranging from 0.2 to 2.0 mmol L1 of phytic acid at a xed potential of
0.6 V vs Ag/AgCl [13]. Recently, amperometric biosensors based
on the immobilization of PhyA into poly(allylamine) hydrochloride/Prussian Blue/ITO electrodes by the Layer-by-Layer technique
were fabricated by our group [14]. These biosensors exhibited a
detection limit of 0.19 mmol L1 and a linear response ranging from
0.5 to 2.0 mmol L1 of phytic acid at 0.0 V vs SCE [14].
The present study provides continuity for this research eld
mainly with the aim of improving properties of the PhyA biosensors. PhyA was immobilized into polypyrrole (PPy) lms by a simple
and fast protocol based on physical interactions that take place
between the enzyme and the polymer matrix. PPy is regarded as a
suitable matrix for most enzyme immobilization once pyrrole oxidizes at relatively low oxidation potentials in both aqueous and
non-aqueous media. Besides, PPy is usually biocompatible [15].
The electronic properties of the PPy lms also allow a fast, direct

V. Cruz Rodrigues et al. / Sensors and Actuators B 160 (2011) 222226

150

PPy/PhyA
100

PPy
50
-2

j ( A cm )

2. Experimental

-150
-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

E (V) vs. SCE


Fig. 1. Cyclic voltammograms of PPy (solid line) and PPy/PhyA (dashed line) in
0.1 mol L1 acetate buffer at pH 5.5; v = 50 mV s1 .

861

PPy/PhyA

700

800

PPy

1442
1480

the PPy lm into a solution of 0.2 mg mL1 PhyA in acetate buffer


at 40 C for 2 h.
The voltammetric responses of the PPy and PPy/PhyA lms were
obtained in a pyrrole-free solution, 0.1 mol L1 acetate buffer at
pH 5.5. The PPy lm exhibited a fairly well-dened voltammetric response (Fig. 1); after modication with PhyA, higher values
of current densities can be noticed in the voltammogram, but still
with the typical electrochemical response of a native PPy matrix.
The differences in current seen for the PPy and PPy/PhyA lms
indicate the occurrence of electrostatic interactions between PhyA
and its matrix, PPy; this is the expectable behavior for lms with
insulating enzymes entrapped into a polymeric matrix [19]. The
voltammogram recorded in the literature for a different matrix (a
metallophthalocyaninemetalloporphyrin complex) without and
with immobilized glucose oxidase [20,21] is very close in shape
to the curves seen in Fig. 1, indicating that the presence of enzyme
has a role in the voltammetric response of the electrode material.
The enhanced capacitive background current seen for the PPy/PhyA
lm can be related to a typically high electron transfer capacity of
this lm, and consequently, its larger specic surface area. However, generally speaking, the voltammetric responses of the PPy
ad PPy/PhyA lms in the blank solution did not show remarkable
differences.
Fig. 2 shows the FTIR spectra of the PPy and PPy/PhyA lms from
650 cm1 to 1750 cm1 since in this spectral range they can be better comparable. The spectrum of the PPy lm obtained by us is

1540

Polypyrrole (PPy) lms were electrodeposited on Pt electrodes


in an aqueous medium containing pyrrole and NaCl up to reach ten
successive voltammetric cycles. During the electropolymerization
process, the current density increases linearly with the number of
cycles indicating the growth of an electroactive lm (PPy) on the
electrode surface. The PPy/PhyA lm was then prepared by keeping

-100

1566

3. Results and discussion

-50

% Reflectance (a.u.)

Phytase (PhyA) from Aspergillus cuum (EC 3.1.3.8)


(1.1 units g1 , IP = 4.5) (SigmaAldrich) and phytic acid (myoinositol hexaphosphate) (Synth) were used as received. Pyrrole
(SigmaAldrich) was freshly distilled before use. PhyA and phytic
acid solutions were prepared by using 0.1 mol L1 acetate buffer at
pH 5.5.
The electropolymerization of pyrrole was performed by cyclic
voltammetry using a potentiostat/galvanostat PAR Versastat II with
a three-electrode cell, a saturated calomel electrode (SCE) as the
reference electrode, a Pt disk as the working electrode, and a Pt
plate as the counter electrode. The electrochemical deposition of
polypyrrole (PPy) lms was carried out by cyclic voltammetry from
0.0 to 0.8 V vs SCE at 50 mV s1 in an aqueous solution containing
0.07 mol L1 pyrrole and 0.1 mol L1 NaCl up to reach 10 complete
cycles. For the PPy lms obtained up to reach 10 cycles, we obtained
a thickness of about 0.8 m which was calculated by an equation
reported in the literature [18]. Smaller numbers of cycles yielded
thinner lms, but not necessarily homogeneous; much thicker
lms, on the other hand, would hinder ionic movements into the
lm and, therefore, offer higher diffusion resistance of the analyte
related to the polymeric matrix.
Freshly prepared PPy lms were washed with water and a
monomer-free electrolyte solution. After that, they were dipped
into a PhyA solution at 0.2 mg mL1 at 40 C, where were kept
for 2 h; this procedure allowed us to physically immobilize PhyA
into the PPy lm. Previously, we veried that the immobilization
of PhyA by the Layer-by-Layer technique occurred efciently at
about 40 C [14]. This temperature has no direct effect on the enzymatic activity, since the optimum temperature of PhyA activity lies
between 37 C and 60 C (at pH 5.5). The geometrical surface area
of the Pt electrodes modied with PPy and PPy/PhyA lms was
0.6 cm2 .
Chronoamperometry was used for the detection of phytic acid
at a xed potential of 0.0 V vs SCE. The current was monitored as a
function of the time after successively adding aliquots of 50 L of
0.1 mol L1 phytic acid at 10 mL acetate buffer at pH 5.5.
The morphology of the PPy and PPy/PhyA lms on Au coatings was studied by scanning electron microscopy (SEM) (Digital
Scanning Microscope, DSM 960, Zeiss, West Germany). The
infrared spectra of these lms on Pt substrates were obtained
using a Thermo NicoletNexus 470/FT-IR spectrophotometer from
400 cm1 to 2000 cm1 after 64 scans.

915

electron charge transfer between the polymer lm matrix and the


immobilized enzyme, thus yielding sensitive and selective signals
[16,17].
Previously, we have already shown the viability of biosensors
based on the immobilization of PhyA onto LbL lms [14]. However, the use of a polymeric matrix appoints as other alternative for
obtaining simpler and cheaper biosensors, in particular when they
are miniaturized, and require minimum sample amounts. Here,
we describe the build of PPy/PhyA lms and their characterization
by electrochemical measurements, scanning electron microscopy
(SEM), and Fourier transform infrared spectroscopy (FTIR).

223

900 1000 1100 1200 1300 1400 1500 1600 1700

Wavenumber (cm-1)
Fig. 2. FTIR spectra of PPy (dashed line) and PPy/PhyA (solid line).

224

V. Cruz Rodrigues et al. / Sensors and Actuators B 160 (2011) 222226

Fig. 3. SEM images of PPy and PPy/PhyA with a 10.000 resolution.

changes in the voltammetric responses were veried for the PPy


lm after adding phytic acid ranged from 0.0 to 4.0 mmol L1 (about
9.5% for the current densities obtained a xed potential). For the
PPy/PhyA lm, the differences in current densities are higher than
54% as a strong evidence of the catalytic effect of the enzyme immobilized into the PPy matrix. After successive injections of phytic
acid into the buffer solution, the PPy/PhyA lm responded rapidly,
and both anodic and cathodic currents varied. The substrate specicity of PhyA was also veried by using phenyl phosphate disodium
salt, buffer, inositol, and dipalmitol phophatidyl choline (DPPC),
however, no signicant changes in the current densities were also
obtained by using these solution, which corroborates the conclusion that the PPy/PhyA lm is only capable of detecting phytic acid
and, consequently, free phosphorous ions.
Fig. 5 shows the chronoamperometric curves obtained for the
PPy/PhyA lm at a xed potential of 0.0 V vs SCE in 0.1 mol L1
acetate buffer at pH 5.5. After adding successive aliquots of
0.1 mol L1 phytic acid (50 L each) in 10 mL of acetate buffer, the
current density increased with the concentration of phytic acid up
the fourth addition at 2.0 mmol L1 . After that, the current density decays abruptly due to a saturation effect of the active sites of
PhyA (denaturation) in the presence of phytic acid. After a longterm experiment, one expects modications in temperature and
pH of the lm surface, with PhyA modifying its three-dimensional

75
-2

j (m A cm )

400
300
200
-2

j (A cm )

in accordance with the literature [2225]. The immobilization of


PhyA into the PPy matrix was conrmed by the presence of sharp
peaks shifted in the spectra of the PPy/PhyA lm. Among them,
the peak assigned to the CH out-of-plane deformation of the pyrrole units [23] appeared at 915 cm1 for the PPy lm and shifted
to 861 cm1 in the spectra of the PPy/PhyA lm. The two peaks at
1480 cm1 and 1540 cm1 , which are typical of the PPy lms, indicate the extent of the conjugation of this polymer [25]. They have
been attributed to the symmetric and anti-symmetric ring stretching modes of PPy, and were shifted to 1442 cm1 and 1566 cm1
for the PPy/PhyA lm and 1540 cm1 for the PPy lm. The peak at
1700 cm1 , which has been usually attributed to the presence of
carbonyl groups in the PPy structure [22,24], is seen in the spectra of the PPy/PhyA lm with a low intensity, indicating a slightly
overoxidized polymer structure. The changes in the spectra of the
PPy and PPy/PhyA lm strongly evidence the occurrence of electrostatic interactions between negatively charged PhyA (IP 4.5) and its
matrix, a positively charged PPy.
The PPy and PPy/PhyA lms were also studied by scanning electron microscopy (SEM). Fig. 3 reveals the typical morphology of
the PPy matrix, a spheroid structure with dispersed spheres with
sizes of ca 0.4 m. For the PPy/PhyA lm, the nodular structure
from the polymeric matrix was maintained, but a narrower distribution of smaller sphere sizes of ca 0.15 m is also evident. Besides,
the spheres are distributed more uniformly over the surface of the
PPy/PhyA lm as an indicative of the presence of immobilized PhyA
on smooth PPy/PhyA lms. Previously, it was shown that the morphology of the polymeric matrix (PPy) modies, usually in a small
extent, after introducing an insulating, bulky protein (glucose oxidase) into the electropolymerization medium [26]. Polymer lms
with entrapped enzymes generally exhibit smoother morphologies
with low defect sites and absent enzyme clusters [27]; these differences have been related to the inuence of the enzyme on the
whole growth process of the polymer lm.
Fig. 4 shows the electrochemical responses of the PPy/PhyA lm
in 0.1 mol L1 sodium acetate buffer at pH 5.5 containing different phytic acid concentrations. Between 0.2 V and +0.6 V vs SCE,
it is clearly evident that the current density increases with the
phytic acid concentration (see the inset of Fig. 4 for values of current densities obtained at a xed potential, 0.0 V vs SCE). A linear
increase of the current density is clearly seen from 0.5 mmol L1
to 2.0 mmol L1 of phytic acid; after that, it is evident a saturation
regime. These results indicate that the PPy/PhyA lm can be used
to detection of phytic acid up to reach 2.0 mmol L1 phytic acid at
0.0 V vs SCE where effects of interferents are expected to be negligible. The electrochemical responses of the PPy/PhyA biosensors
are based on the monitoring of inorganic phosphorous compounds,
which are produced by the enzymatic reaction (see Section 1).
Without the presence of the immobilized enzyme, no signicant

100

50

25

0.0

1.0
2.0
3.0
4.0
-1
[phytic acid] (mmol L )

5.0

-0.3

0.0

0
-100
-200
-300
-400
-0.6

0.3

0.6

E (V) vs SCE
Fig. 4. Cyclic voltammograms of PPy/PhyA in acetate buffer at pH 5.5 and different
concentrations of phytic acid ranging from 0.0 to 4.76 103 mol L1 . Inset: current
density as a function of the concentration of phytic acid obtained at a xed value of
potential, 0.0 V.

V. Cruz Rodrigues et al. / Sensors and Actuators B 160 (2011) 222226

225

Table 1
Characteristics of phytic acid biosensors based on entrapped phytase (PhyA).
Transducer

LOD (mM)

Linear range (mM)

Interferents tested

Matrix

Ref.

Amperometry

0.15

0.52.0

PPy(PhyA)

This work

Amperometry

0.002

0.22.0

Phosphate ions, inositol, and phenyl


phosphate

[13]

Amperometry

0.19

0.22.0

Voltammetry
Impedance

0.004

6.050

Poly(carbamoylsulphonate) hydrogel
(PhyA and pyruvate oxidase)
Poly(allylamine) hydrochloride/Prussian
Blue (PhyA)
Phospholipid LB lms (PhyA)
Poly(vinylsulfonate) (PhyA)

Phosphate ions, inositol, DPPC, and


phenyl phosphate

-3.5
j (A cm -2)

-3.6
-3.7
-3.8

-2

j (A cm )

-3.0

-3.5

0.0

0.5 1.0 1.5 2.0 2.5


-1
[phytic acid] (mmol L )

3.0

-4.0

500

1000

1500

2000

2500

3000

3500

Time (s)
Fig. 5. Amperometric curves for PPy/PhyA at 0.0 V vs SCE in acetate buffer at pH 5.5.
The arrows indicate the addition of aliquots of 50 L of a 0.1 mol L1 phytic acid in
10 mL of acetate buffer solution. Inset: maximum values of the current density as a
function of the concentration of phytic acid as indicated by the arrows.

structure and, consequently, its properties. Since this process is


irreversible, the current density decay is, in fact, very fast.
The PPy and PPy/PhyA lms were tested when in the presence
of phytic acid, inositol, and phosphate ions (Fig. 6). For inositol,
no current signal was noticed in the amperometric curves for both
lms, PPy and PPy/PhyA; for phosphate ions, both lms gave current signals, and for phytic acid, only the PPy/PhyA lm detected its

-3
3-

3-

PO4

PO4

inositol

phytic acid
PPy

j (A.cm )

-4

3-

PO4

[14]
[28]
[29]

presence in solution. These results indicate that the amperometric


response of the biosensor can be in fact related to the adsorption
of phosphate into the polymer matrix, as it was veried before for
Prussian Blue/PAH/PhyA biosensors [14].
We veried that both voltammetric and amperometric detection methods show good performances in detecting phytic acid by
using the PPy/PhyA lms. The detection limit obtained for these
lms was 0.15 mmol L1 (three times the noise), with a linear
range between 0.5 and 2.0 mmol L1 of phytic acid at a low operating potential. The sensitivity of the PhyA/PPy lm towards phytic
acid was calculated by slope of the linear correlation curve, and
gave a value of 0.2 A mmol1 cm2 . Apparent MichaelisMenten
constant (KM ) was found to be 2.44 mmol L1 for the PhyA immobilized into the PPy lm, a value similar to that obtained by Mak
et al. [13], 2.25 mmol L1 . These results are consistent with our
previous results from the use of PhyA immobilized in Layer-byLayer lm onto ITO-coated glass and modied with Prussian Blue
[14] and also, bi-enzyme sensors based on the co-immobilization
of PhyA and pyruvate oxidase [13]. In Table 1, we comparatively
show recent results on biosensors fabricated from PhyA entrapped
into different matrices. Although the detection limit obtained by
us is typically lower than that obtained previously [14], the use
of PPy/PhyA lms provide a suitable means of obtaining low-cost
biosensors. Furthermore, the Pt electrode used for the electrodeposition of the PPy/PhyA lms is reusable and requires only the
presence of PhyA and pyrrole in the electrolyte solution, decreasing
costs and fabrication steps.
4. Conclusions
PPy/PhyA biosensors were successfully fabricated by the
electrochemical synthesis of the PPY matrix with physically immobilized PhyA. Techniques such as cyclic voltammetry, SEM, and FTIR
allowed us to determinate differences in the responses of the PPy
and PPy/PhyA lms. We obtained for the PPy/PhyA lm a detection limit of 0.15 mmol L1 and a linear range of concentrations
between 0.5 and 2.0 mmol L1 of phytic acid. These results indicate
that the PPy/PhyA biosensors are an alternative for the detection of
phytic acid in solution with relatively low costs and at low operating
potentials.

-5

Acknowledgements
inositol

-6

PhyA

3-

3-

PO4

inositol

PO4

phytic acid
-7

200

400

The authors would like to acknowledge nancial support from


FAPESP, CAPES and CNPq (Brazil).
References

600

800

1000

Time (s)
Fig. 6. Amperometric curves for PPy and PPy/PhyA lms at 0.0 V vs SCE in acetate
buffer at pH 5.5 after adding phytic acid, phosphate ions, and inositol. The arrows
indicate the additions of aliquots of 50 L of each solution at 0.1 mol L1 in 10 mL of
acetate buffer solution.

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Biographies
Valquria da Cruz Rodrigues obtained her bachelor degree in Chemistry at Instituto
de Qumica de So Carlos, Universidade de So Paulo (Brazil) in 2002 and her Master
Degree in the eld of biosensors in 2009 at the same university. She is Ph.D. student
under the supervision of Dr. D. Goncalves, and her areas of research include the
fabrication and development of biosensors based on the use of conducting polymers.
Marli Leite de Moraes obtained her bachelor degree in Chemistry in 2001, Master
degree in Biophysics in 2003, and Ph.D. in Physical Chemistry in 2008 at Universidade
de So Paulo, So Carlos SP (Brazil). Her elds of interest are nanostructured thin
lms for biosensing fabricated from enzymes, peptides, polymers, and liposomes,
and spectroscopic, electrochemical and electrical (impedance) characterization of
these lms. She is a researcher at Universidade Federal de So Carlos, Sorocaba SP
since 2009.
Andr Brisolari obtained his graduation in Sciences (habilitation in Chemistry) in
2005 and Master degree in Materials Science in 2008 in the eld of Physical Chemistry of solid surfaces. He is Ph.D. student under the supervision of Dr. D. Goncalves,
and his main areas of research include the fabrication and development of novel
biosensors based on the use of conducting polymers.
Juliana Coatrini Soares obtained her bachelor degree in Chemistry in 2002, Master
degree in Materials Science in 2006, and Ph.D. in Materials Science in 2011 at Universidade de So Paulo, So Carlos SP (Brazil). Her elds of interest are fabrication
and development of biosensors and electrosynthesis of conducting polymers. She is
a researcher at Universidade de So Paulo, So Carlos SP (Brazil).
Marystela Ferreira obtained her bachelor degree in Chemistry in 1993, Master
degree and Ph.D. in Physical Chemistry in 1996 and 2000, respectively, at Universidade de So Paulo, So Carlos SP (Brazil). Her main elds of interest are preparation
and characterization of nanostructured thin lms for sensing, and Layer-by-Layer
and LangmuirBlodgett lms of polymers for sensing different analytes in environmental and biological samples. She is teaching and researching at Universidade
Federal de So Carlos, Sorocaba SP since 2007.
Dbora Goncalves obtained her bachelor degree in Chemistry in 1990, Master
degree in Physical Chemistry in 1993, and Ph.D. in Physical Chemistry in 1997 at Universidade Federal de So Carlos, So Carlos SP (Brazil). Her main elds of interest
are polymer lms as matrices for immobilizing enzymes, and fabrication and development of biosensors for environmental purposes. She is teaching and researching
at Instituto de Fsica de Carlos, Universidade de So Paulo, So Carlos SP since 1998.