Electrochemical Biosensors for the Determination of the
Antioxidant Capacity
M. Cortina, C. Calas-Blanchard, J.-L. Marty
BIOMEM group, Université de Perpignan
52 Avenue Paul Alduy, 66860 Perpignan Cedex, France
[email protected]
Abstract
An amperometric cytochrome c-based electrode was developed and applied to the quantification of
the scavenging capacity of antioxidants. The enzymatic biosensor was constructed by covalently
immobilizing both cyt c and XOD on a mercaptoundecanoic acid/mercaptoundecanol mixed SAMmodified screen-printed gold electrode. The concentration which induces a 50% inhibition of the
superoxide level (IC50) has been determined for different substances, for instance pure substances
such as ascorbic acid or Trolox, and natural sources of antioxidants, particularly orange juices and
garlic extracts.
Keywords: Antioxidants, Cytochrome c electrodes, Superoxide radicals, Electrochemical detection
amperometric biosensors for the detection of
mono and polyphenols (the main antioxidant
compounds in food) have been developed on
the basis of enzymes such as tyrosinase,
laccase or peroxidase [6]. These configurations
allow the evaluation of the usually named “total
phenol content”. On the other hand, biosensors
for the assessment of the antioxidant capacity
are based on the free radical scavenging
activity.
1. Introduction
Reactive oxygen species (ROS), naturally
generated during the metabolism, can damage
biological structures such as proteins, lipids or
DNA. Inside the human body, the antioxidant
defensive system prevents their effect but
sometimes these natural defenses are
overwhelmed by an excessive generation of
ROS and a situation of oxidative stress occurs.
In this case, cellular and extracellular
macromolecules can suffer oxidative damage,
causing tissue injury [1, 2]. Antioxidants are
synthetic or natural substances that prevent or
delay the oxidative damage by scavenging the
free radicals. Fruits and vegetable have
received particular attention because they
contain high amounts of known antioxidants,
which are thought to contribute to prevent
several illnesses such as cardiovascular
diseases and cancer [3].
In order to assess the antioxidant capacity of a
substance based on the measurement of the
●superoxide radical (O2 ) concentration, two
main types of biosensors have been developed,
using cytochrome c (cyt c) or superoxide
dismutase (SOD) enzyme.
The main objective of this work was the
development of a cyt c-based biosensor for the
determination of the antioxidant capacity against
O2●-.
Several methods have been proposed for the
detection of antioxidants. Although photometric,
fluorimetric and chromatographic techniques
have been widely used, in the last years
electrochemical biosensors have become
promising tools, since they provide the
advantage of rather simple equipment and
operation protocols [4, 5]. Whereas in the
medical field the main objective is the evaluation
of the ability of some compounds to scavenge
free radicals, the food science research aims to
detect and quantify them. In this sense, two
different kinds of biosensors are reported in the
antioxidant domain. On one hand, several
2. Materials and methods
2.1 Reagents
All reagents were of analytical grade unless
specified. Cyt c from horse heart , hypoxanthine
(HX), xanthine oxidase from buttermilk (XOD,
EC 1.17.3.2), catalase from bovine liver (EC
1.11.1.6),
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), 11-mercapto-1-undecanol
(MU), 11-mercaptoundecanoic acid (MUA),
ascorbic
acid,
potassium
ferrocyanide,
103
potassium ferricyanide and components of
buffers were supplied by Sigma (St. Quentin
Fallavier, France). N-hydroxysuccinimide (NHS)
is supplied by Fluka and (±)-6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid (Trolox)
by Aldrich. All solutions were prepared using
Milli-Q water.
mM K-PBS pH 7.0, for 30 minutes. Finally, the
cyt c-modified electrodes were rinsed again with
the same buffer to remove non-attached
substances. Electrochemical behaviour of the
immobilized cyt c was analysed using cyclic
voltammetry.
2.5 Measurement of the superoxide scavenging
activity
2.2 Preparation of antioxidant samples
Four Spanish brand name orange juices and a
fresh one were centrifuged at 10000 g for 5 min
at room temperature and the supernatant was
used for analysis.
The principle of detection of these biosensors is
based on the redox reaction of cyt c (Figure 1).
Briefly, the immobilised cyt c is reduced by the
superoxide radical and immediately regenerated
on the gold surface of the electrode polarised at
the oxidation potential. The oxidation current is
proportional to the superoxide concentration in
solution [7]. The addition of antioxidants reduces
the radical concentration and thus the oxidation
current, allowing the quantification of the
antioxidant capacity.
Alliin and allicin extracted from garlic bulbs were
kindly provided by Dr. E. Touloupakis from the
University of Crete. The first one was diluted in
50 mM sodium phosphate buffer (Na-PBS) pH
6.5 and the second one in 96% ethanol (Carlo
Erba, Italy).
S
Gold electrode
2.3 Apparatus
Cyclic voltammetries were carried out by an
AUTOLAB PGSTAT12 Potentiostat (Eco
Chemie, BV, The Netherlands). Amperometric
measurements were made using a 641VA
potentiostat (Metrohm, Switzerland), connected
to a BD40 (Kipp & Zonen, The Netherlands) X-t
recorder.
S
S
S
S
S
COO-
Cyt. c
Heme (Fe3+)
O2
COOH
COO-
e-
COOH
COOCOOH
Cyt. c
Heme (Fe2+)
O2x -
(hypo)xanthine
XOD
O2 + H2O
catalase
H2O2
uric acid
O2
•2
Figure 1. Scheme of the detection principle of O produced
by hypoxanthine-XOD enzyme system, using a cyt cmodified electrode.
Electrochemical measurements were carried out
2
using 12.6 mm gold screen-printed electrodes
(DropSens, Spain) as working, a platinum wire
as a counter and a double-junction Ag/AgCl
electrode (Thermo Orion 900200) as a
reference.
All amperometric experiments were carried out
under constant stirring in 0.1 M Na-PBS with
100 µM EDTA pH 7.5. The working electrode
was polarized at a potential of +150 mV. The
XOD-catalyzed oxidation of HX to uric acid with
superoxide and H2O2 as secondary products
was used as radical source [8].
2.4 Electrode preparation
Electrodes were electrochemically cleaned by
cycling the potential between 0 and +1.4 V at
the scan rate of 100 mV s-1 in 0.1 M H2SO4 until
the characteristic cyclic voltammogram for a
clean gold electrode was obtained. After a
washing step with water and ethanol, the
cleaned electrodes were placed in a solution of
MU:MUA (3.75:1.25 mM) in ethanol and left for
72 h in the ethanolic solution. After three days,
the electrodes were washed by rinsing with
ethanol and water. The MU:MUA monolayer
was next activated incubating the electrode in
an aqueous solution of EDC (200 mM) and NHS
(50 mM) for 30 min. The excess of EDC and
NHS was eliminated by cleaning with 5 mM
potassium phosphate buffer (K-PBS) pH 7.0.
The cyt c and XOD were then immobilized on
the active surface by incubating the electrodes
-1
in a 50 µM cyt c + 50 mU mL XOD solution in 5
Before the start of the measurement, a catalase
solution (final concentration in the cell 10 U mL1
) was added in order to avoid interferences
from H2O2. Once the stable baseline was
established, an HX solution was added into the
cell (final concentration 100 µM) and a steadystate superoxide level was recorded. Stock
solutions of HX were prepared in 50 mM K-PBS
+ 0.01 M KCl + 0.5 mM EDTA pH 7.5.
In the experiments aimed at investigation of the
role of superoxide scavengers, aliquots of the
samples with antioxidant activity to be measured
were added after the addition of the catalase
solution and the current decrease was recorded.
All antioxidant solutions were freshly prepared.
104
2.6 Colorimetric method for the quantification of
ascorbic acid in commercial samples
those where the XOD remained in solution [9,
10].
A standard colorimetric method (Roche
Diagnostics, Mannheim, Germany) was used for
the quantification of ascorbic acid in commercial
samples. In this case, the ascorbic acid reduces
the
tetrazolium
salt
MTT
[3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide] in the presence of the electron carrier
PMS (5-methylphenazinium methosulfate) at pH
3.5 to a formazan (MTT-formazan), which is
determined by means of its light absorbance at
578 nm.
Figure 3 shows the cyclic voltammogram of the
covalently immobilized cyt c. A pair of welldefined redox peaks appeared in 5 mM K-PBS
pH
7.0.
The
quasi-reversible
redox
transformation of cyt c is the basis of the
amperometric superoxide detection.
3. Results and Discussion
3.1 Characterization of the modified electrode
surface
In order to check the blocking characteristics of
the prepared SAM, cyclic voltammetry was
performed with hexacyanoferrate solution.
Figure 2 illustrates the voltammetric behavior of
the bare and the modified electrodes. The bare
Au electrode in hexacyanoferrate solution (1 mM
34[Fe(CN)6] /[Fe(CN)6] in 0.1 M K-PBS pH 7)
showed a well defined faradaic response. In
contrast, the cyclic voltammograms for
electrodes prepared with the mixed SAM
showed effective blocking for this redox couple.
It is attributed to repulsing interaction of the
negative charge of the SAM to negatively
charged redox couple ([Fe(CN)6]3-/[Fe(CN)6]4-).
Figure 3. Cyclic voltammogram of covalently immobilized cyt
c on a SAM-modified gold electrode in 5 mM K-PBS pH 7.0.
-1
Scan rate: 100 mV s .
3.3 Characterization of antioxidants
The antioxidant capacity of both pure
substances such as ascorbic acid and Trolox
and natural sources of antioxidants, particularly
orange juices and garlic extracts was
determined.
Addition of small aliquots of the antioxidant
stock solution resulted in a reduced current
signal. This sensor signal decrease was
normalized to the initial ROS signal. The
relativity of this approach is advantageous in
order to minimize effects of varying sensitivities
of individual sensors. A quantification of their
antioxidant capacity was performed by
calculating the concentration necessary for 50%
signal inhibition (IC50). This value was used for
the comparison of the different antioxidants.
Hence, a low IC50 corresponds to a high
antioxidative ability.
Figure 2. Cyclic voltammograms of the bare Au electrode
(solid line) and the SAM-modified gold electrode (dotted line)
34in 0.1 M K-PBS pH 7 containing 1 mM [Fe(CN)6] /[Fe(CN)6]
-1
. Scan rate: 100 mV s .
3.3.1 Antioxidative properties of ascorbic acid
and Trolox
Ascorbic acid and Trolox are well-characterized,
hydrophilic antioxidants and are often used as
standards for other antioxidative substances. In
Figure 4 the plot current decrease versus the
acid ascorbic concentration is given. Such
behaviour corresponds to the empirical Hill
equation which can describe a variety of sensor
responses [11]:
3.2 Electrochemistry of the immobilized cyt c
Enzymatic biosensors were constructed by
covalently immobilizing both cyt c and XOD on a
mixed long-chain thiol modified gold electrode.
The immobilization of both species on the
electrode surface increased at least 15 times the
sensitivity of the system in comparison with
105
y=
B⋅x
C+x
(1)
where y corresponds to the percentage of signal
inhibition, x corresponds to the antioxidant
concentration in solution and B and C are
constant values.
Figure 5. Representation of the signal inhibition with the
ascorbic acid concentration by 4 commercial orange juices
and a natural one. The pure ascorbic acid curve is included
as reference.
3.3.3 Antioxidative properties of garlic extracts
The developed biosensor was also applied to
the determination of the antioxidant capacity of
alliin and allicin extracted from garlic bulbs.
Figure 4. Representation of the dependence of the signal
depression of the superoxide sensor on ascorbic acid
concentration (error bars are deduced form three repeated
measurements).
It is known that aroma components of Allium
species
are
pharmacologically
active
substances since they exhibit antioxidant and
antitumor activities.
The IC50 of ascorbic acid was calculated to be
6.0 ± 0.9 µg mL-1. On the other hand, Trolox
was found to be less effective with an IC50 value
of 40.8 ± 0.7 µg mL-1.
However, only alliin showed antioxidant capacity
against O2●-, while allicin had prooxidant
properties. For alliin, the IC50 determined using
the Hill equation was equal to 100 ± 8 µg mL-1.
3.3.2 Antioxidative properties of orange juices
In order to show the ability of the system to
allow quantification of antioxidative properties
even in complex media, 5 orange juices were
tested.
4. Conclusions
An amperometric biosensor for the quantification
of the scavenging capacity of antioxidants has
been developed. An MUA/MU-modified gold
electrode with immobilized cyt c and XOD has
been characterized and applied to the
antioxidants analysis. The immobilization of both
species on the electrode surface increased the
sensitivity of the system in comparison with
those where the XOD remained in solution. The
concentration which induces a 50% inhibition of
the superoxide level has been determined for
different substances. The applicability of this
method has been shown by analyzing the
antioxidant capacity of ascorbic acid, Trolox, 5
oranges juices and 2 garlic extracts.
Orange juice contains an array of potent
antioxidants including flavonoids (hesperetin
and naringenin predominantly as glycosides),
carotenoids
(xanthophylls,
cryptoxanthins,
carotenes), and vitamin C in addition to other
beneficial phytochemicals, such as folate. Thus,
the addition of orange juice samples caused a
decrease in current signal, allowing a
quantification of their antioxidant capacity.
Figure 5 shows the orange juices calibration
plots. In each case, the ascorbic acid
concentration was obtained through the
colorimetric method. As can be seen, the
antioxidant capacity of orange juices was always
bigger than the one obtained with the pure
ascorbic acid. As previously commented, this
fact may be understood by considering that
natural and commercial juices contain other
antioxidant substances.
Acknowledgments
The authors greatly acknowledge the European
Commission for financial support through the
project
“Nutra-Snacks”
(FOOD-CT-2005023044).
106
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