Current Analytical Chemistry, 2012, 8, 428-435

428

Electrochemical Biosensors for the Determination of the Antioxidant

Capacity in Foods and Beverages Based on Reactive Oxygen Species
Carole Calas-Blanchard1*, Montserrat Cortina-Puig2, Lise Barthelmebs1 and Thierry Noguer1
1

Univ. Perpignan Via Domitia, Institut de Modlisation et d'Analyse en Go-Environnement et Sant, EA 4218,
F-66860, Perpignan, France
2

Escola Universitria Salesiana de Sarri, C/ Sant Joan Bosco 74, 08017, Barcelona, Spain
Abstract: Reactive oxygen species (ROS) are very unstable molecules generated during the metabolism. They can be
produced in excess, contributing to cellular dysfunctions. Antioxidants are substances that play an important role as a
health-protecting factor, limiting the lesions caused by ROS. Two kinds of electrochemical biosensors based on ROS have
been described to evaluate the antioxidant capacity in the food industry. The first one involves hydroxyl radicals and
studies their effect on DNA alterations. The second one consists of the superoxide radicals scavenging ability, radicals
being essentially generated by the xanthine/xanthine oxidase system. These sensors are commonly based on either
cytochrome c or superoxide dismutase, even though recent strategies are emerging. Whatever the involved ROS, all the
described biosensors possess similar advantages such as simplicity, rapidity and low cost and are successfully applied for
the assessment of antioxidant properties in various foods, additives or beverages.

Keywords: Antioxidant Capacity, Beverages, Biosensors, Food, Hydroxyl Radicals, Reactive Oxygen Species, Superoxide
Radicals.
1. INTRODUCTION
Antioxidants, or anti-oxidation agents, are substances
that may protect human cells from the damage caused by a
large spectrum of oxidants, mainly reactive oxygen species
(ROS). ROS include both oxygen-centered non-radical
molecules and oxygen-centered radical compounds. These
free radicals are highly unstable molecules with available
electrons, generated in vivo during metabolic processes.
However, their production can be accentuated under the
influence of external factors such as pollution, sunlight
exposure, cigarette smoking, excessive alcohol consumption,
etc. or simply by a malfunction of the antioxidant
production. An excessive production of ROS induces the socalled oxidative stress producing damage to lipids,
proteins or DNA, impeding normal cell functioning [1].
These biochemical alterations are implicated in the aging
process, as well as in a growing list of human diseases, such
as cancer [2] and Alzheimer's disease [3]. Studies have
shown that eating a diet rich in antioxidant-containing foods,
such as plants, fruits, vegetables and whole grains, has been
linked to a reduced risk of cardiovascular diseases [4]. In
addition to natural products, new antioxidant compounds as
food additives and nutraceuticals have recently received
remarkable attention [5].
In this context, the determination of antioxidants and free
radicals has been widely investigated in the food technology.
Traditional techniques such as gas or liquid chromatography,
*Address correspondence to this author at the IMAGES EA 4218, Bat S.,
Universit de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860
Perpignan Cedex, France; Tel.: +33 468 66 22 56; Fax: +33 468 66 22 23;
E-mail: calas@univ-perp.fr

1-/12 $58.00+.00

spectrophotometry and fluorescence [6] have been massively

developed either for the quantification of antioxidants or for
their action as radical scavengers. In that case, several
analytical methods have been proposed to characterize the
action of antioxidants and quantify their activity in preventing or delaying the oxidation of reactive species such as
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)
(ABTS) or 2,2-diphenyl-1-picrylhydrazyl (DPPH) [7-8].
Nevertheless, these spectrophotometric methods are not
based on ROS and the information that they contain is not
easily transposed in terms of the protection degree provided
by antioxidants against an attack by a specific ROS.
Nowadays, all these conventional techniques are being
replaced by other innovating technologies. In this direction,
electrochemical biosensors are promising tools, suitable for
fast analysis, based on inexpensive instrumentation and
simple operation protocols. In food science, research
generally aims to detect and quantify compounds supposed
to have antioxidant properties. In this direction, several
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 [9]. These configurations allow the
evaluation of the usually named total phenol content. More
recently, another category of biosensors is reported in the
antioxidant field. Its main objective is the evaluation of the
ability of some compounds to scavenge free radicals. This
article is focused on this kind of biosensors allowing the
assessment of the antioxidant capacity based on the free ROS
scavenging ability. Although many electrochemical biosensors have been developed, only few of them have been
2012 Bentham Science Publishers

Determination of the Antioxidant Capacity Using ROS-based Biosensors

applied to foods or beverages. All biosensors developed for

this purpose use ROS in their configurations. ROS are not
commercially available because of their highly reactive
nature and their very short lifetime. Consequently, the first
step in the development of such biosensors is their in vitro
generation. In the following sections, the biochemical,
chemical and physico-chemical in vitro processes to produce
these radicals are also listed. Afterwards, all the biosensors
already described for the assessment of the antioxidant
capacity in the food industry are critically reviewed.
2. REACTIVE OXYGEN SPECIES
ROS are chemically reactive molecules containing
oxygen, including singlet oxygen, hydrogen peroxide and
several oxygenated radicals. In living systems, the main ROS
generated as normal products of cellular metabolism are the
superoxide anion radical (O2-) and the hydroxyl radical
(OH) [10]. Their in vivo production can be accentuated by
the influence of external factors resulting in an imbalance
between ROS formation and the capacity of the antioxidant
defense system called oxidative stress. Due to the presence
of unpaired valence shell electrons, ROS are highly reactive
compounds and possess a very short lifetime. For these
reasons, the conception of biosensors able to determine the
protective effect of antioxidants against ROS requires
systematically the ROS generation during the assay. All the
described biosensors are based on O2- or OH  radicals, even
though H2O2 is sometimes the detected compound. For their
in vitro production, several processes are frequently used in
biosensor devices and are described in the following sections
(3.1 and 4.1). Table 1 summarizes the biosensors based on
the determination of either O2- or OH radicals that have
been applied in the food industry.
3. MONITORING HYDROXYL RADICALS
3.1. Generation of OH
Hydroxyl radicals (OH) can be generated by using
several chemical processes. During the Fenton reaction (Eq.
1), reduced transition metal cations (Men+), such as Fe2+, Cu +
or Cr2+, react with H2O2 in a one-electron redox reaction
producing OH and hydroxide anion:












 





(Eq. 1)

Furthermore, OH radicals can be generated by the

photocatalytic oxidation of water by TiO2 nanoparticles.
Absorption of the near-UV light by TiO2 is followed by
electron (e-)-hole (h+) pair generation (Eq. 2) CB represents
the conduction band and VB, the valence band. Then, the
trapped hole can react with chemisorbed OH- or H2O to
produce OH radicals (Eq. 3 and 4). In aerated systems, O2
acts as an efficient electron scavenger to trap the conduction
band electron to yield O2- radicals (Eq. 5), which then form
more OH  (Eq. 6) [11]:

TiO2

hv

eCB + h+VB

(Eq. 2)

h+VB + H2 O  OH
+ H+

(Eq. 3)

h+VB +

(Eq. 4)

2OH-  OH- + OH

Current Analytical Chemistry, 2012, Vol. 8, No. 4

-

-

eCB + O2  O2

-

O2 + 2H2 O  2OH
+ 2OH- + O2

429

(Eq. 5)
(Eq. 6)

In more specific cases, redox active metal complex

compounds, associated with DNA and in the presence of
both a reductant and dissolved oxygen, are able to form
hydroxyl radicals in close vicinity of the DNA backbone
[12]. Among these metallic compounds denoted as chemical
nucleases, a copper complex with 1,10-phenanthroline and
an iron complex with EDTA are often used in DNA cleavage
studies.
3.2. DNA-biosensors Based on OH Damaging Agent
Nucleic acids can be oxidized by various oxidants and in
particular by OH radicals. Cellular DNA oxidation by ROS
always results in DNA bases lesions, a significant source of
mutations leading to cancer, premature aging and other
degenerative diseases [13-15]. For this reason, the evaluation
of the protective effect of antioxidants can be achieved by
examining the integrity of DNA nucleobases. In this
direction, electrochemical DNA-based biosensors have been
described in order to simulate DNA damages caused by
ROS. The oxidative lesions are assessed on double stranded
DNA (dsDNA), purine DNA bases or single stranded DNA
(ssDNA) and are frequently studied by in vitro generation of
OH radicals. The different strategies reported in the
literature for DNA-biosensors are illustrated in Fig. (1).
The simplest strategy is based on the immobilization of
dsDNA, usually from Calf thymus, on screen-printed carbon
electrodes by simple adsorption and the detection of the
guanine oxidation peak by square wave voltammetry. Since
the peak current intensity is proportional to the guanine
concentration, the immersion of the DNA-modified electrode
into a Fenton solution produces a signal decrease in the peak
current intensity. The introduction of antioxidants into the
Fenton solution results in the scavenging of some OH 
radicals. Consequently, after immersion of the electrode into
this solution, the peak current intensity is very close to the
original one, which demonstrates the DNA integrity. The
results demonstrated that the DNA-based biosensor was
suitable as a rapid screening test for the evaluation of the
antioxidant properties of different plant extracts [16].
Another strategy to monitor antioxidants via the
determination of DNA damage uses an electrochemical label
able to interact with dsDNA. This interaction is based on an
intercalation phenomenon (predominantly at high ionic
strength) or on an electrostatic interaction (predominantly at
low ionic strength). The amount of redox label bound to the
DNA layer, which can be measured by differential pulse
voltammetry, decreases proportionally to the concentration
of radicals present into the sample [17-18]. As before, the
presence of antioxidants recovers the electrochemical signal.
By using this strategy, Bukov et al. [19] used
[Co(phen)3]3+ as a redox marker and [Cu(phen)2]2+ or
[Fe(EDTA)]- as DNA cleavage agents to evaluate the
antioxidant capacity of different yeast polysaccharides. The
same approach was used by Labuda et al. [20] to assess the
antioxidant activity of plant extracts containing phenolic
compounds.

430 Current Analytical Chemistry, 2012, Vol. 8, No. 4

Calas-Blanchard et al.

Table 1. Biosensors Applied to the Determination of the ROS Scavenging Capacity in Foods and Beverages

Bioreceptor

Immobilization

ROS Generation

Detection
Probe

Electrode

Sample

References

dsDNA

Physical
adsorption

Fenton reaction

OH

Carbon SPE

Plant extracts

[16]

dsDNA

Physical
adsorption

[Cu(Phen)2] 2+ or
[Fe(EDTA)]-

OH

Carbon SPE

Yeast polysaccharides

[19]

dsDNA

Physical
adsorption

[Fe(EDTA)]-

OH

SPE

Plant extracts

[20]

dsDNA

Covalent
bonding

Fenton reaction

OH

GCE

Sericin

[21]

Guanine/adenine

Physical
adsorption

Fenton reaction

OH

GCE

Flavored waters

[26]

ssDNA
(adenine)

Physical
adsorption

Fenton reaction

OH

CPE

Beverages

[27]

Guanine/adenine

Physical
adsorption

XOD

O2
-

GCE

Flavored waters

[57]

ssDNA
(adenine)

Physical
adsorption

XOD

O2
-

CPE

Ascorbic acid

[58]

Cyt c

SAM

XOD*

O2
-

Gold SPE

Garlic samples

[41]

Cyt c

SAM

XOD*

O2
-

Gold SPE

Orange juices

[34]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Red and white wines

[45]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Plant extracts

[46]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Tea samples and herbal products

[47]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Aromatic herbs, olives and fresh fruit

[48]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Dry spices

[49]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Algae samples

[50]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Phytotherapeutic diet integrators

[51]

SOD

Physical
adsorption

XOD

H2O2

Pt electrode

Papaya fruit and papaya-based food

integrators

[52]

Encapsulation
sol-gel

XOD*

H2O2

Pt SPE

Acetylsalicylic acid

[55]

HRP

Os gel polymer

XOD* (separately)

H2O2

Carbon SPE

Non-alcoholic beverages

[56]

SOD

Physical
adsorption

O2

Clark-type
electrode

Extra virgin olive oil

[54]

KO2 in DMSO

SAM: Self-assembled monolayer; SPE: Screen-printed electrode; dsDNA: double stranded DNA; ssDNA: single stranded DNA; GCE: Glassy carbon electrode, CPE: Carbon paste
electrode; XOD: Xanthine oxidase, XOD*: immobilized xanthine oxidase.

The immobilization of DNA strands on the electrode

surface is one critical step in the preparation of the DNA
biosensors. Qian et al. have recently developed a DNA
biosensor using dsDNA (herring sperm DNA) immobilized
on dendrimer-encapsulated Au-Pd bimetallic nanoparticles
with chitosan on a glassy carbon electrode (GCE) [21]. This
method could enhance the immobilized amount of DNA

strands and increase the stability of the biosensor. According

to the authors, the use of Au-Pd nanoparticles could enlarge
the electrochemical signal of DNA indicator and increase the
sensitivity for DNA detection. The oxidative damage of
immobilized DNA generated by OH
radicals was evaluated
by square wave voltammetry. The proposed method allowed
assessing the antioxidant capacity of sericin, a globular

Determination of the Antioxidant Capacity Using ROS-based Biosensors

Current Analytical Chemistry, 2012, Vol. 8, No. 4

431

Fig. (1). Schematic diagram of the DNA-based biosensors principle reported by (1) Qian et al. [21], (2) Kamel et al. [26] and (3, 4 and 5)
Barroso et al. [27, 57-58]. A: adenine, AO: antioxidant.

protein that has potentially been used for food additives. The
drawback of this technique is related to the potential OH

radical scavenging activity of metallic nanoparticles that
could lead to a global overestimation of the antioxidant
capacity [22-24].
Recently, the oxidative damage of purine DNA bases
(guanine and adenine) by the OH
radical was evaluated. All
DNA bases can be used for the electrochemical study, but
purine bases are more sensitive for detection and present
lower potential peaks than pyrimidine bases [25]. Kamel et
al. [26] immobilized guanine and adenine DNA bases by a
simple electrochemical treatment of the GCE surface to
determine the antioxidant capacity of flavored water
samples. The method relies on monitoring the changes of the
intrinsic anodic response of the surface-confined guanine and
adenine species, resulting from its interaction with free
radicals from Fenton-type reaction in absence and presence
of antioxidants.
On the other hand, Barroso et al. have developed an
electrocatalytic voltammetric method to assess the
antioxidant capacity using DNA-modified carbon paste
electrodes (CPEs). In this work, short ssDNA was used to
obtain a higher oxidation current at the electrode surface than
with dsDNA. The adenine-rich oligonucleotide was adsorbed
on the CPE and was partially damaged by OH
radicals
generated in a Fenton-type reaction [27]. Then, the
remaining unoxidized adenine bases were electrochemically
oxidized to give rise to an adsorbed oxidation product that
was able to catalyze the oxidation of NADH in presence of
calcium ions. The presence of antioxidant compounds
scavenged OH
radicals leaving more adenines unoxidized,
and thus increasing the electrocatalytic current of NADH
measured by differential pulse voltammetry. This DNA
biosensor was applied to the determination of the antioxidant
capacity of lemon flavored waters.
DNA sensors are promising devices to perform simple
tests for the routine evaluation of the antioxidant capacity of
samples in an easy way. The use of these biosensors is close
to biological systems occurring in vivo, with a nucleotide
being damaged by free radicals. Moreover, the choice of

screen-printed carbon as electrode material leads to

disposable and low-cost analytical tools.
4. MONITORING SUPEROXIDE RADICALS
Electrochemical biosensors are particularly suitable to
determine superoxide radicals (O2
-) in a real time and
sensitive way. Among them, cytochrome c (cyt c)-based and
superoxide dismutase (SOD)-based biosensors are the
principal ones, even if innovative strategies are nowadays
emerging.
4.1. Generation of O2For the in vitro production of O2
- radicals, an enzymatic
reaction is often employed: xanthine oxidase (XOD)
catalyzes the oxidation of hypoxanthine to xanthine and can
further catalyze the oxidation of xanthine to uric acid with
concomitant reduction of O2 to H2O2. O2
- is formed as an
intermediate of these reactions [28]:

 




























(Eq. 7)








(Eq. 8)

In such a system, it must be highlighted that both O2
and H2O2 ROS are involved when evaluating the antioxidant
capacity.
At fixed experimental conditions, this enzymatic
catalysis allows the production of similar quantities of O2
radicals even if it seems impossible to determine precisely
the real amounts produced.
On the other hand, O2
- radicals can be obtained by
adding NaOH to dimethyl sulfoxide (DMSO) [29]. Finally,
the simple injection of KO2 in aprotic organic solvents,
especially DMSO, also results in O2
- generation [30-31]:








(Eq. 9)

There are two main types of electrochemical O2biosensors, based on either cyt c or SOD. The first group is
based on the reduction of the immobilized cyt c by O2-

432 Current Analytical Chemistry, 2012, Vol. 8, No. 4

radicals followed by the electrooxidation of the redox protein

at the working electrode surface. The second group is based
on the specific dismutation of O2- by SOD to O2 and H2O2.
The electrochemical oxidation of H2O2 usually occurs at a
high potential (> 0.5 V vs. Ag/AgCl), resulting in
interference problems and limiting the practical applications
of this second type of biosensors. Then, several recent and
alternative strategies developed to determine antioxidant
capacities via O2- scavenging abilities are presented.
4.2. Cyt c-based Biosensors
The detection principle of these biosensors is based on
the redox reaction between cyt c-(Fe3+) and O2
- followed by
the further oxidation of the cyt c-(Fe2+) at the electrode
surface, leading to an oxidation current proportional to the
radical concentration in solution [32]. In order to avoid
interference from H2O2, generated as a final product of the
enzymatic reaction or by spontaneous dismutation of the O2
radical, catalase enzyme is sometimes added to the reaction
media. The addition of antioxidants reduces the radical
concentration and thus the oxidation current, allowing the
quantification of the antioxidant capacity.
The need for catalase, when XOD remains in solution, is
not clear and some opposite results can be found in the
bibliography. On the one hand, Tammeveski et al. [32]
stated that no change in the electrode response was seen in
the presence of catalase over the range of XOD
concentrations used, thus eliminating the possibility of H2O2
causing re-oxidation of the reduced cyt c and leading to
overestimation of the response due to superoxide. On the
other hand, Lisdat et al. [33] stated that a stable cyclic
voltammogram during the enzymatic radical production by
the HX/XOD system was only visible in the presence of
catalase. In fact, this enzyme removed the coproduced H2O2
which otherwise would interfere with the measurement
because of the reaction with the reduced cyt c. Therefore,
Tammeveski et al. claim that H2O2 do not react with cyt c
while Lisdat et al. claim that both species can react if
catalase is not present in the reaction media. On the other
hand, when XOD is immobilized on the electrode surface,
catalase is needed to obtain stable and reproducible signal
current responses [34].
The electron transfer between cyt c and the electrode
becomes essential. However, such an electron transfer cannot
be readily obtained at electrodes commonly used in
electrochemistry, such as glassy carbon, gold and platinum.
For this reason, electrodes (especially gold ones) are
modified with self-assembled monolayers (SAMs). These
modifiers are employed as promoters for facilitating the
electron transfer of cyt c. From a practical application point
of view, a free orientation of the cyt c without denaturation is
generally required for achieving an effective electron transfer
between cyt c and the electrode and for the subsequent
biosensing of O2
- radicals. In this case, SAMs of
alkanethiols formed onto gold electrodes are remarkable
because they cannot only facilitate direct electron transfer of
cyt c but also prevent the electrode from the potential
interferents in the solution, such as H2O2 and uric acid.
Although, short-chain alkanethiols show a high efficiency of
communication between cyt c and the electrode [32, 35],

Calas-Blanchard et al.

they cannot form dense films and thus, do not effectively

block the electrode from interfering substances. For this
reason, cyt c has been immobilized on long-chain
alkanethiols-modified electrodes. These cyt c-based
biosensors can be used for O2
- detection and for the analysis
of antioxidant activities [33, 36-37]. In order to increase the
electron transfer rate of cyt c, Ge and Lisdat [30] employed
mercaptoundecanoic acid/mercaptoundecanol mixed SAMmodified gold electrodes. This procedure had already been
described and applied by Gobi and Mizutani [38-39] for the
O2
- sensing. However, they still used short-chain modifiers,
which limited the selectivity. These mixed SAM-modified
biosensors showed more sensitivity to O2
- radicals [40].
Although many researchers have been working on the
development of cyt c-based biosensors for the O2
determination, few applications to the antioxidant capacity
assessment in foods and beverages have been published to
date.
Our research group developed a cyt c-based biosensor for
the determination of the antioxidant capacity of different
orange juices and both alliin and allicin extracted from garlic
bulbs [34, 41]. The developed biosensor was based on the
co-immobilization of both cyt c and XOD on a mercaptoundecanoic acid/mercaptoundecanol mixed SAM-modified
gold electrode. By using this approach, better sensitivities
were achieved since cyt c and XOD were very close and the
generated O2
- radicals could quickly react with cyt c,
avoiding its spontaneous dismutation.
4.3. SOD-based Biosensors
SOD biosensors are shown as a promising alternative to
cyt c biosensors for the evaluation of the antioxidant
capacity. Compared with cyt c, SOD is more specific for
O2
- radicals and the rate of the reaction between O2
- and
SOD is several orders of magnitude higher [31, 42-43]. SOD
has been well addressed for the dismutation of the O2
radical into O2 and H2O2 with strong activity and great
specificity.








(Eq. 10)

First-generation SOD-based biosensors are mainly based

on the detection of the H2O2 oxidation at the electrode
surface, leading to the estimation of the antioxidant capacity
due to the scavenging ability of both O2
- and H2O2 ROS.
Nevertheless, this oxidation reaction occurs at a high
potential (> 0.5 V vs. Ag/AgCl), which results in interference problems, limiting the practical applications of these
biosensors. Strategies to improve the selectivity of the
developed sensors include the use of a H2O2-impermeable
Teflon membrane [44] or two-channel sensors [31]. The
latter method allows the simultaneous determination of O2
radicals and H2O2.
Campanella and co-workers [45-52] have been working
on the development of different SOD-based biosensors for
assessing the antioxidant capacity of several compounds.
Most of their work is based on the amperometric detection of
H2O2. For the production of O2
- radicals, XOD was added to
the solution while SOD was immobilized in a kappacarrageenan gel. The gel containing the enzyme was

Determination of the Antioxidant Capacity Using ROS-based Biosensors

sandwiched between a cellulose acetate membrane, which

improved the selectivity of the electrode by blocking the
access to possible electroactive interferences, and an external
dialysis membrane. The addition of a sample possessing
antioxidant properties produced a decrease in the signal
strength as the antioxidant species reacted with the O2
radical, thus reducing its concentration in solution. There
was a consequent decrease in the released H2O2 and thus also
in the intensity of the amperometric signal. This biosensor
was used to evaluate several products: red and white wines
[45], fresh aromatic herbs, olives and fresh fruit, bulbs and
vegetables, plant products sold by herbalists and/or
pharmacies, tea [46-48], dry spices [49], algae [50],
phytotherapeutic diet integrators [51], and papaya fruit and
papaya-based food integrators [52].
However, it is known that a large number of molecules
with interesting scavenging properties are difficult to
determine because of their very low solubility in water. This
latter point led the same authors to the development of a
biosensor capable of operating in organic solvents, in which
these compounds are more soluble. The developed O2
biosensor, successfully used in a DMSO solution, was based
on SOD entrapped in a cellulose triacetate layer (sandwiched
between two gas-permeable membranes) or in a kappacarrageenan gel layer (sandwiched between an external gas
permeable membrane and an internal cellulose acetate
membrane), coupled to an O2 amperometric transducer [53].
This biosensor was applied to check the free radical
concentration of extra virgin olive oil [54].
4.4. Recent Strategies in the Development of Biosensors
Involving O2- Radicals
Due to the high chemical reactivity of O2
- radicals, the
usefulness of SOD when developing O2
- biosensors has
been contested. Recently, new electrochemical biosensors
have been described with XOD as generator of O2
- radicals
but without SOD. In this sense, our research group [55] has
developed a biosensor for the assessment of the antioxidant
capacity with XOD immobilized in mild conditions by
encapsulation in a sol-gel matrix whereas in most of the
previously cited publications this enzyme remained in
solution. This analytical tool was based on the detection of
H2O2, generated as a final product of the enzymatic reaction
catalyzed by XOD and by the spontaneous dismutation of
O2
- radicals. H2O2 was selectively monitored at +700 mV
vs. Ag/AgCl using a highly selective poly-m-phenylenediamine membrane. This H2O2 perm-selective membrane
was used in order to block the nonspecific oxidation of other
oxidizable molecules avoiding interferences. The applicability of this method was demonstrated by analyzing the
antioxidant capacity of acetylsalicylic acid. Nevertheless,
this biosensor was not used in the analysis of food samples.
On the other hand, their collaborators developed a flow
system for the antioxidant capacity estimation consisting of a
bioreactor with immobilized XOD (for the generation of O2
radicals) coupled with a H2O2 amperometric biosensor [56].
The bioreactor was constructed using Teflon tubing
containing XOD immobilized on cyanogen bromideactivated Sepharose 4B gel, silica gel or controlled-pore
glass. The biosensor was developed separately by
immobilizing electrically wired (via Os-redox polymer)

Current Analytical Chemistry, 2012, Vol. 8, No. 4

433

horseradish peroxidase (HRP) on a screen printed graphite

working electrode. The H2O2, produced during the enzymatic
oxidation of xanthine as well as by the dismutation reaction
of the O2
- radicals, was amperometrically monitored on the
HRP-Os biosensor at -0.1 V vs. Ag/AgCl/KClsat, avoiding
electrochemical interferences. The antioxidant capacity was
estimated for some non-alcoholic beverages.
Even if the oxidative lesions in DNA are frequently
studied by in vitro generation of OH
radicals (3.2), recent
works deal with the effect of O2
- radicals. All the DNAbiosensors principles for the assessment of the antioxidant
capacity are represented in Fig. (1). Barroso et al. have
reported the development of two DNA biosensors for
monitoring the antioxidant capacity of beverages based on
damages caused by O2
- radicals. In the first report, guanine
and adenine bases were firstly immobilized on glassy carbon
electrodes. Then, the DNA biosensor was immersed in an
aqueous O2
- radical solution that was generated by the
enzymatic reaction between XOD and xanthine, leading to
an oxidative damage of the purine bases [57]. The decrease
of the oxidative current of guanine and adenine was recorded
in square wave voltammetry and was used to relate the
extent of oxidative damage. Among the five standard
antioxidants used to protect purine bases, ascorbic acid
presented the highest antioxidant capacity and seemed to be
the most sensitive standard capable to discriminate the
several ingredients added to the flavored tested waters. With
this DNA biosensor, colored samples could be directly used
for the measurements without any pretreatment. In the
second publication, an adenine oligonucleotide was adsorbed
on a carbon paste electrode and was partially damaged by
O2
- radicals also generated by the xanthine/XOD system
[58]. The oxidative lesions were indirectly quantified after
the electrochemical oxidation of the remaining unoxidized
adenine bases to generate a catalyst species that mediates the
oxidation of NADH. The electrocatalytic current of NADH
was measured by cyclic voltammetry. The DNA biosensor
was applied to the determination of the antioxidant capacity
of beverages and lemon flavored waters and the results were
compared with previous reports using OH
. Although the
damage in terms of adenine oxidative lesions was similar
using OH
or O2
- radicals, the scavenging activity of the
tested antioxidant was lower with O2
- radicals due to the
higher concentration of antioxidants needed for similar
efficiencies.
5. CONCLUSIONS
This article is focused on the electrochemical biosensors
developed for the detection of antioxidants in natural
products and additives in food and drink industries. The
antioxidant capacity is deduced from the ability of
compounds to scavenge free radicals. Free radicals have a
very short lifetime, which makes them very hard to measure
with conventional techniques. When developing biosensors
for the determination of the antioxidant capacity, the ROS in
vitro generation facilitates their detection. Multiple
biosensors based on ROS detection are available today for
the evaluation of the antioxidant capacity in foods and
beverages. Due to their major importance in living systems
during cellular metabolism, OH
and O2
- radicals are the
ROS always implicated in such biosensors. Both of these

434 Current Analytical Chemistry, 2012, Vol. 8, No. 4

radicals can be involved in DNA-biosensors while all the

other sensors are based on the O2- radical scavenging
ability. In order to determine OH radicals, mainly generated
by the Fenton reaction, DNA-based biosensors have been
developed. These sensors are based on the damages induced
to DNA by free radicals and they really simulate the DNA
alterations existing in vivo. For the determination of O2radicals, usually generated through the xanthine/XOD
enzymatic system, cyt c and SOD biosensors are commonly
used. Among them, SOD-based biosensors are the most
sensitive and, in principle, the most selective. Nowadays,
simplest and promising strategies are appearing. The
applicability of all these electrochemical biosensors for the
detection of antioxidants in food and beverages is
demonstrated. There is a growing interest in the development
of such devices, as demonstrate the numerous configurations
recently appeared in the literature. Their advantages in terms
of simplicity, rapidity and low cost respect to traditional
techniques promote their investigation and exploitation.
Furthermore, biosensors have also the advantage of being
reversible. Nevertheless, further work is required to avoid
the interferences problem and to consolidate them as
practical and current antioxidant assessment tools.
CONFLICT OF INTEREST

Calas-Blanchard et al.

[11]

[12]
[13]

[14]
[15]

[16]

[17]

[18]

[19]

Declared none.
[20]

ACKNOWLEDGEMENTS
Declared none.
[21]

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Accepted: September 15, 2011