Biosensors and Bioelectronics 49 (2013) 146–158

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Biosensors and Bioelectronics

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

Electrochemical affinity biosensors for detection of mycotoxins:

A review
Juan C. Vidal a,n, Laura Bonel b, Alba Ezquerra a, Susana Hernández a, Juan R. Bertolín a,
Carlota Cubel a, Juan R. Castillo a
a
Analytical Spectroscopy and Sensors Group (GEAS), Institute of Environmental Sciences (IUCA), University of Zaragoza, c/ Pedro Cerbuna, 12,
50009-Zaragoza, Spain
b
CAPHER IDI S.L., c/ Ermesinda de Aragón, 4, c-116, 50012-Zaragoza, Spain

art ic l e i nf o a b s t r a c t

Article history: This review discusses the current state of electrochemical biosensors in the determination of mycotoxins
Received 13 February 2013 in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of
Received in revised form these results in serious human and animal health problems, although it has been only since early 1960s
26 April 2013
when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of
Accepted 2 May 2013
Available online 9 May 2013
agricultural products, most important cereals and cereal-based foods. A majority of countries, mention-
ing especially the European Union, have established preventive programs to control contamination and
Keywords: strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires
Mycotoxins sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, com-
Electrochemical affinity biosensors
bined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and
Immunosensor
faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very
Aptasensor
Amperometry promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or
Electrochemical impedance spectroscopy conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical
biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant
antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals
with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete
literature from the first reports about sixteen years ago.
& 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
2. The importance of mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
2.1. Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
2.2. European legislation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
2.3. Analytical techniques for mycotoxin determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
3. Electrochemical affinity biosensors for mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3.1. Electrochemical transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3.2. Recognition receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3.2.1. Antibodies and antibody fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3.2.2. Aptamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3.2.3. DNA and artificial receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4. Electrochemical biosensors: selected applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4.1. Aflatoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4.2. Ochratoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
4.3. Trichothecenes and other mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
5. Recent advances in electrochemical mycotoxin biosensors and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

n
Corresponding author. Tel.: +34 976 762253; fax: +34 976 761292.
E-mail address: jcvidal@unizar.es (J.C. Vidal).

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bios.2013.05.008
 J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158 147

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

1. Introduction
Mycotoxins are a large and varied group of mold-secondary
metabolites, with common features they are all produced by fungi
and have toxic effects against vertebrates and other organisms.
Filamentous fungi produce thousands of toxic compounds, but the
more important mycotoxins belong to species of Aspergillus,
Fusarium, and Penicillium (Moss, 1996). The most relevant myco-
toxins under a toxicological and legislative point of view are the
aflatoxins, ochratoxins, some trichotecenes (fumonisins, deoxyni-
valenol, T-2, HT-2, and zearalenone), patulin, citrinin, and ergot
alkaloids. The chemical structures of selected molecules are
depicted in Fig. 1 (Bennett and Klich, 2003). Mycotoxins affect a
broad range of agricultural products including cereals, cereal-
based foods, dried fruits, wine, milk, coffe beans, cocoa bakery or
meat products, which are the basis of the economies of many
developing countries (Shephard et al., 2012).
Mycotoxins likely have existed for as long as crops have been
grown, but recognition of the true chemical nature was not known
until recent times. Occurrence has been taken into account from
the recognition of aflatoxins in the early 1960s (Shephard, 2009),
and determination are based upon their occurrence and/or the
severity of the disease they produce (mycotoxicoses), especially if
they are known to be carcinogenic (Richard, 2007).
Official methods for determination of mycotoxins are usually
performed in accredited laboratories with sophisticated instru-
mentation like high-performance liquid chromatography (HPLC)
with fluorescence (FLD) or mass (MS) detectors, but there is a
growing demand for small devices and rapid determinations
preferably in-situ. In about last sixteen years many approaches
have appeared developing new affinity biosensors for mycotoxins,
especially electrochemical biosensors, which are the main subject
of this review. Some excellent reviews are available on analytical
methods (Cigic and Prosen, 2009; Turner et al., 2009; Koppen
et al., 2010; Shephard et al., 2012), general biosensors (Logrieco
et al., 2005; Campas et al., 1055; Maragos and Busman, 2010;
Prieto-Simon et al., 2007) and electrochemical biosensors
(Palchetti and Mascini, 2008; Laschi et al., 2011) for determining
mycotoxins.
The goal of this review is to cover the full scope of electro-
chemical biosensors for mycotoxins. Of particular interest are food
contamination and the presence of conjugated (masked) myco-
toxin derivatives. Analytical determination of mycotoxins in foods
is extremely important due to its high toxicity in humans and
animals, and many countries around the world have established
very strict controls of those foods likely to be contaminated during
harvest or storage. With increasing globalization and world trade,
the mycotoxin problem will significantly grow in coming years.
2. The importance of mycotoxins
2.1. Toxicity
The contamination of food by mycotoxins has become a matter
of great concern, as these are responsible for many diseases
(Richard, 2007). According to an estimate, 25% of the world's crops
are affected by toxigenic fungi, so that mycotoxins have become
part of the food chain. High levels of mycotoxins in the diet can
cause adverse acute and chronic effects on human health and a
variety of species animals. Side effects may particularly affect the
liver, kidney, nervous system, endocrine and immune systems
(Cigic and Prosen, 2009).
Aflatoxin B1 (AFB1) (Aspergillus flavus and Aspergillus parasiti-
cus) was first described in the early 1960s, is a potent human
carcinogen (first hazard class according to the IARC classification)

Fig. 1. Molecular structures of the main mycotoxins.

148 J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158
and primarily responsible for liver cancer in animals. AFB1 occurs
in the low sub-nanogram per gram range in a variety of food
products and owing to carcinogenic properties immediately arose
requirements to meet food safety and new legislative regulations
(Olsen et al., 2006). When AFB1 is ingested with feed by cows, it is
transformed into its hydroxylated metabolite product, aflatoxin
M1 (AFM1), which is then secreted in the milk. Unfortunately,
AFM1 is relatively stable during milk pasteurization and storage,
as well as during the preparation of various dairy products
(Gordon, 2009).
Ochratoxins, especially Ochratoxin A (OTA) (Aspergillus and
Penicillium fungi) was firstly isolated in 1965 and occurs in a large
variety of commodities, mainly in cereals. Because of the persis-
tence of OTA in the food chain and carry-over effect, OTA can also
contaminate milk or poultry meat from animals fed with contami-
nated feed (Van-Egmond et al., 2007). OTA belongs to carcinogenics
of the second hazard class, and is known to be mutagenic,
teratogenic and immunosuppressive. Other metabolites like ochra-
toxin B and ochratoxin C occur rarely in foods.
Fusarium mold fungi produce fumonisins and trichothecenes
(deoxynivalenol, T-2, HT-2, zearalenone), with chemical structures
shown in Fig. 1. Fumonisins are known from the middle 1980s and
are long-chain poly-hydroxy-alkylamines containing tricarboxylic
fragments. At least 13 structurally different fumonisins have been
described. Major health effects in humans of the most important
fumonisins B1 and B2 (FB1 and FB2 respectively) include liver and
kidney tumors and oesophagal cancer (Scott, 2012). Trichothecenes-
type A (deoxynivalenol and nivalenol, named DON and NIV respec-
tively) are cyclic sesquiterpenoids and produce food refusal and
vomiting together with kidney problems. Zearalenone (ZEA, a resor-
cyclic acid lactone), and their metabolites are analog of estrogens
having estrogenic effects and reproductive toxicity. Toxic effects of
fusarium toxins are too severe in animals like pigs, poultry, cattle and
horses. Another area of focus is the use of some trichothecene-type B
(T-2, HT-2) mycotoxins as bioweapons in wars, since mold-toxins are
cheap, easy to access and can be applied to a small group of enemies
(Rai and Varma, 2010).
2.2. European legislation
From the mid of 2000s, approximately 100 countries (covering
approximately 85% of the world's inhabitants) had specific regula-
tions or detailed guidelines for the occurrence of mycotoxins in
food (Van-Egmond et al., 2007). The over-all objective is the
protection of the consumer´s health, together to describing pro-
cedures for decreasing amounts in food commodities. In the
countries of the European Union (EU) since the 1960s regulations
increase based on scientific opinions of authoritative agencies, for
example the EFSA (European Food Safety Authority) FVO (Food
and Veterinary Office) and the CPVO (Community Bureau of Plant
Varieties), giving requirements and recommendations for ade-
quate sampling, alerts and analytical methods (Olsen et al.,
2006; Van-Egmond et al., 2007).
Harmonized regulations in European Union now exist for a
number of mycotoxins-food combinations. One of the most impor-
tant legislation is Comission Regulation 1181/2006 (Commission
Recommendation, 2006) setting maximum levels of aflatoxins, OTA,
patulin, DON, ZEA, fumonisins and unlimited T-2 and HT-2. While the
high toxicity of HT-2 and T-2 is supported, the lack of sufficiently
sensitive analytical methods makes their maximum concentration
levels are waiting to be defined in the EU. Comission Regulation
1126/2007 updates the maximum allowed levels of fusarium myco-
toxins in maize and products maize (DON, ZEA fumonisins) in a
range 50–2000 μg kg−1 (Commission Recommendation, 2007).
Allowed limits depend on the toxicity but also on the kind of food.
For example AFB1 is regulated by legislation in the EU at 2 μg kg−1 in
foods for direct human consumption (Commission Recommendation,
2006). But AFM1, although not as potent as AFB1, is regulated in the
EU at the level of 0.05 μg kg−1, due to the high consumption of milk
by infants and children. OTA is allowed in very small concentrations
in about a range 0.5–10 μg kg−1 also depending on the kind of food
(Commission Recommendation, 2006a). Samplig methods of analysis
for the official control of mycotoxins (Commission Directive, 2005),
recommendations for prevention/reduction (Commission Recom-
mendation, 2006a) and regulatory limits for product intended for
animal feeding (Commission Recommendation, 2006b) are also
available from the EU.
2.3. Analytical techniques for mycotoxin determination
As low ppb´s concentrations are usually involved, very sensitive
analytical methods for mycotoxins are needed (Krska and Molinelli,
2007). Conventional analytical methods are mainly based on
separative instrumental techniques (e.g. high-performance liquid
chromatography, HPLC) and enzyme immunoassays for screening
purposes (e.g. enzyme-linked immunosorbent analysis, ELISAs)
(Koppen et al., 2010).
The analytical techniques currently used in official methods are
HPLC with reverse-phase columns, using molecular fluorescence
(Shephard, 2009), UV absorption or mass-spectrometry detectors
(Turner et al., 2009), affording detection limits of 0.01–5 ng mL−1.
These methods involve preconcentration and cleanup procedures
for the removal of matrix and interferents from samples, for
example using immunoafinity columns comprising anti-mycotoxins
antibodies or solid-phase extraction columns with organic solvents.
Sometimes analyte derivatization is necessary for optical detection.
The intrinsic electroactivity of some mycotoxins has been exploited
to develop amperometric detection in HPLC of e.g. ZEA (Smyth and
Frischkorn, 1980; Campas et al., 2012), adsorptive stripping voltam-
metry of DON, AFB1 and AFB2 at trace levels (Campas et al., 2012),
square-voltammetry of OTA in wines (Perrotta et al., 2011), the
amperometric screening of ZEA and their metabolites α,β-zearalenol
(Zougagh et al., 2008), and a 96-well ELIME array for direct electro-
chemical sensing of DON and nivalenol after microwave hydrolysis
of the samples (Ricci et al., 2009), but all these methods do not
fulfill the sensitivity required for food applications (Prieto-Simon
et al., 2007).
Microtiter plate spectrophotometric ELISAs have become for
over 20 years one of the most useful tools for rapid monitoring of
mycotoxins, especially for the screening of raw materials, getting
sometimes detection limits as low as 0.1 ng g−1 (Zheng et al.,
2006). The technology is based on the ability of specific antibodies
to distinguish the three-dimensional structure of a mycotoxin and
the establishment of a competitive incubation in times of approxi-
mately 1–2 h. Despite high matrix dependence and possible over-
estimation, the great advantages of ELISAs are speed, ease of
operation, sensitivity, and high sample throughput. A number of
commercial ELISAs for mycotoxins are well established and avail-
able for field use (Cigic and Prosen, 2009). A number of other
immunoassay-based techniques have been developed, one of the
simplest and fastest (responses in a few minutes) technologies is
the lateral flow devices, usually in the format of a strip or dipstick,
but only qualitative or testing a cutoff value information can be
given (Maragos, 2004). For example, aflatoxin if present in the
sample extract interacts with colloidal gold conjugated anti-
aflatoxin antibodies at the base of such a stick (Krska and
Molinelli, 2007).
Owing to the control of food safety and the high demand for
rapid and accurate methods to detect mycotoxins, the development
of biosensors is crucial, has increased significantly in the last 20
years, and is currently one of the most active areas of mycotoxin
analytical research (Ricci et al., 2007). Biosensors involve biological
 J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158
recognition elements (such as enzymes, antibodies, nucleic acids
or artificial receptors) in contact with a transducer. Related to
physicochemical properties of micotoxins (e.g. fluorescence) or
the type of transduction, four groups of biosensors are mostly used:
luminescent/colorimetric (Goryacheva et al., 2007), surface plasmon
resonance (SPR) sensors (Maragos, 2004), mass-sensitive quartz-
crystal microbalance (QCM) sensors (Vidal et al., 2009), and
electrochemical sensors (Laschi et al., 2011). Most of the importance
of biosensors relies on their high sensitivity and specifity with
minimum sample treatment. Some other reviews summarize elec-
trochemical (Tombelli et al., 2009; Palchetti and Mascini, 2012),
optical (Goryacheva et al., 2007; Zheng et al., 2006), and other
transducing (Maragos, 2004; Goryacheva et al., 2007; Prieto-Simon
et al., 2007) biosensors for toxin analysis. Electrochemical biosen-
sors are predominant among the above groups and are the main
subject of this review (Farre et al., 2009).
3. Electrochemical affinity biosensors for mycotoxins
Electrochemical biosensors for mycotoxins detection are pro-
minent owing to their sensitivity, selectivity, low cost, simplicity,
and in some cases miniaturization, portability and integration in
automated devices (Farre et al., 2009). Most of them are based on
the high affinity interactions between antigen and specific anti-
bodies, but novel specific ligands (e.g. aptamers) are emerging. To
transform the toxin interaction to an analytical signal, a variety of
electrochemical techniques (amperometric, potentiometric, con-
ductimetric, impedimetric) have been used (Palchetti and Mascini,
2008; Laschi et al., 2011; Grieshaber et al., 2008).
3.1. Electrochemical transduction
Amperomety (and voltammetry) is undoubtedly the most
suitable electrochemical transducer due to their high sensitivity
and working response over a wide range of mycotoxin concentra-
tions. Widely used amperometry is based on the measurement of a
current under a fixed (potentiostatic control) or variable (voltam-
metry) potential. A large amount of research effort has been
directed toward finding electrode configurations or nano-
structurations. Electrodes are commonly made of inert metals
(Pt, Au) or carbon (graphite, glassy-carbon), although the main
drawback is the regeneration between measurements, so today
single-use disposable screen-printed electrodes (SPEs), character-
ized by low-cost and mass-production, are widely used to over-
come this problem. (Ricci et al., 2007; Grieshaber et al., 2008).
Electrochemical impedance spectroscopy (EIS) is a powerful,
very sensitive, nondestructive and informative technique which
allows to study the electrical properties of the sensing device
interface and trace the reactions occurring on it (Laschi et al., 2011;
Zamfir et al., 2011). Label-free EIS immunosensor and aptasensors
follow changes in electron-transfer resistance at the working
electrode surface resulting from complexation of the mycotoxins
in the range 1–20 ng mL−1, with detection limits of about
0.5 ng mL−1. One of the main advantages of EIS biosensors is no
need of conjugating antibodies or mycotoxins with an enzyme to
provide the electrochemical signal (Grieshaber et al., 2008). This
implies modification of the electrode surface, for example using an
Au electrode surface, 4-carboxyphenyl (4-CP) film is grafted by
electrochemical reduction of the corresponding 4-carboxyphenyl
diazonium salt for the covalent linkage of the antibody to
construct a sensitive impedance immunosensor detecting OTA
(limit of detection: LOD ¼0.5 ng mL−1) (Radi et al., 2009b).
Although much less used owing to somewhat lower signal/
noise ratio, potentiometric (Rameil et al., 2010) and conducti-
metric (Liu et al., 2006) immunosensors have also been described
149
for aflatoxin (Rameil et al., 2010; Liu et al., 2006), and DON (Kwon
et al., 2011) toxins. Potentiometric devices detect changes in
potential at zero current, while conductimetric devices detect
changes in conductivity between two electrodes. Chemically
sensitized field effect transistors (CHEMFET) are an important
subgroup of the potentiometric biosensors and have been used
for detecting DON (Kwon et al., 2011).
3.2. Recognition receptors
Both natural (antibodies, DNA) and artificial (engineered anti-
bodies, antibody fragments, aptamers and receptors like molecu-
larly imprinted polymers) have been used for the selective binding
of mycotoxin molecules (Romanazzo et al., 2010; Campas et al.,
2012). Specific antibodies raised for the selected mycotoxins are
the most common (Rasooly and Herold, 2007), but using of
aptamers as recognizing ligands is currently growing in the case
of OTA.
Some authors have used the enzymatic inhibition produced by
mycotoxins to obtain enzymatic biosensors for these analytes.
Nevertheless, the main problem of this kind of biosensors is the
lack of selectivity, as other toxins or molecules from the sample
might also inhibit the enzyme response (Arduini et al., 2007). For
instance, amperometric biosensors were developed for alternaria
mycotoxins (AOH, AME) quantified with mushroom tyrosinase on
a carbon paste electrode (Moressi et al., 1999), for aflatoxin B1
inhibiting acetylcholinesterase (AChE) (Pohanka et al., 2010;
Arduini et al., 2010; Ben-Rejeb et al., 2009; Cuccioloni et al.,
2008) and for OTA acting as substrate of horseradish peroxidase
(HRP) immobilized in a polypyrrole matrix (Alonso et al., 2010).
These sensors remain as a “proof of concept” rather than practical
assays, due to lack of selectivity (e.g. AChE is irreversibly inhibited
by organophosphorus and carbamate pesticides (Vidal et al.,
2008)) and high LODs.
3.2.1. Antibodies and antibody fragments
The synthesis of mycotoxin specific antibodies has allowed a
range of analytical immunoassays which rely on the selective
recognition of any epitope of the mycotoxin. Mycotoxins are small,
non-immunogenic molecules (haptens) and have to be bound to
suitable carrier proteins to elicit adequate immune response. This
implies some kind of possible affinity to this protein, e.g. for BSA
(Vidal et al., 2011). Most of the immunosensors detecting myco-
toxins are based on competitive assays, and typical formats
include direct and indirect schemes (Maragos, 2009), but not
sandwich formats due to small size of these molecules.
From their mode of production, there are three kinds of
antibodies: polyclonal (pAb), monoclonal (mAb) and recombinant
(rAb). Polyclonal are purified from the blood of immunized
animals having the benefits of low cost and easy development.
But it is very important careful purifying of the conjugate (to gain
selectivity) and sometimes appears cross-reactivity to the protein
used for this conjugate (e.g. BSA). MAbs are produced from
positive hybridomas by fusing murine myeloma cells and spleen
cells from immunized mice. Uniformity with same analytical
properties and unlimited production are their benefits. The third
generation in Ab technology are recombinant antibodies (rAbs),
which does not require animals. The functional gene of some Ab cn
be cloned and transmitted into prokaryotic or eukaryotic organ-
isms (genetically modified organisms) from positive hybridoma or
spleen cells with or without immunization. Finally, rAbs can be
expressed, screened and collected (Li et al., 2009).
Antibodies can also be treated with reducing agents or proteo-
lytic enzymes to generate fragments that retain the antigen
binding region (Fab) and increased affinity and specifity (Maragos,
150 J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158
2009). In addition it is possible with recombinant technologies to
isolate the variable portions of the immunoglobulin that are
primarily involved with toxin binding (Maragos, 2009). Fab frag-
ments are shown as an alternative to whole antibodies, and their
smaller size contributes to the minimization of non-specific binding
due to interactions through the Fc fragment and lowering the steric
hindrance in heterogeneous systems (Romanazzo et al., 2010).
Phage-display methods can also be used to identify molecules from
a peptide library with affinity for the mycotoxin, ligands that can be
generated against this toxic antigens which are harmful to animals
and thus with difficulties to produce with traditional antibody-
raising techniques.
3.2.2. Aptamers
Aptamer ligands are short sequences (20–90 oligonucleotides)
of single stranded nucleic acids (DNA, RNA) that can bind with
high affinity and specifity to a wide range of targets, ranging from
large proteins to small molecules like aminoacids or drugs. They
are obtained by an in vitro selection process named SELEX
(systematic evolution of the ligand by the exponential enrichment
process), which was first reported in 1990 (Ellington and Szostak,
1990). Aptamers have the ability in certain physicochemical
conditions to fold into defined three-dimensional conformations,
which facilitate specific interactions with target molecules having
high affinity constants (Hayat et al., 2012). While antibodies have
been the standard for molecular recognition of mycotoxins for
several decades, aptamers have emerged owing to inherent
advantages with respect to antibodies: immunization of animals
is not required, more chemical and thermal stability, less varia-
bility compared to pAbs, or inexpensive in-vitro synthesis. Parti-
cularly, aptamers are not susceptible to denaturation in the
presence of solvents commonly used in the extraction of
mycotoxins.
Aptamers for OTA were selected in 2008, following character-
ization by fluorescence polarization and equilibrium dialysis
(Cruz-Aguado and Penner, 2008a; Cruz-Aguado and Penner,
2008b). The identified aptamers exhibited high specificity to
OTA, and were demonstrated to be useful for the determination
of OTA in wheat grains by affinity chromatography coupled to
molecular fluorescence detection. The binding affinities, deter-
mined by equilibrium dialysis, are in the nanomolar range (Cruz-
Aguado and Penner, 2008a), comparable to or below the binding
constants of antibodies to OTA. As consequence of these two
works, a number of electrochemical OTA aptasensors appeared
from the year 2010, one of the firsts was developed in our
laboratory using magnetic beads (MBs) as solid support for
incubations (Bonel et al., 2011). Aptamer-based electrochemical
sensors are very sensitive, displaying detection limits in the range
0.03–0.8 ng mL−1 OTA (Table 3) usually higher to that obtained
with amperometric immunosensors in validated applications. It is
expected more work in the near future, as new schemes are
possible exploiting the conformational changes of the aptamers
attached to redox mediators. Very sensitive (up to 10−4 ng mL−1
OTA) label free aptasensors were described using electrochemical
impedance spectroscopy (EIS) (Prabhakar et al., 2011; Tong et al.,
2012).
In 2010, six unique aptamer sequences after 18 rounds of SELEX
were shown to bind FB1 with high affinity, one of them (FB1-39)
with a dissociation constant of 100 730 nM. (McKeague et al.,
2010). This work opened the door to new aptasensors and solid
extraction columns, but to date nothing has been reported. As an
initial work, our laboratory has demonstrated the posibility of
using FB1-39 aptamer in future electrochemical aptasensors (Vidal
et al., 2012d). The company Neoventures Biotechnology Inc. has
patented specific aptamers to AB1 and ZEA targets with Kd ¼ 10 nM
(Patent, 2010), and describes commercial aptamer-based devices
of OTA, aflatoxin B1 and zearalenone with lateral-flow technology.
In this situation, we find essential the availability of new
aptamers for mycotoxins issue. To date, only specific aptamers
for OTA, FB1, AB1 and ZEA have been reported, but it is expected a
large increase in the near future.
3.2.3. DNA and artificial receptors
The affinity of aflatoxins to both native and denatured DNA (ss-
DNA and ds-DNA) has been demonstrated (Mascini et al., 2001).
Detection of hybridization reaction between an immobilized probe
and target in solution through oxidation of guanine was modified
with AFB1 non-specifically trapped in the double strand, which
allows determination of AFB1 in the range 10–30 mg L−1. Detection
of changes in the time dependence of the current from DNA
hybridization caused by aflatoxin M1 allow determination within
the range 1.9–20.9 nM (Siontorou et al., 1998). An impedimetric
AFM1 biosensor based on a ss-DNA probe and gold nanoparticles
provides linear response over the concentration range 1–14 ng mL−1
and was applied to milk samples. An electrochemical ds-DNA
biosensor based on the electrochemical oxidation of OTA at a
glassy-carbon electrode was also used to evaluate the possible
interaction between OTA and DNA (Oliveira et al., 2007). Never-
theless, most of these hybridization DNA biosensors have a huge
lack of selectivity and sensitivity for environmental or toxicological
applications, and very few reports are dedicated to these today.
Supramolecular organic structures like cyclodextrins and chlor-
phyllin are well known to strongly bind aflatoxins, but without any
selectivity (Rai and Varma, 2010). On the contrary, molecularly
imprinted polymers (MIPs) and binding peptides obtained by
combinatorial synthesis, are good candidates for affinity ligands
for mycotoxins. MIPs are synthetic crosslinked functional poly-
mers made in the presence of the analyte of interest. After UV or
thermal polymerization, the analyte or template molecule is
removed, thus forming a cavity complementary to the target
molecule in which only the analyte of interest can be fitted very
selectively, acting similar to synthetic antibodies (Blanco-Lopez
et al., 2004). Nevertheless, despite some analytical fluorescent
applications (Baggiani et al., 2008; Vidal et al., 2012a), MIPs have
only been applied to selective cleanup extractive for OTA (Jorn and
Edward, 2007) and thricothechene ZEA+DON (Mizaikoff, 2003), or
in solid phase columns (Jodlbauer et al., 2002) where mycotoxin
selectivity in a very complex food matrix is the main problem,
instead of as synthetic receptors for biosensing applications.
4. Electrochemical biosensors: selected applications.
A variety of electrochemical immunosensors have been
reported with LODs lie in the order of 0.5–20 ng mL−1 and dynamic
ranges up to concentrations of mycotoxins about 1–100 ng mL−1
(Shephard et al., 2012). Recently reported OTA electrochemical
aptasensors have similar or better sensitivity and selectivity to
conventional immunoassays with shorter time for incubations.
Amperometric biosensors are the most common, but EIS are
becoming more frequent. Some recent advances (use of MBs,
nanostructuration and others) are commented in Section 5.
Tables 1–4 summarize a complete bibliography of the reported
electrochemical affinity biosensors for aflatoxins, ochratoxins and
other (trichochetene and others) mycotoxins.
4.1. Aflatoxins
The synthesis of aflatoxin-specific antibodies has produced a
range of competitive and non-competitive immunoassay techni-
ques and a number of commercial ELISAs and immunosensors (Li
Table 1
Summary of electrochemical immunosensors for aflatoxins.

Mycotoxin Assay format/Technique Working range or LOD Sample Sample treatment Reference
sensitivity

AFB1 IC/IP-AMP on 96-well SPEs 0.05–2 ng mL−1 0.03 ng mL−1 Corn Extraction (85% methanol in PBS) Piermarini et al. (2007a)
AFB1 IC/DPV on an 8x ELIME-array 0.8–9 ng mL−1 0.6 ng mL−1 Corn MycoSep clean-up column with acetonitrile/water (84/16) (v/v) Piermarini et al. (2009)
and resuspension in PBS with 1% methanol
AFB1 IC/AMP on SPEs 0.1–10 ng mL−1 0.09 ng mL−1 Barley Extraction with 85% methanol 15% PBS Ammida et al. (2006)
AFB1 DC/AMP on SPEs 0.15–0.25 ng mL−1 0.15 ng mL−1 – – Pemberton et al. (2006)
AFB1 IC on MBs/AMP on ITO electrodes 0.05–12 ng mL−1 0.006 ng mL−1 Red paprika Extraction methanol–water (80:20) (v/v) Tang et al. (2009)
AFB1 IC/DPV on SPEs 0.5–5000 ng mL−1 0.03 ng mL−1 Barley Extraction methanol+PBS+dimethylformamide (70+29+1) (v/v) Nagwa et al. (2004)
AFB1 IC/DPV in GCE doped with AuNPs 0.6–2.4 ng mL−1 0.07 ng mL−1 – – Owino et al. (2008)
AFB1 NC/AMP 0.1–12 ng mL−1 0.05 ng mL−1 Human serum, Dilution with PBS Sun et al. (2008)
Grape
AFB1, T2, IC/IP-AMP on 96-well SPEs array S¼ 1.2 ng mL−1 0.2– Corn Extraction (85% methanol in PBS and 80% methanol in water) Piermarini et al. (2007b)
HT-2 0.3 ng mL−1
AFB1 DC/EIS on silica-gel+ionic liquid films 0.1–10 ng mL−1 0.001 ng mL−1 Bee pollen Extraction (85% methanol in PBS) and centrigugation Zaijun et al. (2010)
(6000 rpm, 10 min)

J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158

AFB1 DC/EIS on electropolymerized PANi-PSSA films 0.1–6 ng mL−1 0.1 ng mL−1 – – Owino et al. (2007)
AFB1 DC/COND on an interdigitated conductimetric 0.5–10 ng mL−1 0.1 ng mL−1 – – Liu et al. (2006)
transducer
AFB1 AChE inhibition/AMP on SPEs modified with PB 10–60 ng mL−1 2 ng mL−1 Olive oil Extraction (methanol) Ben et al. (2009)
AFB1 Enzyme Biosensor (AFOx)/AMP using CNTs 1–225 ng mL−1 0.5 ng mL−1 – – Li et al. (2011)
AFM1 DC on MBs/CRA 0.01–0.5 ng mL−1 0.01 ng mL−1 Milk Centrifugation (15 min, 6000 rpm ) for defatting Paniel et al. (2010)
AFM1 DC/AMP on SPEs 0.001–1.000 ng mL−1 0.039 ng mL−1 Milk Addition 18 mM Ca+2 in PBS Parker et al. (2009)
AFM1 DC/AMP on SPEs 0.030–0.060 ng mL−1 0.025 ng mL−1 Milk Centrifugation (15 min, 6000 rpm) for defatting Micheli et al. (2005)
AFM1 DC/IP-AMP on 96-well SPEs 0.005–0250 ng mL−1 0.001 ng mL−1 Milk Centrifugation (20 min, 6000g) for defatting Neagu et al. (2009)
AFM1 IC/EIS in AuNPs and AgNPs on SPEs 0.015–1 ng mL−1 0.015 ng mL−1 Milk Defatting by centrifugation (10 min, 6000 rpm) and dilution with PBS Vig et al. (2009)
+0.5% (v/v) tween-20
AFM1 DC on SAM/EIS on an Ag wire electrode 0.006–0.100 ng mL−1 0.001 ng mL−1 Milk Defatting by centrifugation (10 min, 6000 rpm) and dilution Bacher et al. (2012)
with PBS+0.5% tween-20 (1/1) (v/v)
AFM1 DNA affinity/EIS on cysteamine SAMs with 1–14 ng mL−1 1 ng mL−1 Milk Deffating by centrifugation (6000g, 15 min) Dinckaya et al. (2011)
AuNPs
−1 −1
AFM1 FI Immunoassay/AMP 0.020–0.500 ng mL 0.011 ng mL Milk Dilution 1:1 with PBS 0.10 M, pH 6.5 Badea et al. (2004)
AFM1 DC/microelectrode Array 10–100 ng mL−1 8 ng mL−1 Milk Defatting (centrigugation 9000 rpm, 5 min) and CaCl2 Parker et al. (2009)
addition to samples
AFM1 DC/POT 0.125–2 ng mL−1 0.040 ng mL−1 Milk No sample treatment necessary Rameil et al. (2010)
AFM1 DNA/AMP 1.9–20.9 nM – – – Siontorou et al. (1998)

Abbreviations: OTA: ochratoxin A; IC: indirect competitive assay; NC: No competitive; DC: direct competitive assay; PVP: poly-(vinylpyrrolidone); AMP: Amperometry; COND: conductimetry; POT: potentiometric; MBs: magnetic
Beads; DPV: differential pulse voltammetry; AMP: amperometry; AFB1: aflatoxin B1; AFM1: aflatoxin M1; EIS: electrochemical impedance spectroscopy; AChE: achetylcholinesterase enzyme; PBS: phosphate buffer solution; ZOL:
zearalenol; T2, HT-2: trichothecene mycotoxins T-2 and HT-2 respectively; AOH: alternariol; AME: alternariol monomethyl ether; CIT: citrinin; CRA: chronoamperometry; DON: deoxynivalenol; NIV: nivalenol; PEG: polyethylene
glycol; FI: flow-injection; ELIME-array: enzyme-linked immuno-magnetic electrochemical array; ITO: indium tin oxide electrode; IP-AMP: intermittent pulse amperometry; GCE: glassy-carbon electrode; AuNPs: gold
nanoparticles; PB: electrocatalyzer prussian blue; CRA: chronoamperometry.

151
 152
Table 2
Summary of electrochemical immunosensors for ochratoxins.

Mycotoxins Assay format/Technique Working range or sensitivity LOD Sample Sample treatment Reference

OTA DC/DPV on SPEs S¼ 6.1 70.1 ng mL−1 0.180 ng mL−1 – – Alarcon et al. (2004)
OTA IC/Amperometry 0.05–0.5 ng mL−1 0.3 ng mL−1 Wine Addition of 0.1 g PVP to 10 mL wine, filtration and adjustment pH to 7.2 Prieto-Simon et al.

J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158

(2008)
OTA DC/CRA 12–60 ng mL−1 12 ng mL−1 – – Radi et al. (2009b)
OTA IC nanostructured (AuNPs)/ 0.3–8.5 ng mL−1 0.20 ng mL−1 Wheat Acetonitrile:water 6:4 (v/v) Bonel et al. (2010)
DPV Vidal et al. (2011)
OTA DC on MBs/DPV 10−4–1000 ng mL−1 0.11 70.01 ng mL−1 Wine Adjusting to pH ¼ 7.5 and adding polyvinylpirrolidone for complexing Vidal et al. (2012)
polyphenols
−1 −1
OTA IC/AMP 0.01–100 ng mL 0.0082 ng mL Corn Extraction (75% (v/v) methanol/water), centrigugation (3000 rpm, 10 min) Liu et al. (2009)
and dilution with 0.01 M PBS
−1 −1
OTA DC on MBs/SWV in SPEs 0.01–20 ng mL 0.008 ng mL Red WIne Dilution with 0.05 M citrate+0.05 M phosphate buffer, pH ¼5.0 Perrotta et al. (2012)
OTA IC/CRA on SPEs 0.01–100 ng mL−1 0.05 ng mL−1 Wine Extraction in Ochraprep IAC Heurich et al. (2011)
OTA IC on MBs/AMP in a 2–30 ng g−1 0.05 ng g−1 Apples Extraction 70% methanol in water Fernandez-Baldo
microfluidic chip et al. (2011)
OTA DC/EIS on a PANI modified Pt 2–10 ng mL-1 0.010 ng kg−1 Roasted coffee, Extraction in hydrogen carbonate buffer 0.13 M Muchindu et al.
electrode wheat, corn (2011)
OTA DC/EIS on several ITO 0.005–0.006 ng mL−1 0.003 ng mL −1
– – Kaushik et al.
modified electrodes (2009a)
Ansari et al. (2010)
Kaushik et al. (2008)
Khan et al. (2009)
OTA DC/EIS 0.005–0.06 ng mL−1 0.0008 ng mL−1 Coffee – Solanki et al. (2010)
OTA NC/EIS on MBs 0.01–5 ng mL−1 0.01 ng mL−1 White wine Dilution with PBS Zamfir et al. (2011)
OTA DC/EIS 1–20 ng mL−1 0.5 ng mL−1 – – Radi et al. (2009a)
OTA DC/EIS Up to 10 ng mL−1 – – – Khan and Dhayal
(2008)
OTA DC/EIS on a PANI–AG modified 0.5– – – – Khan et al. (2011)
ITO 3.0 ng mL−1(S ¼ 12.7 7 0.5 mA ng−1 mL)
OTA Inhibition HRP/ CRA 0.09–0.8 ng mL−1 (0.24–2.06 nM) 0.04 ng mL−1 Beer, Roasted Extraction with chloroform and water (coffee) Alonso et al. (2010)
Coffee

Abbreviations: PVP: poly-(vinylpyrrolidone); CRA: chronoamperometry; IAC: immunoaffinity column; ITO: indium tin oxide electrode; AG: acacia gum; PANI: polyanyline; CV: cyclic voltammetry.
Table 3
Electrochemical aptasensors for ochratoxins.

Mycotoxins Assay format/Technique Working range or sensitivity LOD Sample Sample treatment Reference

OTA DC Apt on MBs/DPV Aptasensor 0.78–8.74 ng mL−1 0.077 0.01 ng mL−1 Wheat Extraction acetonitrile/water (6:4) (v/v) Bonel et al. (2011)
OTA Aptasensor/CV 0.1–20 ng mL−1 0.030 ng mL−1 Red wine Solid-phase extraction Hua et al. (2010)
OTA Aptasensor/DPV 0.005–10 ng mL−1 0.001 ng mL− Wheat startch Extraction acetonitrile:water (60/40) (v/v) Tong et al. (2011)
OTA Aptasensor DC, IC/DPV on MBs and 1–50 ng mL−1 (IC)0.11–15 ng mL−1 0.11 ng mL−1 Wine Adding PVP and adjusting to pH ¼7.2 after Barthelmebs et al. (2011b)
SPEs (DC) filtration
OTA Aptasensor and hybridization/AMP 0. 1–20 ng mL−1 0.03 ng mL−1 Red grape wine Solid phase extraction column Hua et al. (2010)
OTA DC Aptasensor/EIS 0.1–100 nM 0.12–0.4 nM Spiked coffee, flour and 10% (w/v) of sample matrix was spiked with OTA Castillo et al. (2012)
wine
OTA EIS Aptasensor 0.1–10 ng mL−1 0.1 ng mL−1 – – Prabhakar et al. (2011)

J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158

Table 4
Summary of electrochemical immunosensors for trichothecene and other mycotoxins.

Mycotoxins Assay format/Technique Working range or LOD Sample Sample treatment Reference
sensitivity

FB1+FB2 DC/AMP on SPEs 1–1000 ng mL−1 5 ng mL−1 Corn Extraction 70% methanol 30% water Kadir et al. (2010)
DON DC/ EIS 0.001–0.3 ng mL−1 0.0003 ng mL−1 Food samples Addition of PEG, extraction with water and filtering Wei et al. (2011)
DON IC MBs/ELIME array 100–4500 ng mL−1 0.063 ng mL−1 Cereals Extraction with water and with acetonitrile/water (84/16) (v/v) and Romanazzo et al. (2010)
methanol/water (80/20) (v/v)
DON, NIV Hydrolysis and ED/ 96-well 2–20 μg g−1 1.1 μg g−1 Cereals Extraction with acetonitrile/water (84/16) (v/v) Ricci et al. (2009)
SPEs plate
ZEA DC/microfluidics AMP; SPEs EC50 ¼ 0.079 ng mL−1 0.01 ng mL−1 Baby food cereal, Maize, Cereal Extraction (acetonitrile:methanol 50:50, v/v and Hervas et al. (2009a)
and MBs milkshakes acetonitrile:water 75:25, v/v) Hervas et al. (2009b)
Hervas et al. (2010)
Hervas et al. (2011)
ZEA DC/AMP, microfluidic, MBs S¼ 0.463 nA/ng g−1 0.41 ng g−1 Feedstuffs (feedlot cattle) Extraction methanol/water (70/30) (v/v) Lai et al. (2011)
ZEA DC/Microfluidic AMP 0–500 0.77 ng mL−1 Corn silage Extraction with 70% methanol in water Panini et al. (2010)
ZEA, α,β- Amperometric detection Cut-off 0.17 ng g−1 0.016 ng g−1 Maize flour Supercritical CO2 Fluid extraction Zougagh et al. (2008)
ZOL (screening)
AOH, AME Tyrosinase inhibition/ AMP Up to 2.0 M 2.5 ng mL−1 Posibility in water samples Not influenced with 20% (v/v) acetonitrile Moressi et al. (1999)
CIT Microfluidic Immunoassay/ 0.5–50 ng mL−1 0.1 ng mL−1 Rice Extraction in acetonitrile+4% aqueous KCl (9:1) (v/v), pH ¼ 2.00 Arevalo et al. (2011)
AMP

Abbreviations: PEG: polyethylene glycol; ED: Electrochemical Detection; α,β-ZOL: α-zearalenol and β-zearalenol; CIT: citrinin.

153
154 J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158
et al., 2009). Quantitative analysis of aflatoxins usually requires
sample extraction with a suitable aqueous methanol solvent
mixture before the electrochemical biosensor assay. Most occur-
rence are cereals (Ammida et al., 2006), but few articles focused on
special matrices such as red paprika, olive oil, grape and human
serum, and bee pollen (Table 1).
To achieve higher sensitivity than ELISAs and move to the use of
disposable probes, electrochemical AFB1 immunosensors based on
single SPEs were first proposed by Palleschi et al. at the middle of
years 2000s (Nagwa et al., 2004). The same authors have described
an indirect competitive ELISA format for AFB1 using MBs and eight
magnetized SPEs as electrochemical transducers (Piermarini et al.,
2009). Indirect competitive voltammetric (DPV) immunosensors
with mAb–AFB1 and SPEs have been compared with HPLC-FLD and
ELISA for determination of AFB1 in barley (Ammida et al., 2006)
SPEs bearing antibodies against AFB1 in an array configuration
can be used in conjuction with 96-well microtriter plates by diping
the electrodes into the microwells for automated multi-analyte
determinations (Pemberton et al., 2006). In a similar but more
advanced idea, subsequent progress was made with an electroche-
mical immunosensor array using a 96-well screen-printed micro-
plate coupled to a multichannel electrochemical detection system
and an intermittent pulse amperometry technique (IP-AMP). This
system allows the sensitive determination of AFB1 (Piermarini et al.,
2007a) and AFM1 simultaneously for a number of samples (Neagu
et al., 2009), similarly to an indirect competitive ELISA. The same 96-
well array was applied to simultaneous detection of AFB1 and type-
A trichothecenes (T-2 and HT-2) (Piermarini et al., 2007b). Given the
costs, this array results in a cost/electrode 1/5 than for individual SPE
produced by thick film technology and this format allows the
creation of a multianalyte array.
Although the first AFM1 biosensor based on electrochemical
transduction and DNA hybridization inhibition appears yet in 1998
(Siontorou et al., 1998), the firsts AFM1 electrochemical immu-
noassays appeared in year 2004 using flow-injection systems for
AFB1 (Nagwa et al., 2004) and AFM1 (Badea et al., 2004). In almost
all articles of AFM1 (Table 1) the investigated matrices involved
milk and dairy products. Again SPEs are ideal transducers for
detachable AFM1 immunosensors (Parker et al., 2009; Micheli
et al., 2005), and for studying matrix interferences from milk
(Parker and Tothill, 2009).
More recently, very sensitive impedance immunosensors have
been developed for AFB1 based on the formation of a silica gel–
ionic liquid biocompatible film on a glassy carbon electrode (GCE)
(Zaijun et al., 2010) and Pt electrodes modified with polyaniline
(PANi) and polystyrene sulfonic acid (PSSA) films (Owino et al.,
2007). Impedance measurements have also resulted in AFM1
biosensors using immobilized layer-by-layer ss-DNA probes that
specifically bound the toxin (Dinckaya et al., 2011), nanostructured
with Au and Ag SPE immunosensors (Vig et al., 2009) and an
immunosensor on a Ag wire electrode (Bacher et al., 2012).
Conductimetric for AFB1 (Liu et al., 2006) and potentiometric for
AFM1 (Rameil et al., 2010) direct competitive immunosensors have
been reported. A recombinantly expressed aflatoxin-oxidase
enzyme allows a catalytic biosensor for AFB1 by measuring the
hydrogen peroxide generated enzymatically (Li et al., 2011).
4.2. Ochratoxins
OTA contamination occurs in very low nanograms per gram
amounts in a large variety of commodities (cereals, beans, ground-
nuts, spices, dried fruits, coffe, beer or wine), therefore, very highly
sensitive electrochemical biosensors and selectivity from matrices
are necessary to achieve the limits of regulatory legislation.
Electrochemical biosensors of OTA are usually applied to wine
(Vidal et al., 2012b), wheat (Bonel et al., 2010), roasted coffe, beer,
corn, and other cereals (see Tables 2 and 3), although also in such
unusual specimens as in apples (Fernandez-Baldo et al., 2011). One
typical problem with wine samples are potential electroactive
interferents (e.g. polyphenolic compounds), which can be removed
with complexing agents like PVP (poly-(vinylpyrrolidone)) (Vidal
et al., 2012b; Prieto-Simon et al., 2008).
There have been many reports of OTA immunosensors from
about the year 2004, in which Palleschi et al. immobilized pAb–
OTA on screen-printed electrodes (SPCEs) (Alarcon et al., 2004).
Both monoclonal (mAb–OTA) and polyclonal (pAb–OTA) antibodies
against OTA have been compared (Bonel et al., 2010; Vidal et al.,
2011), showing in some cases at least one-order of magnitude
lower IC50 values when working with mAbs (Prieto-Simon et al.,
2008). Transducers of OTA immunosensors are usually SPEs
(Perrotta et al., 2012; Vidal et al., 2011), glassy-carbon, or metallic
electrodes, and methods of immobilization include diazonium-
functionalized gold electrodes (Radi et al., 2009a), gold colloid
layers for enhancing surface loading of conjugated OTA (Liu et al.,
2009), passive adsorption (Heurich et al., 2011), coupling to a
carboxymethylated dextran hydrogel on a gold electrode (Heurich
et al., 2011), or carbodiimide chemistry covalent procedures (Bonel
et al., 2010). Malhotra et al. have carried out the immobilization of
generic rabbit antibody (r-IgGs) in a variety of nanomaterials
such as a nanocrystalline TiO2–chitosan (Khan and Dhayal, 2008),
anyline coupled to acacia gum (Khan et al., 2011), fumed silica
NPs (nano-SiO2) with chitosan (CH) films (Kaushik et al., 2009a),
sol–gel derived cerium oxide films (Kaushik et al., 2009b),
CH–iron oxide nanocomposites (Kaushik et al., 2008), CH–polyani-
line hybrid conducting biopolymer films (Khan and Dhayal, 2008),
CS–titanium nanocomposite films (Khan and Dhayal, 2009)
and sol–gel derived zinc oxide films (Ansari et al., 2010), all
having led to OTA immunosensors. Nevertheless, the use of
generic r-IgGs antibodies with no specificifity to OTA precludes
their selectivity. Our group of research have developed competi-
tive direct and indirect electrochemical immunosensors to OTA on
SPE transducers (Bonel et al., 2010; Vidal et al., 2011), showing
improvements owing to nanostructuration with AuNPs in an
indirect assay format and the use of magnetic beads (MBs)
(Vidal et al., 2012b).
The intrinsic sensitivity and advantages of EIS have led to the
development of a number of impedimetric immunosensors for
OTA all having very small LODs (Table 2). In most cases, a simple
ferricyanide/ferrocyanide charge-transfer mediator is only neces-
sary to obtain impedance measurements, without need for
enzyme labels or any bounded electrochemical mediator (Radi
et al., 2009a). The main challenge is to find the proper method for
immobilization on the electrode changing initial electrochemical
electron-transfer impedance and produce large changes upon
binding OTA with an antibody or aptamer. For this purpose,
electropolymerization of sulfonated-aniline (Muchindu et al.,
2011), diazonium organic salt modified gold electrodes (Radi
et al., 2009b) and co-imobilization with BSA onto C-11 SAMs
(Solanki et al., 2010) have been reported Zamfir et al. have
compared EIS with SPR OTA detection using magnetic nanoparti-
cles on a gold electrode (Zamfir et al., 2011). While SPR measure-
ment showed a larger response range (1–50 ng mL−1 OTA) than EIS
(0.01–5 ng mL−1), the LOD in SPR (0.94 ng mL−1) was higher than
with impedance detection (0.01 ng mL−1). Analytical results were
in accordance with standard ELISA test kits in all cases.
The use of aptamers against OTA has been the subject of a
number of papers published during the last two years (Table 3).
These aptamers are inexpensive and can be easily labeled with a
variety of molecules such as enzymes, biotin or electroactive
mediators, which has enabled the development of a variety of
detection methods (Laschi et al., 2011). Usually, the presence of
 J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158
calcium ions is essential for OTA binding to aptamers since these
ions favor the quadruplex structure of the aptamer improving their
affinity (Castillo et al., 2012). EIS is a well suited technique for OTA
aptasensors, so several assay schemes have been reported (Castillo
et al., 2012; Prabhakar et al., 2011). The first aptamer-based electro-
chemical sensor for wines had a sensing range 0.1–20 ng mL−1 using
methylene blue as redox mediator and a DNA hybridization reaction
to amplify the sensing signal (Hua et al., 2010). Barthelmebs et al.
studied the performance of various aptamers and investigated the
use of both direct and indirect competitive enzyme-linked aptamer
assays (ELAA) for detection of OTA in wine (Barthelmebs et al.,
2011a). As a follow-up of that work, the same authors reported
a novel electrochemical aptasensor based on competitive assay on
MBs having a LOD¼ 0.11 ng mL−1 (Barthelmebs et al., 2011b). In
another work, an exonuclease-catalyzed target recycling strategy
achieved amplified electrochemical aptasensing of OTA (Tong et al.,
2011). A ferrocene labeled probe DNA was hybridized with the
aptamer on a GCE electrode, then the presence of the OTA results in
the dissociation and transformation of the probe DNA into a hairpin
structure leaving the ferrocene in close proximity to the electrode
surface after liberated from the aptamer–OTA complex due to
exonuclease (Tong et al., 2011). Competitive direct and indirect
aptasensors can be carried out in a similarly to spectrophotometric
ELISAs (Barthelmebs et al., 2011b). We have reported for the first
time a competitive aptasensor for OTA based on a DNA biotinylated
aptamer, coupled to paramagnetic beads with electrochemical
detection, able to be used for the determination of OTA in wheat
well below the maximum level allowed in the European Union for
cereals (3 μg kg−1). The LOD was 0.07 ng mL−1 and negligible cross-
reactivity of the aptamer with ochratoxin B was observed (Bonel
et al., 2011)
4.3. Trichothecenes and other mycotoxins
Apart from aflatoxins and OTA, very few electrochemical
biosensors have been developed for other important mycotoxins
(Table 4), which means it is a labor camp for exploring. To our
knowledge, the only reported electrochemical immunosensor for
FB1+FB2 uses a monoclonal antibody modified screen-printed
gold electrode and monitoring the reaction with TMB by chron-
oamperometry, obtaining a LOD ¼5 ng mL−1 of FB1+FB2 with a
dynamic range from 1 to 1000 ng mL−1 (Kadir and Tothill, 2010).
Some reports point to DON electrochemical immunosensors (Vidal
et al., 2012d), using composites such as fullerene C-60, an ionic
liquid and ferrocene on a chitosan film at a GCE (Wei et al., 2011),
a recombinant Fab antibody fragment (Romanazzo et al., 2010) and
a 96-well electrochemical plate for the fast detection of DON and
nivalenol after a microwave hydrolysis procedure and direct
electrochemical sensing (Ricci et al., 2009).
Escarpa et al. have developed some ZEA electrochemical
immunosensors using MBs (Hervas et al., 2009a) and SPCEs
(Hervas et al., 2010) for determination in baby foods. Some of
these ZEA immunosensors were integrated into an electrokinetic
microfluidic system using MBs (Hervas et al., 2009b, 2011), which
allowed sensitivity, extremely low sample and reagent volumes
and the incubation enzymatic reaction to be manipulated in-situ.
Raba et al. also reported ZEA electrochemical immunosensors in
microfluidic devices (Lai et al., 2011; Panini et al., 2010), obtaining
lower LODs than with ELISA. The same authors have also devel-
oped a microfluidic electrochemical immunosensor for determin-
ing citrinin in rice samples (Arevalo et al., 2011). Our group of
research has been developing successfully for the last year DON
and FB1 amperometric immunosensors using SPCEs and MBs, and
our early results have already been presented to the scientific
community prior to publication (Vidal et al., 2012c, 2012d).
155
5. Recent advances in electrochemical mycotoxin biosensors
and future trends
Several scientific advances over conventional biosensors have
overcome some of the first difficulties in the determination of
mycotoxins and improved their analytical properties. One of the
most active areas is the use of nanomaterial composites for
modifying electrodes (Palchetti and Mascini, 2012; Farre et al.,
2009). Nanomaterials have a number of features for electroche-
mical biosensors, such as high surface area, stability, biocompat-
ibility, easy functionalization, unique size and shape, easy
dispersability and rapid fabrication, usually producing enhanced
surface concentrations of the ligands and increased currents
(Tothill, 2011). The main challenge is they have composition-
dependent physical and chemical properties requiring careful
optimization. Metallic nanoparticles (NPs), especially gold nano-
particles (AuNPs), together with carbon nanotubes (CNTs) are the
most used materials (Campas et al., 2012; Palchetti and Mascini,
2012; Tothill, 2011). For example, AuNPs were coupled with
electropolymerized films of polithionine (Owino et al., 2012) and
an ionic liquid in nafion for determining AFB1 (Sun et al., 2008).
AuNPs can be directly generated onto a glassy-carbon surface for
the microfluidic determination of citrinin in rice samples (Arevalo
et al., 2011). Coupling of SAMs (self-assembled monolayers from
alkane-thiols) with AuNPs allowed OTA immunosensors after
hapten-immobilization (Liu et al., 2009; Vidal et al., 2009).
Nanomaterials also increase the measurement of the charge-
transfer resistance on the electrode in EIS (Campas et al., 2012)
and using conjugation of ligands (aptamer or antibodies) with
nanomaterials (Palchetti and Mascini, 2012). For example, AuNPs
with a DNA probe for AFM1 (Dinckaya et al., 2011) or in conjuction
with silver NPs in an AFM1 immunosensor (Vig et al., 2009). We
have used gold nanostructuration of antigen OTA conjugated with
BSA (bovine serum albumin protein) for a synergistic effect of OTA
voltammetric immunosensors (Vidal et al., 2011). Nanoparticles
act as spacer matrix to extend the OTA–BSA away resulting in
binding sites more available to anti-OTA antibodies. Biotinylation
of the antibody and conjugation of the enzyme tracers with
extravidin also eliminated one incubation step, e.g. with a labeled
secondary antibody, to obtain the amperometric currents (Vidal
et al., 2011). Other uses of NPs are as labels for electrochemical
detection, but often amplifying enzymes are preferable for this
purpose (Tothill, 2011).
As in the case of NPs, CNTs have been used to increase the surface
area of working electrode, electrocatalytic properties (activity of the
edge-plane-like graphite sites), and to incorporate specific redox
probes (Campas et al., 2012; Tothill, 2011). A microfluidic immuno-
sensor to ZEA (Panini et al., 2010) and an enzymatic aflatoxin-
oxidase biosensor (Li et al., 2011) use multiwalled CNTs deposited on
glassy-carbon and Pt electrodes respectively. Novel graphene elec-
trode material has not been exploited up to date in mycotoxin
electrochemical biosensors. Quantum-dot semiconductor nanocrys-
tals have generated much interest for optical biosensors, but there
are not applications in electrochemical biosensors for mycotoxins,
despite their potential use as electrodiverse population of tags for
electrochemical multiplexed bioanalysis or in conjuction with strip-
ping voltammetry (Palchetti and Mascini, 2012).
The transducer itself also provides potential for further sensi-
tivity enhancement, for instance with interdigitated electrodes
formed from metals in EIS (Laschi et al., 2011), modified SPCEs
(Vidal et al., 2011) or a micro-comb conductimetric electrode for
AFB1 (Liu et al., 2006). Biosensor arrays are more expected in
future for detecting multiple mycotoxins in the same sample as
they can save time and to detect synergistic effects than affect the
individual determination of each mycotoxin (Prieto-Simon et al.,
2007). For example, a microarray immunosensor with 35 gold
156 J.C. Vidal et al. / Biosensors and Bioelectronics 49 (2013) 146–158
elements and on-chip reference and counter electrodes was
developed for determination of AFM1 in milk samples, with high
sensitivity that arises from the enhanced mass-transport at the
micro-electrodes when hemispherical diffusion layers are formed
(Parker et al., 2009). We used in our laboratory a multisensory
device combined with a multiplexed potentiostat that conducts
eight in-parallel electrochemical measurements which allows
multiple samples, replicates and all the corresponding controls
be measured at the same time (Vidal et al., 2012c).
Other prevalent advance is using magnetic beads (MBs). The
possibility of functionalization and separation of the MBs under an
external magnetic field markedly improve the performance of
classical immunosensors and aptasensors (Laschi et al., 2011). MBs
are particles (d≈1–3 mm) made from a dispersion of magnetic
material (Fe2O3 and Fe3O4) and covered with a thin shell of
polymer that serves to define a surface area for the adsorption
or coupling a large variety of molecules (mycotoxins, antibodies or
aptamers). Commercial MBs with a variety of functionalizations
are easily available. Multifunctional MBs have been used for
affinity supports of AFB1 conjugated to BSA (bovine serum
albumin) magnetically attached to an indium tin oxide (ITO)
electrode (Tang et al., 2009), a ZEA immunosensor (Hervas et al.,
2009b; Hervas et al., 2010), in detection of AFM1 in milk (Paniel
et al., 2010), or in EIS/SPR detection of OTA (Zamfir et al., 2011). We
have demonstrated in our laboratory a significant decrease of the
nonspecific adsortions of revealers and wine matrix interferences
due to paramagnetic microparticles, also permitting the precise
placement of the enzyme labels on a magnetized working elec-
trode surface in an OTA immunosensor (Vidal et al., 2012b).
“Lab-on-a-chip” refers to integrated microfabricated fluidic
systems designed to perform the multiple steps of an immuno-
sensor or aptasensor (Laschi et al., 2011). These devices can be
made with chambers for incubations or many channels and
allowing electrochemical multi-analyte detection with the pre-
sence of micro-electrodes in each channel for the direct quantifi-
cation of the affinity reaction (Parker et al., 2009). Electrochemical
microfluidic circuits and immunochip sensors have been designed
for AFM1 (Parker et al., 2009), ZEA (Hervas et al., 2009b; Liu et al.,
2006), OTA (Fernandez-Baldo et al., (2011)) and citrinin (Arevalo
et al., 2011). The small dimensions of these devices and large
relative surface area within the microfluidic system reduce incu-
bation times, the amount of reagents, labor and costs. Moreover,
this technology can advantageously and easily be combined with
the use of MBs (Hervas et al., 2009a, 2011; Lai et al., 2011).
In our opinion, one of the most exciting fields for improving
mycotoxin biosensors is development and synthesis of new
affinity ligands. Undoubtedly, new recombinant antibodies and
today inexistent new aptamers (e.g. DON, trichothecenes) will
open new opportunities. Conformational changes of labeled apta-
mers can be used to modulate the distance of the electroactive
labels to the electrode, thereby altering redox currents in the
design of new aptamer-based assays. Other possibilities are the
exploiting of new competitive assay schemes (Vidal et al., 2011),
reduced time-consuming incubation steps (Vidal et al., 2011), or in
conjuction with tightly packed nanosurfaces (Bonel et al., 2010)
and MBs (Vidal et al., 2012b). Nevertheless, sandwich assays with
two ligands are usually not possible due to the small size of the
mycotoxin molecules (Palchetti and Mascini, 2012).
Other aspects that must be improved in future in our opinion
are: (i) the identification of individual biomarkers of mycotoxin
exposure, involving analysis of metabolites of AFB1 (Shephard,
2009) or FB1 (Scott, 2012) in blood, urine or liver samples, very
useful in determining human exposure; (ii) Determination of
extractable masked (covalently and non-covalently bounded)
mycotoxins, with toxic properties but hidden to the analytical
methodology used. Some authors warn high occurrence of these
derivatives in foods for what they should be considered in
evaluating exposure to mycotoxins (Goryacheva and De Saeger,
2012); and (iii) More emphasis in multiple-sensing instruments,
possibly integrated into microfluidic devices, in a clear evolution
from single to multiple mycotoxin determinations (Zhang et al.,
2011; Goryacheva et al., 2007).
6. Conclusions
The numerous examples in the literature illustrate the high
potential of the electrochemical biosensors in mycotoxin analysis,
contributing to their sensitive determination in a variety of food and
commodities. Electrochemical biosensors are a serious alternative to
more complex official instrumental techniques such as HPLC coupled
to FLD or MS detectors, and provide additional benefits allowing
reduce costs, shortening analysis time and mobility due to portable
instrumentation. These biosensors can achieve the sensitivity and
selectivity required for the very strict regulatory limits from legislation.
Miniaturization of these devices also enables in-situ integration into
various food production equipment or into Hazard Analysis and
Critical Control Point programs.
Under an electrochemical point of view, label-free and direct
binding detection in techniques such as EIS are attractive features
that open wide the door to new analytical applications. Possibly future
research will be directed to simplifly assay schemes and devices in
applications performed under real conditions, synthesis of new
recombinant antibodies and aptamers, integration into novel technol-
ogies (e.g. microfluidic devices) and new nanostructuration schemes
of the sensor surface for improving (electro)analytical properties. New
applications are also expected with other mycotoxins to be studied,
multi-mycotoxin analysis, and the study of biomarkers and masked
mycotoxins to evaluate toxicology and human exposure to these
toxins. Electrochemical biosensors undoubtedly will contribute in near
future to have a major impact on quality control in food processing,
improving product quality and safety with minimal investment.
Acknowledgments
Spanish Science and Innovation Ministry for the contracts PTQ-
10-03580 (Torres Quevedo 2010) and the Project SERIBIO IPT-
2011-1766-010000. Spanish Education Ministry for the grant
AP2010-4609 (FPU 2010). University of Zaragoza for the Projects
UZ2011-CIE-07 and UZ-2012-CIE-14.
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