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Nanomaterial-based sensors for mycotoxin analysis in food


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Mycotoxins are low-molecular weight natural products produced as secondary metabolites by filamentous fungi and more than 500 mycotoxins with different physicochemical properties are currently known. Contamination by mycotoxins can result in liver and kidney diseases, nervous system damage, immunosuppression, and carcinogenicity. Consequently, the food safety authorities have set strict maximum permissible limits for mycotoxins. Over the past two decades numerous analytical techniques have been developed for mycotoxin detection in food. Recently, the application of nanotechnology in biosensors can range from transducer device to recognition ligand, label, and running systems. The application of nanomaterials in sensor development provides excellent advantages such as miniaturization of devices and signal enhancements. Such nanomaterial-based sensor devices can be sensitive, cost effective, and highly beneficial for the food industry ensuring on-site safety and preservation of food quality. This chapter reviews some recent developments on nanomaterial-based sensors developed for analysis of mycotoxin in food.
Content may be subject to copyright.
Novel Approaches of Nanotechnology in Food.
Copyright © 2016 Elsevier Inc. All rights reserved.
Bing Huei Chen, Baskaran Stephen Inbaraj
Fu Jen University, Department of Food Science, Taipei, Taiwan
1 Introduction
Mycotoxins are organic compounds produced as secondary
metabolites by fungi. Filamentous fungi produce thousands of
toxic compounds, but the more important mycotoxins belong
to species of Aspergillus, Fusarium, and Penicillium (Vidal
et al., 2013). From the toxicological and legislative point of view,
the mycotoxins of interest include aflatoxins, ochratoxins, fu-
monisins, deoxynivalenol, T-2, HT-2, zearalenone, patulin, citri-
nin, and ergot alkaloids (Bennett and Klich, 2003). The presence
of mycotoxins in foods and feeds not only retards the interna-
tional trade but also represents a health hazard for humans and
animals ( Shephard et al., 2012; Vidal et al., 2013). Storage of food
and feed under high temperature and humidity conditions favor
the growth of filamentous fungi (Campas et al., 2012). In addition,
agricultural products, particularly a wide variety of grains in the
field and during storage after harvest, are the main source of my-
cotoxin production in the food chain (Prieto-Simon et al., 2007).
Mycotoxins affect a broad range of agricultural products includ-
ing cereals, cereal-based foods, corn, rice, rye, wheat, barley, oats,
sorghum, soybeans, buckwheat, peanuts, malt, beer, dried fruits,
wine, milk, baby and infant foods, coffee beans, cocoa, bakery,
and meat products, all of which play a vital role for economy in
many developing countries (Prieto-Simon et al., 2007; Shephard
et al., 2012). Only since 5 years mycotoxins attracted worldwide
interest as a result of serious economic losses due to deleteri-
ous impact on human health, animal production, domestic and
international commerce (Bhat et al., 2010). However, their exis-
tence has been as long as crops have grown on earth. Mycotoxins
can produce acute and chronic effects causing serious impact on
central nervous, pulmonary, and cardiovascular systems (Bhat
et al., 2010). Consequently, to protect consumers and maintain
sustainability of agriculture, the maximum permissible limits
(MPLs) in several matrices as well as detection limits of analytical
methods have been established by several organizations includ-
ing World Health Organization (WHO), United States Food and
Drug Administration (USFDA), European Commission (EU), and
Food and Agriculture Organization (FAO) (Campas et al., 2012;
Vidal et al., 2013). The EU has recommended the MPL for fusar-
ium mycotoxins in maize products to be in the range from 50 to
2000 µg/kg (Vidal et al., 2013). However, the MPLs may vary de-
pending on the toxicity and variety of food. For example, aflatoxin
B1 is regulated by EU legislation with MPL at 2 µg/kg in foods for
human consumption, while aflatoxin M1, which is not as toxic as
aflatoxin B1, is regulated with MPL at 0.05 µg/kg owing to the high
consumption of milk by infants and children (Vidal et al., 2013).
Thus, it is imperative to develop analytical methods for rapid and
sensitive determination of mycotoxins with capability for multi-
plexing and on-site analysis.
Analyses of mycotoxins have been difficult because of their het-
erogeneous distribution in agricultural products and complexity
of mycotoxin-containing food matrices (Koppen et al., 2010). Ac-
cordingly, the selection of detection method strongly depends on
the mycotoxin and the matrix to be analyzed (Campas et al., 2012).
Moreover, the MPLs of mycotoxins set by the legislation have
necessitated development of analytical methods for qualitative
and quantitative determination of mycotoxins in both food and
feed samples (Valdes et al., 2009). While thin-layer chromatogra-
phy (TLC) has been widely used to detect and identify the pres-
ence of mycotoxins, some other methods with superior analytical
performance are also currently used for quantitative determina-
tion of mycotoxins (Turner et al., 2009). For instance, most of the
official methods are based on high-performance liquid chro-
matography (HPLC) coupled with ultraviolet (UV), fluorescence
(FL), or mass spectrometry (MS) detectors (Krska et al., 2005;
Shephard, 2008). Detection methods based on gas chromatogra-
phy (GC) coupled with MS detectors are not widely used due to
interference drawbacks (Koppen et al., 2010). Of the various meth-
ods, liquid chromatography-tandem MS (LC-MS/MS) have been
used as a valuable analytical technique for simultaneous multi-
toxin determination. Though these techniques provide very low
detection limits, their cost and need of skilled personnel along
with the incapability to perform in situ analysis have consider-
ably reduced their application in many parts of the developing
countries (Campas et al., 2012). Routine screening of mycotoxins
is commonly performed by enzyme-linked immunosorbent assays
(ELISA), which is capable of analyzing a large number of samples
in a single assay (Tothill, 2011; Malhotra et al., 2014). Yet, the dis-
advantages are laborious and tedious procedures, as well as cross-
reactivity resulting in false positive results especially for single
mycotoxin determination. Extract cleanup and/or dilution and ad-
dition of detergents are some of the pretreatment steps carried out
to minimize the matrix effects (Shephard, 2008). For rapid analy-
sis of mycotoxins, both quantitative and semiquantitative ELISA
kits as well as immunochromatography test kits are commercially
available (Campas et al., 2012). Emerging methods for mycotoxin
analysis include fluorescence polarization assays and Fourier
transform near-infrared spectroscopy (FT-NIR) (Cruz-Aguado and
Penner, 2008a; Chun et al., 2009; Tripathi and Mishra, 2009). In
addition, new receptors such as recombinant fragments of anti-
bodies, DNA aptamers or molecularly imprinted polymers (MIPs)
have been proposed for incorporation into high-throughput anal-
ysis (Romanazzo et al., 2010; Cruz-Aguado and Penner, 2008b;
Navarro-Villoslada et al., 2007; Maragos, 2009). Over the past two
decades, many approaches for developing new affinity biosen-
sors for mycotoxin analysis have been evaluated and some excel-
lent reviews have been published on analytical methods (Cigic and
Prosen, 2009; Koppen et al., 2010; Shephard et al., 2012), general bi-
osensors (Logrieco et al., 2005; Maragos and Busman, 2010; Prieto-
Simon et al., 2007) and electrochemical biosensors ( Palchetti and
Mascini, 2008; Laschi et al., 2011) for mycotoxins.
Biosensors are valuable devices for detection of many differ-
ent target toxins including mycotoxins in a wide range of matrices.
Among different sensors such as enzyme sensors, electrochemical
sensors, optical immunosensors, tissue biosensors, quartz crystal
sensors, and surface plasmon resonance (SPR) biosensors, electro-
chemical sensors have been used for a long time, which consists
of a transducer and recognition receptor (Brett and Oliveira-Brett,
2011). Several transducers widely used in conventional electro-
chemical sensors include amperometric, voltammetric, impedi-
metric, potentiometric, and conductometric transducers ( Tothill,
2011; Vidal et al., 2013). For recognition receptors, antibodies,
DNA, antibody fragments, aptamers, and molecularly imprinted
polymers are commonly employed (Prieto-Simon et al., 2007;
Tothill, 2011). The application of nanotechnology in biosensors
can range from the transducer device to the recognition ligand,
the label and the running systems (Campas et al., 2012; Malhotra
et al., 2014). The application of nanomaterials in sensor develop-
ment provides excellent advantages such as miniaturization of de-
vices and signal enhancements (Tothill, 2011). Moreover, the use
of nanoparticles as labels can not only result in high precision and
accuracy as well as signal amplification, but also the high surface-
to-volume ratio of nanomaterials can enhance sensitivity and al-
low single molecule detection of toxins (Malhotra et al., 2014). The
small size of nanomaterials also shorten the time of assay, decrease
the distance of electron travel, lower both power and voltage, im-
prove component density, and enhance chip functionality for
multiplexing capability (Tothill, 2011; Malhotra et al., 2014). This
chapter provides an overview on various nanomaterial-based sen-
sors used for detection of mycotoxins in food and/or feed. Fig. 12.1
shows the chemical structures of various mycotoxins reviewed in
this chapter.
2 Aflatoxins
Aflatoxins are a group of secondary fungal metabolites mainly
produced by Aspergillus flavus and A. parasiticus under certain
optimum conditions (Vidal et al., 2013). They are the most widely
spread group of toxins resulting in contamination of agricultural
and food products (Ye et al., 2010). Aspergillus molds grow mostly
on crops such as grains and nuts as well as in milk, cheese, corn,
peanuts, cottonseed, almonds, figs, spices, and a variety of other
foods and feeds (Malhotra et al., 2014). Besides, milk, eggs, and
meat products are sometimes contaminated due to animal con-
sumption of aflatoxin-contaminated feed. The Aspergillus species
can grow well under warm temperature and moisture level 7%
(Malhotra et al., 2014). Of more than 20 aflatoxins identified, the
major ones are AFB1, AFB2, AFG1, and AFG2, with AFB1 (2,3,6a,9a-
[1]-benzopyran-1,11-dione) being the most toxic one, followed by
AFG1, AFB2, and AFG2 (Fig. 12.1) (Delmulle et al., 2005). The Inter-
national Agency for Research on Cancer (IARC) has categorized
AFB1 to be under group 1 carcinogen, which can cause liver can-
cer in animals (Vidal et al., 2013). The EU has regulated its level at
2.0 µg/kg in foods for direct human consumption and 5 µg/kg for
all kinds of animals (Delmulle et al., 2005). Therefore, there is an
urgent need to develop sensitive and selective analytical methods
for rapid detection of aflatoxins in food and feed samples.
2.1 Detection of Aflatoxin B1
An immunoassay based on bioelectrocatalytic reaction was
developed by Liu et al. (2006), who fabricated a microcomb
electrode by self-assembling horseradish peroxidase (HRP) and
AFB1 antibody molecules on gold nanoparticles (GNPs). A good
conductometric response relative to AFB1 concentration was
shown in a linear range from 0.5 to 1.0 ng/mL with a low detec-
tion limit of 0.1 ng/mL at 3δ. The developed immunoassay shows
high accuracy and is comparable with that obtained by ELISA,
with the interassay coefficient of variation being 7.1 and 4.6% for
1.0 and 8.0 ng/mL AFB1, respectively. The presence of GNPs pro-
vides a congenial environment for the immobilized biomolecules
and decreases the electron transfer impedance leading to a direct
Figure 12.1. Chemical structures of some mycotoxins.
electrochemical behavior of the immobilized HRP. In principle,
a barrier of direct electrical communication generated between
the immobilized HRP and the electrode surface through the for-
mation of antibody–antigen reaction of immobilized anti-AFB1
and AFB1 in samples cause variation in local conductivity, which
is measured by the HRP bioelectrocatalytic reaction in 0.02 M
phosphate buffer solution (pH 7.0) containing 80 µM hydrogen
peroxide, 0.05 M potassium iodide, and 0.15 M sodium chloride.
However, a two-fold decrease in detection limit (0.05 ng/mL) with
a wide linear dynamic range (1.0–12 ng/mL) was attained in a later
study (Sun et al., 2008), in which ionic liquid 1-ethyl-3-methyl im-
idazolium tetrafluoroborate was immobilized on the surface of a
glassy carbon electrode (GCE) through titania sol and Nafion film,
followed by GNPs adsorption onto titania surface and HRP-labeled
anti-AFB1 antibodies on GNPs surface. Likewise, Owino et al. (2008)
developed an electrochemical immunosensor by immobilizing
AFB1-bovine serum albumin (BSA) on a polythionine/GNPs conju-
gate-modified glass carbon electrode, with the surface of AFB1-BSA
being covered with HRP for prevention of nonspecific binding of
ions onto the sensor. The detection was based on competition be-
tween free AFB1 and the immobilized AFB1-BSA conjugate for the
binding sites of free anti-AFB1 antibody. The response by differen-
tial pulse voltammetry dropped following an increase in the level of
AFB1 in the linear working range of 0.6–2.4 ng/mL with a detection
limit of 0.07 ng/mL being attained.
Application of CNTs is becoming increasingly important for
construction of biosensors because of their unique physical and
chemical characteristics, especially the high specific superficial
area can increase the amount of enzymes immobilized, enlarge the
reaction areas between enzyme and substrate, elevate the electrical
conductivity and enhance the biosensor response. Li et al. (2011)
constructed an amperometric biosensor by embedding aflatoxin-
oxidase (AFO) in sol–gel and linking to multiwalled carbon nano-
tubes MWCNTs-modified Pt electrode and demonstrated that the
covalent linkage between AFO and MWCNTs could retain enzyme
activity for sensitive detection of AFB1 oxidation. The biosensor ex-
hibited good affinity for AFB1 as shown by the apparent Michaelis–
Menten constant value of 7.03 µmol/L. Moreover, the sensor
was sensitive over the wide linear range from 3.2 to 721 µmol/L
(1–225 ng/mL) with the limit of detection of 1.6 nmol/L and aver-
age response time of 44 s. A high activation energy (18.8 kJ/mol) ob-
tained for this method demonstrates significant catalyzation of AFO
for oxidation of AFB1. However, in a recent study dealing with evalu-
ating carboxylated MWCNTs (c-MWCNTs)-based biosensor, Singh
et al. (2013) attained a much lower detection limit (0.08 ng/mL) in
the linear dynamic range of 0.25–1.375 ng/mL for AFB1 by electro-
phoretically depositing c- MWCNTs onto indium tin oxide (ITO)
glass, followed by functionalizing with monoclonal AFB1 antibody
and analyzing using an electrochemical technique. The method
showed high sensitivity (95.2 µA/ng mL/cm2 and improved detection
limit (0.08 ng/mL), with low association constant (0.0915 ng/mL)
indicating high affinity of the immunoelectrode toward AFB1.
2.2 Novel On-Site Immunological Techniques
for Detection of Aflatoxins AFB1 and AFB2
Due to consumers’ demand for food safety, a one-step assay
is necessary for determination of mycotoxin residues. After re-
searching on many techniques, scientists have developed a novel
concept of immunochromatography (IC), which has several ben-
efits including user-friendly format, short assay time, long-term
stability over a wide range of climates and cost effectiveness
(Delmulle et al., 2005; Shim et al., 2007). This IC technique, also
known as lateral-flow assay, utilizes antigen, and antibody prop-
erties for the rapid detection of toxin residues (Xiulan et al., 2006).
For fast screening of AFB1, Xiulan et al. (2005) developed an IC
technique by using a nanogold-labeled polyclonal antibody probe
and a detection limit of 2.5 ng/mL was attained. Compared to the
conventional ELISA method, a 6- to 10-fold reduction in assay
time and two-fold decrease of detection limit was achieved by this
IC method. The entire analysis can be completed within 10 min
and the technique is simple for easy on-site application even by
an untrained personnel. By adopting a similar IC technique, the
same research group employed colloidal gold-labeled polyclonal
antibody specific to AFB1 as marker, AFB1-BSA conjugate as com-
petitive antigen and goat-anti-rabbit antibody as control for AFB1
detection in 67 naturally contaminated food samples (Xiulan
et al., 2006). This IC method shows a comparable detection limit of
2.5 ppb as the above method by visual observation while a much
lower value of 0.05–0.1 ppb by photometric strip reader. However,
the detection limit in food extract matrix increased to 2 ng/mL,
with the recovery data of AFB1-spiked rice, corn, and wheat rang-
ing from 80.79 to 110.56%. A high correlation (R2 = 0.93) between
IC and ELISA kit test was shown for all the 67 food samples. Like-
wise, Delmulle et al. (2005) employed a membrane coated with
two capture reagents, namely, AFB1-BSA conjugate and rabbit
antimouse antibodies, conjugate pad saturated with GNPs as de-
tection reagent and absorbent pad for rapid detection of AFB1 in
pig feed. The AFB1 in the sample competes with AFB1 immobilized
on the membrane for binding to limited amounts of antibodies
in the detection reagent with line color intensity of positive AFB1
being visually distinguishable from that of AFB1-negative sample.
The whole analysis can be completed within 10 min, but the vi-
sual detection limit (VDL) of 5 ng/mL is high when compared to
the two aforementioned methods. However, in a later study, Shim
et al. (2007) reported a much lower VDL of 0.5 ng/mL by using an
IC strip test with nanocolloidal gold-monoclonal antibody as a
probe. Application of both IC and HPLC methods to 172 grain and
feed samples showed comparable results for detection of AFB1.
However, a cross reactivity to AFB2, AFG1, and AFG2 can occur for
IC assay, which should be overcome in the future study.
Immunoassay-based lateral flow dipstick (LFIDS) method is
also gaining considerable interest in recent years for on-site detec-
tion of mycotoxins in food and feed. Moreover, besides employ-
ment of one kind of nanoparticle, composite nanoparticles are also
used as nanomaterial supports in sensors based on an immunoas-
say technique. For example, Tang et al. (2009) developed a novel
membrane-based LFIDS assay for fast screening of AFB2 in foods
by using magnetic nanogold microspheres (nano-Fe2O3 as core
and GNPs as shell) biofunctionalized with monoclonal anti-AFB2
antibodies. A VDL (0.9 ng/mL) three-fold lower than the GNPS-
based conventional immunostick test was obtained. Qualitative
results can be obtained within 15 min with no false negative results
and without need of any expensive equipment. An excellent corre-
lation of results was shown as HPLC for AFB2-spiked food samples
such as peanuts, hazelnuts, pistacia, and almonds. Likewise, Liao
and Li (2010) used a monoclonal antibody immobilized nanopar-
ticles with a silver core and a gold shell (Ag/Au) in a LFIDS assay.
The Ag/Au dipstick exhibited a lower VDL of 0.1 ng/mL for AFB1,
with sensitivity, reproducibility, and stability being higher than
that for pure GNPs. Also, the assay was validated for application to
naturally contaminated samples including rice, wheat, sunflower,
cotton, chilies, and almonds.
Similar to IC and LFIDS, dot-immunogold filtration (DIGF)
assay is a new technique, which uses nitrocellulose membrane as
solid phase support and GNPs as label. Ye et al. (2010) demon-
strated rapid detection (15 min) of AFB1 in 45 different food sam-
ples with a VDL of 2 ng/mL and high specificity for AFG1, AFG2, and
AFM1 could be attained. A high correlation of results was obtained
as HPLC for all the samples. However, a slight cross reactivity with
AFB2 is claimed to be a pitfall of this method. Overall, these immu-
nological techniques (IC, LFIDS, and DIGF) have the potential to
be used as user-friendly, rapid, and cost-effective screening tools,
especially for preliminary screening of mycotoxin detection in food
and agricultural products within a very short time. In addition,
these techniques enable on-site application by an untrained per-
sonnel providing long-term stability over a wide range of climates.
2.3 Simultaneous Determination of AFB1, AFB2,
AFG1, and AFG2
Usually aflatoxins of more than one variety exist together in
the contaminated foods (Zhang et al., 2009). Although more than
20 aflatoxins have been identified, the major aflatoxins of great
concern include AFB1, AFB2, AFG1, and AFG2 owing to their clas-
sification under group 1 human carcinogens by the International
Agency for Research on Cancer (Cavaliere et al., 2007; Ventura
et al., 2006). Moreover, the current maximum level set by the
EU is 4 ng/g for total aflatoxins (B1 + B2 + G1 + G2) in groundnuts,
nuts, dried fruits, and cereals (Kolosava et al., 2006). To meet the
growing demand in rapid monitoring of total aflatoxins in agro-
products, Zhang et al. (2011) developed an ultrasensitive immu-
nochromatographic assay (ICA) for simultaneous detection of
4 aflatoxins AFB1, AFB2, AFG1, and AFG2 in peanuts. The ICA is
based on a competitive format whose sensitivity is improved by us-
ing a novel criterion to screen the optimal amount of MAb labeled
onto nanogold particles. Initially, antiaflatoxin MAb was conju-
gated with nanogold solution for preparation of nanogold probe,
followed by preparation of IC strip containing a sample pad, con-
jugate release pad, absorbent pad, and nitrocellulose membrane.
AFB1-BSA conjugate and rabbit antimouse IgG were used for test
and control lines, respectively, and coated onto the nitrocellulose
membrane using the BioDot XYZ3050 platform, nanogold probe
on conjugate pad and no treatment on absorbent pad. Then,
the coated membrane, conjugate pad, sample pad, and absor-
bent pad were laminated, pasted to a plastic scaleboard and the
assembled scaleboard was cut vertically for dividing into strips
and storing in a desiccator at 4°C. After dipping the test strip in
sample solution, the released nanogold probe moved along the ni-
trocellulose membrane chromatographically due to capillary ac-
tion and the different shades of red color depending on aflatoxin
concentration formed after 15 min were visually compared. In the
peanut matrix, all the four aflatoxins could be detected in the lin-
ear working range of 0.03–2.0 ng/mL for AFB1 and AFB2 as well as
0.06–4.0 ng/mL for AFG1 and AFG2, with the VDL being 0.03, 0.06,
0.12, and 0.25 ng/mL, respectively. The results were also shown to
be in good agreement with those obtained by HPLC for analysis
of aflatoxins in peanuts, demonstrating practical application of
ICA in real samples. It is worth pointing out that ICA possesses
several advantages such as procedure simplicity, rapid operation,
immediate results, low cost and no requirement of skilled techni-
cians or expensive equipment (Cui et al., 2008; Zhang et al., 2011).
Owing to these characteristics, ICA is suitable for online, in-field,
and immediate applications. However, until now, only a few stud-
ies have focused on application of ICA-based technique in analy-
sis of total aflatoxins.
2.4 Detection of Aflatoxin M1
Aflatoxin M1 (AFM1) is the hydroxylated metabolite of AFB1 and
the mammals ingesting AFB1 contaminated diets excrete AFM1
into milk, resulting in its presence in a large variety of dairy prod-
ucts (Fig. 12.1) (Stoloff, 1980; Radoi et al., 2008). It is relatively
stable during pasteurization, storage, and preparation of various
dairy products, thereby raising significant threats to human health
especially to children as they are the major consumers of milk
(Stubblefield and Shannon, 1974; Wang et al., 2011). Several stud-
ies have demonstrated the toxic and carcinogenic effects of AFM1,
which led WHO-IARC to change its classification from group 2 to
group 1 for carcinogens (IARC, 2002). Also, the European Com-
munity Legislation has limited the concentration of AFM1 in milk,
dried and processed milk products intended for adults and infants
to be at 0.050 and 0.025 ppb, respectively (ECR, 2001, 2004).
A superparamagnetic nanoparticle-based enzyme-linked im-
munosorbent assay was developed for AFM1 detection by Radoi
et al. (2008), who tested five different clones of antibodies (1C6,
3G11, 6G4, ATX9, and ATX2) in a direct competitive ELISA format
and compared with the classical indirect and direct competitive
ELISA formats aiming to reduce the time consumed during coating,
blocking, and competition steps. Of the five antibody clones, the
best IC50 value of 15 ng/L in buffer and 41 ng/L in milk was shown
by the clone 3G11 in the linear working range of 4–250 ng/L with
recovery ranging from 93 to 98% depending on the spiked AFM1
concentration (30–120 ng/L). In addition, the precision data for
replicate measurements showed a relative standard deviation rang-
ing from 3 to 7%. The conjugation of superparamagnetic iron oxide
nanoparticles (SPIONs) with protein G and antimouse IgG reduced
the coating time from 12–14 h (in conventional ELISA methods) to
20 min. Also, the competition time was reduced with complete skip-
ping of blocking step. The application of SPIONs enabled fast, reli-
able, and easy detection of AFM1 in milk with their separation from
the bulk solution being accomplished by using a simple magnet.
In a later study, Vig et al. (2009) developed an impedimetric
immunosensor based on colloidal gold and silver electrodeposi-
tion for detection of AFM1. An indirect competitive ELISA assay
performed on screen-printed electrodes (SPE) in the presence
of anti-AFM1 gold-labeled antibodies and chronoamperomet-
rically electrodeposited silver provided good response signal
with the calculated charge transfer resistance correlating well
with AFM1 concentration. The linear working range achieved
by this method was from 15 to 1000 ng/L with a detection limit
of 15 ng/L. However, in milk sample matrix, a wide range of
linearity (25–125 ng/L) and higher detection limit (25 ng/L) was
shown, which may be attributed to absorption of protein com-
pounds on gold-labeled AFM1. A high mean recovery of 116% was
achieved for milk samples spiked with different concentrations
of AFM1 (25, 50, and 100 ng/L). Dinckaya et al. (2011) developed
an impedimetric AFM1 biosensor based on DNA probe and
GNPs. A self-assembled monolayer of cysteamine and GNPs
were prepared layer-by-layer on gold electrodes by monitoring
using EIS and CV techniques and then a thiol-modified single
stranded DNA probe was immobilized to specifically bind AFM1.
The biosensor used K3[Fe(CN)6]/K4[Fe(CN)6] as a redox probe
for electrochemical measurements providing a linear response
over the concentration range of 1–14 ng/mL and detection limit
of 0.4 ng/mL. Both cysteamine and GNPs enhanced the charge
transfer of the redox probe through the electrode surface. Over-
all, the biosensor exhibited good analytical characteristics such
as high affinity of AFM1 toward the DNA probe, low quantifica-
tion limit (0.4 ng/mL), high recovery (107%), reproducibility (R2
ranging from 0.9244 to 0.9913), and repeatability (coefficient of
variation = 6.2%).
Immunochromatographic strip method using GNPs and
electrochemical immunochip sensor is becoming popular for
detection of low molecular weight toxins (Liu et al. 2008; Paek
et al., 2000). This technique involves comigration of test samples
and antibody–gold nanoparticle conjugates along membrane
strips for binding interactions to occur and comparison of color
intensity visually provides efficient and simple on-site detection
in less than 10 min without requirement of any skilled person-
nel or sophisticated instruments (Parker et al., 2009). Wang et al.
(2011) developed a gold nanoparticle immunochromatographic
strip method for effective on-site detection of AFM1 providing
linear response in the concentration range of 0–10 ng/mL in milk
samples with detection limit at 0.5–1.0 ng/mL, which is close to the
Taiwan FDA and United States FDA regulatory limit of 0.5 ng/mL.
Also, of the 144 milk samples analyzed in Taiwan, only 1 sample
(0.054 ng/mL) was found to slightly exceed the regulatory limit
of the European Union, suggesting that the milk products sold in
Taiwan are safe for human consumption.
3 Ochratoxin A
Ochratoxin A (OTA) is a ubiquitous mycotoxin produced
by fungi of improperly stored food products. Of at least nine
ochratoxins identified so far, only three (OTA, OTB, and OTC)
contribute mainly to overall proportion with OTA being the
most prevalent and toxic (Bonel et al., 2011). Structurally, OTA is
4-dihydro-3R-methyl-isocumarin (Fig. 12.1) (Kaushik et al., 2009a).
It is commonly found in cereals, barley, wheat, corn, rye, coffee,
wines, dried fruits, nuts, baby foods, and animal feeds as well as in
blood of animals and humans (Bonel et al., 2011). Also OTA is found
in tissues and organs of animals and is known to produce nephro-
toxic, teratogenic, carcinogenic, and immunotoxic effects, mainly
associated with the Balkan endemic nephropathy and urinary
tract tumors in human (Pfohl-Leszkowicz and Manderville, 2007).
The IARC has classified OTA as Group 2B human carcinogen and
the mode of action is possibly by induction of oxidative DNA dam-
age (Kaushik et al., 2009a; Vidal et al., 2013). Pfohl-Leszkowicz and
Manderville (2007) have presented an overview on OTA’s toxicity
and carcinogenicity in animals and humans. Consequently, the
EU has imposed a strict permissible limit of 3–5 µg/kg in cereals
and cereal products (Bonel et al., 2011). Thus, the development of
analytical methods for detection of OTA with improved sensitivity
is important.
For detection of OTA using GNPs, Bonel et al. (2010) developed
competitive electrochemical immunosensors based on dispos-
able SPE through physical adsorption of OTA onto BSA, followed
by binding of OTA-BSA on GNPs-modified working electrode
surface and detecting by differential-pulse voltammetry using
α-naphtyl-phosphate as substrate. The secondary IgG antibody
labeled with alkaline phosphatase enhances the detection signal.
The response signal changes linearly in the concentration range
from 0.3 to 8.5 ng/mL with the detection limit being 0.20 ng/mL,
which is about four-fold lower than that obtained by OTA-BSA
based immunosensor without GNPs. Application of this sensor to
OTA-spiked wheat sample provides a high recovery of 104–107%,
implying no matrix interference on OTA detection. In a later study,
compared to the previous method, a further reduction in detec-
tion limit to one-half (0.10 ng/mL) was achieved by an improved
competitive immunosensor employing biotinylated monoclo-
nal antibody capture probe and GNPs. The differential pulse
voltammetric measurements show linear response with OTA
concentration in the range of 0.15–9.94 ng/mL. A high recovery
(101–105%) of OTA from OTA-spiked wheat sample demonstrates
high accuracy of the proposed immunosensor method. Through
covalent immobilization via amine coupling to carboxymethylated
dextran hydrogel- modified gold working electrode, Heurich et al.
(2011) reduced further the detection limit to 0.05 ng/mL, which
remained unaltered in the wine matrix, while a linear dynamic
response in the wide concentration range from 0.01 to 100 ng/mL
was obtained. Electrochemical detection was performed using
3,3,5,5-tetramethylbenzidine and hydrogen peroxide with HRP
as the enzyme label. The generated signal was detected by chrono-
amperometry at 150 mV using onboard screen-printed Ag-AgCl
as pseudoreference electrode. Earlier to the previously mentioned
methods, Liu et al. (2009) constructed a more sensitive immuno-
sensor by loading OTA-ovalbumin conjugate on self-assembled
monolayer of 1,6-hexanedithiol assembly on GNPs and a much
lower detection limit of 8.2 × 103 ng/mL in the linear working
range between 0.01 and 100 ng/mL was obtained. After competi-
tion of the limited anti-OTA mouse monoclonal antibody between
immobilized hapten and OTA in sample, alkaline phosphatase-
labeled horse antimouse IgG antibody was selectively bound on
electrode surface, thereby providing response signal inversely
proportional to OTA concentration in the sample. For OTA deter-
mination in corn samples, negligible matrix effect and good recov-
ery (85–105%) were obtained, revealing feasibility of the proposed
method for application to real sample matrix.
Instead of antibodies, recently aptamer-based biosensors are
developed for detection of OTA. Aptamers are functional nucleic
acids selected from combinatorial oligonucleotide libraries
through in vitro selection to bind to specific target molecules such
as drugs, proteins, peptides, vitamins, and other organic/inorganic
compounds. Aptamers have several advantages over antibod-
ies due to their small size. Also, they can provide greater surface
density for receptors and bind a wide range of targets including
nonimmunogenic targets. They are thermally stable, reusable, and
easily modified to facilitate immobilization of reporter molecules
and functional groups. By making use of SPIONs, Barthelmebs
et al. (2011) developed a novel electrochemical aptasensor on dis-
posable SPE for sensitive detection of OTA. More specifically, the
competition between free OTA and labeled alkaline phosphatase
(ALP)-OTA for binding to the DNA aptamer immobilized on mag-
netic beads generated signals from the ALP enzyme for measure-
ment by differential pulse voltammetry (DVP). The OTA can be
detected at a level as low as 0.11 ng/mL in buffer and 0.15 ng/mL in
wine samples, with a high recovery of spiked OTA (0.5–10 ng/mL)
ranging from 94 to 98%. In another study, a much lower detection
limit (0.07 ng/mL) in the linear range from 0.78 to 8.74 ng/mL can
be attained by using superparamagnetic beads functionalized with
an DNA biotinylated aptamer specific to OTA, which can compete
with OTA-HRP conjugate and free OTA (Bonel et al., 2011). The
modified-magnetic beads are then localized on disposable SPE
under a magnetic field for OTA detection by DPV. Spiking of OTA
in wheat samples yielded average recoveries ranging from 102 to
104% with the relative standard deviation (RSD) being <8%. Fab-
ricated aptamer-based electrochemical “signal-off” sensor was
developed by Kuang et al. (2010), who immobilized three single-
stranded DNA molecules (including aptamer) on the surface of
an electrode and electrochemically measured the change in redox
current of methylene blue produced by binding of OTA target to
the aptamer. A detection limit as low as 0.03 ng/mL in the linear
working range of 0.1–20 ng/mL is attained due to sensitive detec-
tion of aptamer by OTA and signal enhancement caused by GNPs-
functionalized DNA molecules. A highly sensitive and selective
fluorescent aptamer sensor after modification with carboxyfluo-
rescein was constructed by Guo et al. (2011) using single-walled
carbon nanotubes ( SWCNTs) as fluorescence quencher. The fluo-
rescence measurements provide a linear response over the OTA
concentration range from 0.025 to 0.2 ng/mL, while the lowest de-
tection limit was 0.024 ng/mL. This method has been successfully
applied to real sample such as OTA-spiked beer sample. Thus, the
integration of nanostructures with the use of specific recognition
molecules such as nucleic acids (aptamers) to recognize and bind
target analytes in the sample matrix is promising for application to
a wide range of toxins and pathogens in food and feed.
Of the various metal oxides, zinc oxide nanoparticles (ZNPs) has
been of interest for immunosensor applications due to their unique
properties such as high isoelectric point (9.5) and biocompatibility
facilitating immobilization of an enzyme and protein having low
isoelectric point via electrostatic interactions. Moreover, ZNPs not
only provide a friendly microenvironment for immobilizing nega-
tively charged rabbit antibodies (r-IgGs) to retain its bioactivity, but
also accelerate electron transfer communication between protein
and the electrode to a large extent. For detection of OTA, Ansari et al.
(2010) deposited ZNPs onto indium-tin-oxide (ITO) glass plate
and coimmobilized with r-IgGs and BSA. The electrochemical im-
pedimetric response of BSA/r-IgGs/ZNPs/ITO immune electrode
obtained as a function of OTA concentrations showed a linear re-
sponse ranging from 6 × 106 to 1 × 105 ng/mL, detection limit
of 6 × 106 ng/mL and response time of 25 s. In an earlier study,
Kaushik et al. (2009a) also employed a similar immunoelectrode
(BSA/r-IgGs/CNPs/ITO) using cerium oxide nanoparticles (CNPs)
instead of ZNPs and developed an immunosensor for detection of
OTA with a detection limit of 2.5 × 105 ng/mL in the linear work-
ing range of 5 × 105–6 × 104 ng/mL. In addition, a high affinity
of the electrode toward OTA is evident as shown by a high associa-
tion constant (0.9 × 1011 L/mol). Similar to ZNPs, CNPs also possess
unique properties of high mechanical strength, oxygen ion con-
ductivity, high isoelectric point, biocompatibility, high absorption,
and high oxygen storage capacity. Several advantages of using CNPs
in biosensors are low temperature processing, tunable physical
parameters, optical transparency, chemical inertness, thermal sta-
bility, and insignificant swelling in both aqueous and nonaqueous
solutions for immobilization of enzymes.
The surface functionalization of nanoparticles with organic
moieties can result in controlled release and improved molecular
recognition capabilities of nanoparticles for biosensing applica-
tion. Kaushik et al. (2009b) utilized silica nanoparticles (SiNPs)
after functionalizing with chitosan (CH) and coimmobilized with
r-IgGs and BSA (BSA/r-IgGs/CH-SiNPs/ITO) for detection of OTA.
A rapid response (25 s) with a detection limit of 3 × 105 ng/mL
in the linear dynamic range of 5 × 105–6 × 104 ng/mL was ob-
tained. It is worth pointing out that SiNPs with controlled par-
ticle size, morphology, porosity, and surface area, along with its
chemical, thermal, and easy functionalization properties make
SiNPs suitable for several technological applications including
biosensing. Besides, their biocompatibility, nontoxic nature, high
ionic conductivity and surface-to-volume ratio are also suitable
for construction of biosensors. In another study, Khan and Dhayal
(2008) also conjugated chitosan with titanium oxide nanoparticle
(CH/TNPs) and immobilized with r-IgGs and proteins for detec-
tion of OTA with response signal by electrochemical impedance
spectroscopy increasing linearly to 10 ng/mL. The sensitivity of
CH/TNPs conjugate was shown to be four-fold higher than that
by using only CH.
3.1 Simultaneous Determination of AFB1 and OTA
Analyzing more than one mycotoxin using the same method
at the same time is important as multiplexing capability reduces
both time and cost. Shim et al. (2009) developed a one-step simul-
taneous immunochromatographic assay for rapid analysis of AFB1
and OTA in feed samples by using colloidal–monoclonal antibody
conjugate and a VDL of 0.5 ng/mL for AFB1 and 2.5 ng/mL for OTA
was obtained. The matrix interference from the feed extracts was
significantly reduced by appropriate dilution with buffer and the
detection limit for the feed spiked with different AFB1/OTA mixtures
were 10 and 50 µg/kg for AFB1 and OTA, respectively. In a later study,
Wu et al. (2011) further lowered the detection limit of both AFB1 and
OTA by 50-fold by employing antigen-modified magnetic nanopar-
ticles as immunosensing probes and antibody functionalized
upconversion nanoparticles (UCNPs) as signal probes for simulta-
neous detection of AFB1 and OTA. The signal probes were prepared
by functionalizing NaY0.78F4:Yb0.2, Tm0.02, and NaY0.28F4:Yb0.7,Er0.02
UCNPs with immobilized anti-AFB1 and anti-OTA antibodies. The
detection limit of both AFB1 and OTA was 0.01 ng/mL in the lin-
ear range from 0.01 to 10 ng/mL. Both high sensitivity and selec-
tivity obtained by this method can be attributed to the magnetic
separation and concentration effect of magnetic particles, the high
sensitivity of UCNPs and the different emission lines of Yb/Tm and
Yb/Er doped NaYF4 UCNPs by 980 nm laser. These two methods
have been successfully applied for simultaneous determination
of AFB1 and OTA in naturally contaminated feed samples or maize
samples, with the results being comparable with those obtained by
conventional ELISA and HPLC methods.
4 Sterigmatocystin
Sterigmatocystin (SMC) with a molecular formula (C18H12O6)
is a mycotoxin with toxicity second to AFB1 (Versilovskis and De
Saeger, 2010; Georgianna and Payne, 2009). Being a biogenic
precursor of AFB1, both SMC and AFB1 possess a similar struc-
ture consisting of a xanthone nucleus attached with a bifuran
(Fig. 12.1) ( Versilovskis and De Saeger, 2010). Several Aspergillus
species responsible for the production of SMC include A. versicolor,
A. nidulans, A. chevalieri, A. ruber, A. amstelodami, A. aureolatus,
A. quadrilineatus, and A. sydowi (Versilovskis and De Saeger,
2010). Some other fungal species such as Penicillium, Bipolaris,
Chaetomium, Emiricella are also capable of producing this myco-
toxin. The severity of SMC’s toxicity is mainly due to its xerophil-
ic nature, that is, the tendency to grow even at low water activity
(<0.8) (Versilovskis and De Saeger, 2010). In addition, it can grow
in a wide range of temperature between 4 and 40°C. It commonly
contaminates foods and crops such as wheat, corn, peanut, barley,
pecan nuts, soybeans, coffee beans, ham, and cheese (Table 12.1)
(Prieto-Simon et al., 2007). Like aflatoxins, SMC is carcinogenic to
humans and has been classified as group 2B by the IARC (Chen
et al., 2010). SMC is acutely toxic to the liver and kidney and closely
associated with the occurrence of hepatocellular, gastric and
esophagus carcinoma (Purchase and van der Watt, 1970). Moreover,
SMC is found to be one of the predominant contaminating myco-
toxins in highly incident areas of malignant tumors in China (Tian
and Liu, 2004). Although many countries do not have regulations
Table 12.1 Source, Harmful Effects as Well as Food
Products and Animals Affected by Certain Mycotoxins
Mycotoxins Sources
Food Products
Animals Harmful Effects
(B1, B2, G1, G2,
M1, M2)
Aspergillus flavus,
Aspergillus nominus,
Aspergillus parasiticus
Corn, rice, pasta,
Brazil nuts, peanuts,
peanut butter,
pistachios, cassava,
tobacco, cottonseed
cake, oilseeds,
figs, milk, cheese,
yoghurt, spices
dogs, birds,
Humans: hepatomegaly,
jaundice, hepatitis,
cirrhosis, liver cancer, Reye’s
syndrome, kwashiorkor,
Animals: weight loss,
reduced reproductive
function, teratogenesis,
mutagenesis, kidney, cancer,
liver cancer, death
Ochratoxin A Aspergillus alliaceus,
Aspergillus auricomus,
Aspergillus carbonarius,
Aspergillus glaucus,
Aspergillus melleus,
Aspergillus niger,
Aspergillus ochraceus,
Penicillium cyclopium,
Penicillium verrucosum,
Penicillium viridicatum
Corn, rice, rye,
wheat, buckwheat,
barley, millet, oats,
grapes, raisins,
currants, nuts,
coffee, cocoa,
spices, wine, beer,
pork, cheese
Humans: nephropathy (Balkan
endemic nephropathy)
Animals: reduced
reproductive function,
teratogenesis, nephrotoxicity,
genotoxicity, mutagenesis,
Aspergillus nidulans,
Aspergillus versicolor
Corn, wheat, barley,
peanuts, pecan nuts,
soya beans, green
coffee beans, ham,
Cows, mice,
rats, zebra
Animals: diarrhea, lactation
loss, teratogenesis, pulmonary
tumors, liver lesions,
hepatomas, liver tumors, liver
cancer, kidney lesions, skin
tumors, mutagenesis, cancer,
Zearlenone Fusarium culmorum,
Fusarium equiseti,
Fusarium graminearum,
Corn, wheat,
wheat flour, bread,
breakfast cereals,
noodles, rice, barley,
oats, sorghum,
walnuts, milk, corn
beer, meat, animal-
feed products
Cows, mice,
pigs, sheep
Humans: cervical cancer
Animals: vaginitis, sterility,
infertility, abortion, neonatal
mortality, reduced litter size
and weight, hyperestrogenism,
earlier sexual maturity, prolonged
estrus, reduced lactation,
Mycotoxins Sources
Food Products
Animals Harmful Effects
Deoxynivalenol F. culmorum, F.
Corn, popcorn,
rice, rye, wheat,
wheat flour, bread,
buckwheat, barley,
barley products,
oats, sorghum,
triticale, breakfast
cereals, noodles,
baby and infant
foods, malt, beer
Pigs, horses Humans: throat irritation,
gastrointestinal and abdominal
pains, nausea, vomiting,
diarrhea, hemorrhages,
dizziness, fever, headache
Animals: appetite loss, weight
loss, vomiting, diarrhea,
necrosis, teratogenesis,
Citrinin Aspergillus carneus,
Aspergillus niveus,
Aspergillus terreus,
Penicillium citrinum,
P. verrucosum,
Penicillium expansum
Corn, rice, rye,
wheat, barley, oats,
peanuts, fruits,
mice, pigs,
rabbits, rats
Humans: fatal kidney disease,
Balkan endemic nephropathy,
Shoshin-kakke beriberi
Animals: fetotoxicity, abortion,
teratogenesis, kidney damage,
vasodilatation, bronchial
constriction, liver damage,
increased muscular tone,
nephropathy, necrosis
(B1, B2)
Aspergillus alternate,
Fusarium anthophilum,
Fusarium dlamini,
Fusarium moniliforme,
Fusarium naphiformel,
Fusarium nygama,
Fusarium proliferatum,
Fusarium verticillioides
Corn, polenta,
corn-based breakfast
cereals, snack
products, rice,
sorghum, mung
beans, navy beans,
asparagus beer
horses, pigs,
Humans: esophageal cancer
Animals: appetite loss,
reduced litter weight, low
bone development in fetus,
fetal mortality, respiratory
problems, porcine pulmonary
edema, lethargy, hydrothorax,
hepatic lesions, fibrosis,
hepatocellular carcinoma,
neurotoxicity, equine
Plants: cell-membrane damage,
reduced chlorophyll synthesis,
disrupted sphingolipids synthesis
Table 12.1 Source, Harmful Effects as Well as Food
Products and Animals Affected by Certain Mycotoxins
on SMC, Czech Republic and Slovakia have set 5 µg/kg for rice, veg-
etables, potatoes, flour, poultry, meat, and milk as well as 20 µg/kg
for other foods (Stroka et al., 2004). For humans, the California
Department of Health Services has recommended no significant
risk level of 8 µg/kg bw per day for a 70 kg adult (Versilovskis and De
Saeger, 2010). Consequently, the EU-based European Food Safety
Authority (EFSA) has suggested that analytical methods for SMC
determination in foods should attain a limit of quantification of
<1.5 µg/kg (EFSA, 2013) (Table 12.2).
Novel electrochemical biosensors were designed incorporat-
ing SWCNTs and MWCNTs and enzymes. Ever since MWCNTs
came into use in 1991, the manufacture of enzyme biosensors
have increased substantially as MWCNTs can facilitate large
quantities of enzymes to be immobilized, thereby enlarging the
reactive area between enzyme and substrate, elevating the elec-
trical conductivity and enhancing the biosensor response (Yao
et al., 2006). Yao et al. (2004) conducted a preliminary study on
SMC detection using a triplet electrode enzyme-biosensor sys-
tem, in which Ag/AgCl is used as reference electrode, while Pt and
Au deposited on MWCNTs and aflatoxin-detoxifizyme (ADTZ) are
used as counter and working electrode, respectively. The response
signals are shown to be substrate correlative with linear working
range and detection limit being 8.32 × 105–66.56 × 105 mg/mL
and 8.32 × 105 mg/mL (0.268 µM) respectively. However, the nar-
row linear range and low sensitivity are claimed to be the disad-
vantages of this method. In a later study, the same research group
improved the sensitivity and widened the linear working range of
this method by saturating the ADTZ with SMC prior to covalently
immobilizing on the surface of MWCNT-modified electrode ( Yao
et al., 2006). Initially, the MWCNTs are oxidized by refluxing with
aqua regia and the MWCNTs-modified electrode are prepared by
dropping 10 µL of MWCNTs suspension on the gold electrode and
evaporating the solvent in a 60°C oven. Then, the SMC- saturated
ADTZ is immobilized onto MWCNTs-modified electrode through
covalent bonding. More specifically, upon oxidation of MWCNTs,
its surface area is enhanced and the hydrophobic surface of
MWCNTs are converted into hydrophilic ones favoring attach-
ment with hydrophilic sites on ADTZ enzyme and thereby the
attachment of hydrophobic sites on ADTZ with the hydropho-
bic biosensor substrate (SMC) remains undisturbed. Differential
pulse voltammetric measurements revealed the linear working
range of 0.134–4.29 µM and a detection limit of 0.134 µM could
be achieved with two-fold improvement in sensitivity com-
pared to the aforementioned method using randomly absorbed
ADTZ-MWCNTs electrode. The improvement in sensitivity may
Table 12.2 Some Nanoparticle-Based Biosensors
and Their Analytical Performance for Detection
of Certain Mycotoxins
Dynamic Range Sample References
GNPs AFB1Anti-AFB1 Ab;
0.1 ng/mL;
0.5–10 ng/mL
Buffer Liu et al. (2006)
0.05 ng/mL;
0.01–100 ng/mL
Buffer, wine Heurich et al.
OTA Aptamer to OTA; CV 0.03 ng/mL;
0.1–20 ng/mL
Buffer Kuang et al. (2010)
OTA Anti-OTA MAb; EI 0.008 ng/mL;
0.01–100 ng/mL
Corn Liu et al. (2009)
AFB1 and
MAb; ICA AFB1 = 0.5 ng/mL;
OTA = 2.5 ng/mL
Feed sample Shim et al. (2009)
MAb; ICA AFB1 = 0.03 ng/mL;
AFB2 = 0.06 ng/mL;
AFG1 = 0.12 ng/mL;
AFG2 = 0.25 ng/mL
Peanuts Zhang et al. (2011)
AFB1Anti-AFB1 MAb; EI 0.05 ng/mL;
0.1–12 ng/mL
Sun et al. (2008)
ssDNA; EIS and CV
1–14 ng/mL Milk Dinckaya et al.
and LSV
0.015 ng/mL;
0.015–1 ng/mL
Buffer and
Vig et al. (2009)
OTA Anti-OTA MAb; DPV 0.15–9.94 ng/mL;
0.10 ng/mL
Cereals Vidal et al. (2011)
AFB1PI; ICIA 0.01 ng/mL;
0.1–10 ng/mL
Milk Jin et al. (2009)
0.1 ng/mL;
0.5–50 ng/mL
Rice Arevalo et al.
AFB1Anti-AFB1 Ab; LFIDS 5 ng/mL; 2–10 ng/mL Pig feed Delmulle et al.
and CV
0.07 ng/mL;
0.6–2.4 ng/mL
Buffer Owino et al. (2008)
Dynamic Range Sample References
OTA Anti-OTA PAb; DPV 0.86 ng/mL;
0.3–8.5 ng/mL
Wheat Bonel et al. (2010)
AFM1Anti-AFM1 PAb;
1.0 ng/mL;
0.01–10 ng/mL
Milk Wang et al. (2011)
AFB1Anti-AFB1 PAb; ICA 2.5 ng/mL; 0.5–5 ng/mL Buffer Xiulan et al. (2005)
AFB1Anti-AFB1 PAb; ICA 2 ng/mL; 2–50 ng/mL Rice, corn,
Xiulan et al. (2006)
AFB1Anti-AFB1 MAb;
2 ng/mL;
0.5 – 10 ng/mL
45 Food
Ye et al. (2010)
AFB1Anti-AFB1 MAb; ICA 0.5 ng/mL;
0.1–10 ng/mL
172 Grain and
feed samples
Shim et al. (2007)
5 ng/mL; 1–1000 ng/mL Corn Kadir and Tothill
CNTs SMC Aflatoxin-
electrode; EIS and
0.13 ng/mL;
0.13–4.29 ng/mL
Buffer Yao et al. (2006)
ZRL Anti-ZRL Ab; EI 0.77 ppb; 0–500 ppb Corn, food and
feed samples
Panini et al. (2010)
AFB1Anti-AFB1; EI 0.08 ng/mL;
0.25–1.38 ng/mL
Buffer Singh et al. (2013)
1.6 ng/mL;
1–225 ng/mL
Buffer Li et al. (2011)
SMC Aflatoxin-oxidase;
3 ng/mL;
10–1480 ng/mL
Buffer Chen et al. (2010)
OTA Aptamer-
conjugate; FS
0.024 ng/mL;
0.025–0.2 ng/mL
Beer Guo et al. (2011)
Table 12.2 Some Nanoparticle-Based Biosensors
and Their Analytical Performance for Detection
of Certain Mycotoxins (cont.)
Dynamic Range Sample References
MNPs AFB1 and
Anti-AFB1 Ab and
Anti-OTA Ab
0.01 ng/mL;
0.01–10 ng/mL
for AFB1 and OTA
Maize Wu et al. (2011)
AFM1Anti-AFM1 Ab; ELISA 0.004 ng/mL;
0.004–0.25 ng/mL
Milk Radoi et al. (2008)
OTA Alkaline
aptamer; DPV
0.11 ng/mL; 0.11–15
Wine Barthelmebs et al.
MBs ZRL r-IgG MAb;
0.011 ng/mL Maize, baby
Hervas et al. (2009)
ZRL EI using carbon SPE;
0.007 ng/mL Baby foods Hervas et al. 2010)
ZRL MAb; ELISA and EIS 0.4 ng/mL Solid and
liquid samples
Hervas et al. (2011)
OTA Aptamer to OTA;
0.07 ng/mL;
0.78–8.4 ng/mL
Wheat Bonel et al. (2011)
DON Anti-DON Ab;
63 ng/mL;
100–4500 ng/mL
Wheat, cereal,
and baby food
Romanazzo et al.
CNPs OTA r-IgG Ab; CV and EIS 0.0025 ng/mL;
0.005–0.06 ng/mL
Buffer Kaushik et al.
ZNPs OTA r-IgG; EIS 6 × 106 ng/mL;
6 × 106
1 × 105 ng/mL
Buffer Ansari et al. (2010)
TNPs OTA r-IgG Ab; EIS 0–10 ng/mL Buffer Khan and Dhayal
SiNPs OTA r-IgG Ab; CV 0.003 ng/mL;
0.005–0.06 ng/mL
Buffer Kaushik et al.
Table 12.2 Some Nanoparticle-Based Biosensors
and Their Analytical Performance for Detection
of Certain Mycotoxins (cont.)
be attributed to the protection of ADTZ’s active centers and its
appropriate orientation provided by saturation of ADTZ and di-
rect attachment of ADTZ to MWCNTs. However, several param-
eters such as sample temperature, ion intensity, and acidity are
still required to be optimized for further improvement in sensitiv-
ity and applicability.
Recently, Chen et al. (2010) developed a simple, rapid, and
highly sensitive electrochemical biosensor for SMC based on
a new enzyme aflatoxin-oxidase (AFO), which is cloned from
Armillariella tabescens and expressed in Pichia pastoris. The
chitosan-coated SWCNTs are prepared first by dispersing acid-
functionalized SWCNTs in 0.2% CH solution by weight. Then, for
preparation of CH-AFO-SWCNTs/Au electrode, the purified AFO is
ultrasonicated with CH-SWCNTs suspension to obtain black- color
CH-AFO-SWCNTs solution containing 5 mg/mL AFO, followed by
Dynamic Range Sample References
AFB2Anti-AFB2 MAb;
0.9 ng/mL Peanuts,
Tang et al. (2009)
AFB1Anti-AFB1 MAb;
0.1 ng/mL Rice, wheat,
cotton, chilies,
Liao and Li (2010)
LOD, limit of detection; AFB1, aflatoxin B1; AFB2, aflatoxin B2; AFG1, aflatoxin G1; AFG2, aflatoxin G2; OTA, ochratoxin A; FMN, fumonisin;
CIT, citrinin; SMC, sterigmatocystin; ZRL, zearalenone; DON, deoxynivalenol; GNPs, gold nanoparticles; CNTs, carbon nanotubes;
MNPs, superparamagnetic iron oxide nanoparticles; SNPs, silver nanoparticles; CNPs, cerium oxide nanoparticles; ZNPs, zinc oxide
nanoparticles; TNPs, titanium oxide nanoparticles; QDs, quantum dots; SiNPs, silica nanoparticles; GNPs@MNPs, gold nanoparticles
coated over magnetic iron oxide nanoparticles; GNPs@SNPs, gold nanoparticles coated over silver nanoparticles; Ab, antibody; MAb,
monoclonal antibody; PAb, polyclonal antibody; r-IgG, rabbit immunoglobulin G; CMD, carboxymethylated dextran; ssDNA, single-
stranded DNA; EI, electrochemical immunosensor; FS, fluorescence spectroscopy; PI, piezoelectric immunosensor; EIS, electrochemical
impedance spectroscopy; DPV, differential pulse voltammetry; LSV, linear sweep voltammetry; ICIA, indirect competitive immunoassay;
LFIDS, lateral-flow immunodipstick; CV, cyclic voltammetry; ELISA, enzyme-linked immunosorbent assay; ICA, immunochromatographic
assay; DIGFA, dot-immunogold filtration assay; SPGE, screen printed gold electrode; SPCE, screen printed carbon electrode; EMC,
electrochemical microfluidic chips; and ITA, immunoturbidimetric assay.
Table 12.2 Some Nanoparticle-Based Biosensors
and Their Analytical Performance for Detection
of Certain Mycotoxins (cont.)
dropping 5 µL of CH-AFO-SWCNTs on the gold electrode surface
and drying at 4°C for 24 h. The AFO enzyme is immobilized onto
CH-SWCNTs-modified Au electrode through electrostatic and hy-
drophobic interactions. The CH-SWCNTs nanocomposite matrix
could provide a favorable microenvironment for AFO to retain its
native conformation and accelerate direct electron transfer be-
tween protein and underlying electrode surface. Accordingly, the
CH-AFO-SWCNTs/Au electrode exhibiting excellent electrocata-
lytic response, long-term stability, and high reproducibility was
successfully prepared for detection of SMC. An SMC response of
95% is reached within 10 s with the linear concentration range
from 10 to 1480 ng/mL and the detection limit (3 ng/mL) obtained
is comparable to that obtained by the conventional GC-MS and
LC-MS methods. The kinetic parameters such as charge transfer
coefficient, electron transfer rate constant, the apparent Michae-
lis–Menten constant and activation energy obtained for this meth-
od are 0.4, 0.9 ± 0.02/s, 7.8 µM, and 38.8 kJ/mol, respectively. The
biosensor also showed high operational stability, reproducibility,
and long-term stability, which can be attributed to three major
aspects: (1) electrostatic interaction between positively charged
CH-SWCNTs and negatively charged AFO can favor AFO immobi-
lization; (2) the 3-D-porous structure of CH-SWCNTs biofunction-
al film can prevent leaching of the enzyme; and (3) the biopolymer
chitosan can provide desirable environment around the enzyme
for maintenance of AFO’s bioactivity. In addition, the 3-D-porous
structure of composite film can also accelerate the direct electron
transfer between protein and electrode surface.
5 Zearalenone
Zearalenone (ZRL), also known as F-2 toxin, is a nonsteroidal
estrogenic mycotoxin biosynthesized through a polyketide path-
way by several Fusarium species including F. graminearum, F. cul-
morum, F. sporotrichioide, F. cerealis, F. equiseti, F. crookwellense,
and F. semitectum, all of which are common contaminants of
cereal crops worldwide in both temperate and warm regions
( Zinedine et al., 2007; Bennett and Klich, 2003). It belongs to a
phenolic resorcyclic acid lactone mycotoxin chemically described
as 6-[10-hydroxy-6-oxo-trans-1-undecenyl]-B-resorcyclic acid
lactone (Fig. 12.1) (Panini et al., 2010). Under the moist and cool
field conditions, ZRL can contaminate several food products such
as maize, corn, wheat, bread, cereals, noodles, rice, oats, barley,
sorghum, walnuts, milk, meat, rye, and animal-feed products
( Table 12.1) (Prieto-Simon et al., 2007). More specifically, the high-
est concentrations of ZRL are reported to be found in wheat bran,
corn and corn products, grains and grain-based foods as well as
vegetable oil, all of which contribute largely to the overall ZRL
exposure (EFSA, 2011). The worldwide contamination of food and
feed products is estimated to range from a minimum of 180 ng/kg
in Qatar and maximum of 6 × 108 ng/kg in India ( Zinedine
et al., 2007). Although steroidal estrogens and ZRL are structurally
dissimilar, several derivatives (mainly ZRL and zearalenol) exhibit
estrogenic activity comparable to diethylstilbestrol through ap-
propriate folding to orient hydroxyl groups and facilitate attach-
ment to estrogen-binding tissue receptors (Zinedine et al., 2007;
Panini et al., 2010). It is frequently associated with reproductive
disorders of farm animals and hyperestrogenic syndromes in hu-
mans (Diekman and Green, 1992; Farnworth and Trenholm, 1983;
Green et al., 1990). The biotransformation of ZRL in animals in-
volves formation of two ZRL metabolites, namely, α-zearalenol
and β-zearalenol, both of which conjugate with glucuronic acid
(Long and Diekman, 1986). Zinedine et al. (2007) have presented
an overview of acute, subacute, and chronic toxicity along with re-
productive and developmental toxicity, carcinogenicity, genotox-
icity, and immunotoxicity of ZRL and its metabolites. The mean
dietary intake for ZRL is 20 ng/kg bw per day for people in Canada,
Denmark, and Norway, while a higher intake of 30 ng/kg bw per
day is estimated among Americans (Zinedine et al., 2007). The
joint FAO/WHO Expert Committee on Food Additives (JECFA)
established a maximum tolerable daily intake of 0.5 µg/kg bw for
ZRL (JECFA, 2000).
For rapid detection of ZRL in corn silage samples, an
electrochemical-based method was developed by Panini et al.
(2010) by coupling an immunosensor with GCE modified with
MWCNTs and integrating with a continuous-flow system. The
MWCNTs-modified GCE was prepared by dropping a 20 µL aliquot
of MWCNTs dispersed in 0.1% Nafion solution onto the GC elec-
trode surface and drying under infrared lamp for solvent evapo-
ration. Next, the immunosensor was designed by immobilizing
mouse monoclonal anti-ZRL antibody to ZRL on 3-aminopropyl-
modified controlled-pore glass, which was previously spread
smoothly on a double-coated tape affixed to the disk surface.
The immobilized antibodies were finally washed with phos-
phate buffer (pH 7) and stored at 5°C until further use. The ZRL
in corn sample was then allowed to compete immunologically
with HRP-bound ZRL for the immobilized antibodies. HRP in the
presence of hydrogen peroxide catalyzes the oxidation of 4-tert-
butylcatechol, followed by measuring the back electrochemical
reduction on MWCNTs-modified glass carbon electrode at 0.15 V
by an amperometric technique. The developed electrochemical
immunosensor is rapid (15 min) and enables detection of ZRL
in the linear working range of 0–500 ppb with detection limit at
0.77 ppb, recovery from 93 to 106.5%, coefficient of variation for
within-assay precision at <2.42% and between-assay precision at
<4.36%. Compared to the conventional ELISA method, a higher
sensitivity could be achieved by 3.2-fold reduction in detection
limit. The main advantages of this method are enhancement of
electrochemical responses by MWCNTs, minimization of expen-
sive reagents, physical and chemical stability of immunosensor,
low background noise, wide working potential range and accuracy.
Application of magnetic beads in electrochemical immu-
noassay for detection of ZRL was explored by Hervas et al.
(2009, 2010, 2011). In an amperometric analysis based on direct
competitive immunoassay involving antibody-coated magnetic
beads as immobilization support and HRP as enzymatic label, ZRL
could be detected in baby food samples with low detection limit
(0.011 µg/L) and high recovery (95–108%) (Hervas et al., 2009).
However, in a later study, a remarkably lower detection limit
(0.007 µg/L) and a good recovery (101–111%) are achieved by an
electrochemical immunosensor consisting of magnetic beads cou-
pled with disposable carbon SPE (Hervas et al., 2010). In addition,
a very low systematic error (<4%) and excellent reproducibility
(RSD = 6%) are obtained by this method. After the immunochemi-
cal reaction, the modified paramagnetic beads were confined by
a magnet on the surface of SPE where the electrochemical detec-
tion is accomplished using hydrogen peroxide substrate and hy-
droquinone mediator for the HRP enzyme.
Microfluidic technology has now become a novel sensing
platform in which different analytical steps, biological recogni-
tion materials and suitable transducers are cleverly integrated to
yield a generation of efficient sensors for analytical applications.
Hervas et al. (2011) reported a novel “lab-on-chip” strategy in-
tegrating an electrokinetic magnetic bead-based electrochemi-
cal immunosensor on a microfluidic chip for reliable control of
permitted levels of ZRL in infant foods. Immunological reaction
is performed using a competitive enzyme-linked immunosorbent
assay where the ZRL and enzyme-labeled derivative compete for
the binding sites of the specific monoclonal antibody immobi-
lized onto protein G coupled with magnetic beads. The back elec-
trochemical reduction obtained by hydrogen peroxide–mediated
HRP catalysis of hydroquinone oxidation into benzoquinone was
measured at +0.1 V. This method was performed in less than 15 min
attaining a limit of detection of 0.4 µg/L well below the legislative
requirements, low systematic error of 2%, and good recovery of
101% for solid samples and 103% for liquid samples. This novel
microfluidic technology involved creative use of simple channel
layout of double-T microchip for sequential performance of im-
munointeraction and enzymatic reaction through application of
electric fields suitably connected to reservoirs for driving the flu-
idics to different chambers for specific reactions. In addition, the
nonspecific adsorption is avoided in a simple and elegant way by
operating both zones through application of magnetic field.
6 Deoxynivalenol
Deoxynivalenol (DON) is a naturally occurring trichothecene-
type B mycotoxin mainly produced by two fungi F. graminearum
and F. culmorum (Kushiro, 2008; Sobrova et al., 2010). DON is also
named as “vomitoxin” because of its strong emetic effects after
consumption and was first diagnosed among Japanese men in
1972 who consumed moldy barley containing Fusarium fungi
(Ueno, 1988; Sobrova et al., 2010). It is commonly found in cere-
als, posing serious threat for the safety of cereal-based foods and
feedstuffs (Romanazzo et al., 2010). Besides, DON is also present
in food products such as corn, popcorn, rice, rye, wheat, buck-
wheat, barley, oats, sorghum, noodles, malt, beer, baby and in-
fant foods (Table 12.1) (Prieto-Simon et al., 2007). Structurally, it
is a polar compound with the chemical name 12,13-epoxy-3,7,
15-trihydroxytrichothec-9-en-8-on and the presence of three free
hydroxy groups are responsible for toxicity of DON (Fig. 12.1), and
the possible detoxification route of DON in human is cytochrome
P450 (Nagy et al., 2005). The most critical physicochemical prop-
erty of DON lies its ability to withstand high temperatures (up
to 350°C), which intensifies its risk of occurrence in foods/food
products (Manthey et al., 2004; Sobrova et al., 2010). However,
owing to its high solubility in water, its level is considerably re-
duced during food processing (Sugita-Konishi et al., 2006; Visconti
et al., 2004). Consequently, the European Union has recommend-
ed a maximum permissible level (MPL) of DON to range from 50
to 2000 µg/kg depending on the type of cereal and cereal products
(Romanazzo et al., 2010). However, most of the countries have set
the MPL to be 750 µg/kg, with the provisional tolerable daily in-
take recommended by JECFA being 1 µg/kg bw per day (FAO, 2003;
Chen et al., 2012). Thus, it is important to develop reliable analyti-
cal methods for analysis of DON in food products.
For detection of DON in food samples, a cost-effective
enzyme-linked immunomagnetic electrochemical assay (ELIME)
was developed by Romanazzo et al. (2010), who employed a novel
anti-DON fragment antibody (Fab) and immunomagnetic beads
combined with 8 magnetized SPE as electrochemical transducers.
The entire ELIME assay is performed in three major steps with
first step involving washing of magnetic beads coating with DON
conjugate and stored at 4°C until use. In the second step, the
coated particles are subjected to immunological chain, followed
by pipetting particles on the surface of eight magnetized SPE for
electrochemical measurement. Based on the cross-reactivity of
antigen-binding fragment toward different trichothecene myco-
toxins, a good selectivity was shown for DON with the exception
of 3-acetyldeoxynivalenol in the working range between 100 and
4500 ng/mL with an EC50 (amount of DON required to produce
50% decrease in the response signal) of 380 ng/mL. Application to
real food samples showed precision and recovery values of 9–24%
and 82–110% for breakfast cereals, respectively, and 10–33%
and 97–108% for baby foods. This ELIME assay is claimed to be
extremely robust and insensitive to sample matrix interference
as compared to some other techniques such as SPR, fluorescent
array biosensor and electronic olfactory system. Additionally, a
simple water extraction was used for wheat, while acetonitrile-
water extraction coupled with a drying step can be employed for
breakfast cereal and baby food.
7 Citrinin
Citrinin (CIT) or (3R,4S)-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-
6-oxo-3H-2- benzopyran-7-carboxylic acid is a secondary metabo-
lite produced by the three fungal genera Aspergillus (A. carneus,
A. niveus, and A. terreus), Penicillium (P. citrinum, P. verruco-
sum, and P. expansum), and Monoscus (M. ruber) (Fig. 12.1) (Flajs
and Peraica, 2009; Bragulat et al., 2008). It usually contaminates
maize, wheat, rye, rice, corn, barley, oat, peanut, fruit, and sausage
( Table 12.1) (Prieto-Simon et al., 2007). In addition, CIT contami-
nation has been reported in numerous agricultural commodi-
ties, foods, feedstuffs, and biological fluids from a wide variety of
geographical origins (Flajs and Peraica, 2009; Arevalo et al., 2011).
Though CIT possesses antibiotic property against Gram-positive
bacteria, it is seldom used as a drug due to its high nephrotoxic-
ity (Gupta et al., 1983). Besides kidney, the other target organs of
CIT include liver, and bone marrow (Flajs and Peraica, 2009). The
decomposition products (CIT-H1 and CIT-H2) obtained by heating
CIT with water at 140–150°C are more toxic than CIT as the decom-
position temperature declines from 175 to 140°C in the presence
of water (Kitabatake et al., 1991; Xu et al., 2006). Since CIT is com-
monly found along with OA, it was shown to increase the toxicity of
the latter either additively or synergistically causing a kidney dis-
ease called “Balkan endemic nephropathy” in human (Speijers and
Speijers, 2004; Klaric et al., 2013). The IARC (1986) classified CIT in
Group III of carcinogens due to limited evidence in animals and no
evidence in human. The scientific opinion published by EU on be-
half of EFSA (2012) has recommended no-observed-adverse-effect
level (NOAEL) of 20 µg/kg bw per day for both rats and pigs, while
the NOAEL between 9 and 53 µg/kg was suggested for high con-
sumers as well as 19 and 100 µg/kg for average consumers.
Recently, Arevalo et al. (2011) have developed an electrochemi-
cal immunosensor incorporated in a microfluidic cell for quan-
tification of CIT in rice samples. The assay involved competition
of monoclonal anti-CIT (mAb-CIT) IgG antibody in solution with
CIT in rice samples as well as CIT immobilized on a gold surface,
which is previously electrodeposited over a GCE and modified
with a cysteamine self-assembled monolayer. An excess of rab-
bit IgG (H + L)-labeled HRP added reacts with mAb-CIT on the
electrode surface. The HRP catalyzes the oxidation of catechol to
benzoquinone in the presence of hydrogen peroxide and its elec-
trochemical reduction back to catechol is measured on the GCE at
0.15 V versus Ag/AgCl by amperometry. The measured current is
directly proportional to the enzyme activity and inversely propor-
tional to the amount of CIT present in rice samples. Detection of
CIT by this assay is fast (2 min for detection and 45 min for total
assay) and sensitive in the linear working range between 0.5 and
50 ng/mL, with the LOD and LOQ being 0.1 and 0.5 ng/mL, re-
spectively, while the coefficient of variation for both intraday and
interday assays was <6%. The main advantages of this immuno-
sensor assay are higher sensitivity and shorter analysis time com-
pared to other conventional chromatographic methods.
8 Fumonisins
Fumonisins (FMNs) are naturally occurring mold toxins pro-
duced mainly by Fusarium verticillioides, F. proliferatum, and
Gibberella fujikuroi (Leslie, 1996; Ghali et al., 2009). Though there
are seven structurally similar FMNs, only FMN-B1, FMN-B2, and
FMN-B3 are the most abundant ones contaminating food and feed
worldwide with FMN-B1 (molecular formula C34H59NO15) contrib-
uting about 75% of the total FMs content (Fig. 12.1) (Branham and
Plattner, 1993). It is reported to occur in several human foods in-
cluding maize, maize-based products, barley, wheat, rice, and sor-
ghum (Table 12.1) (Jimenez et al., 1997; Mateo and Jimenez, 2000;
Prieto-Simon et al., 2007). FMNs are potentially hazardous to hu-
mans and animals causing various diseases such as immunosup-
pression, neurotoxicity, carcinogenicity, liver and kidney toxicity
(Branham and Plattner, 1993; Gelderblom et al., 1994). Moreover,
the exposure to FMNs has been closely associated with disruption
of sphingolipids metabolism causing leukoencephalomalacia
in equines and pulmonary edema in pigs (Voss et al., 1990; Colvin
and Harrison, 1992; Ross et al., 1993). In humans, FMNs are sus-
pected to cause esophageal cancer among certain South African
and Chinese people (Pitt, 2000), and the IARC (1993) has classi-
fied FMN-B1 as 2B carcinogen. The USFDA (2001) has enforced
permissible limits on total FMNs to be ranged from 0.2 to 4 ppm
for direct human consumption of food products, while a level of
5–100 ppm recommended for animal feed.
An electrochemical affinity sensor for detection of FMNs in
foods was developed by Kadir and Tothill (2010), who employed
a gold SPE modified with monoclonal antibody and silver–
silver chloride pseudoreference electrode. Initially, a direct
ELISA assay was developed, followed by transferring the plat-
form to the surface of a gold SPE, monitoring the reaction of
3,3’,5,5’- tetramethylbenzidine dihydrochloride (mediator) and
hydrogen peroxide (substrate) catalyzed by HRP at 100 mV and
detecting FMNs by chronoamperometry. Compared to conven-
tional ELISA method, this immunosensor assay attained a 20-fold
reduction in detection limit of 5 µg/L in the linear working range
of 1–1000 µg/L, which is lower than the detection limit (2–4 mg/L)
of FMNs required by European Union legislation. The sensor also
showed >1% cross reactivity in the presence of some other my-
cotoxins like aflatoxin B1 and ochratoxin A, implying that a good
selectivity could be achieved by this method. Also the application
to ground corn samples spiked with four different concentrations
(50, 250, 500, and 2500 µg/kg) of FMN-B1 and FMN-B2 mixture
showed a recovery of 101.7, 76.04, 112.6, and 104.64%, respec-
tively. The advantages of this electrochemical assay include low
matrix interference, high sensitivity and reproducibility, as well as
rapid detection for on-site application.
9 Conclusions and Future Perspective
Recent advancements in the field of nanotechnology has en-
abled improved sensitivity, on-site analysis, and multiplexing ca-
pability of electrochemical sensors for detection of mycotoxins.
This chapter has reviewed some applications of nanomaterial-
based sensors for detection of aflatoxins (B1, B2, G1, G2, and M1),
ochratoxin A, sterigmatocystin, zearalenone, deoxynivalenol,
citrinin, and fumonisins in food/feed samples. However, more
research still needs to be dedicated to the application of other
nanomaterials including metal and metal-oxide nanoparticles
as analytical sensors for mycotoxin detection. In addition, the
nanomaterial-based detection should be extended to mycotoxins
other than aflatoxin and ochratoxin A. More studies should also
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... F ood safety is a vital health-related issue and many countries including Taiwan have established a law for critical control of food safety and sanitation with a main goal to achieve ultimate protection for human health [1,2]. Mycotoxins (MYTs) are a class of low molecular weight food toxins (300e700 Da) produced as secondary metabolites by fungi during pre-and post-harvest of various agricultural products including cereals, cereal-based foods, corn, rice, rye, wheat, barley, oats, sorghum, soybeans, buckwheat, peanut, malt, beer, dried fruits, wine, milk, baby and infant foods, coffee beans, cocoa, bakery and meat products [2,3]. ...
... F ood safety is a vital health-related issue and many countries including Taiwan have established a law for critical control of food safety and sanitation with a main goal to achieve ultimate protection for human health [1,2]. Mycotoxins (MYTs) are a class of low molecular weight food toxins (300e700 Da) produced as secondary metabolites by fungi during pre-and post-harvest of various agricultural products including cereals, cereal-based foods, corn, rice, rye, wheat, barley, oats, sorghum, soybeans, buckwheat, peanut, malt, beer, dried fruits, wine, milk, baby and infant foods, coffee beans, cocoa, bakery and meat products [2,3]. The MYTs are most commonly produced by filamentous fungi such as Aspergillus, Fusarium and Penicillium species with their levels increasing during storage under high temperature and humidity conditions [4]. ...
... The presence of MYTs in foods and feeds not only result in serious economic loss but also cause deleterious, acute and chronic effects on human health. The fungal source, occurrence in food/feed and their toxicity have been elaborately reported elsewhere [2,5]. Most importantly, the MYTs are not only carcinogenic, teratogenic, mutagenic and/or hepatotoxic, but also affect the pulmonary, cardiovascular and central nervous systems [6,7]. ...
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Mycotoxins (MYTs), a class of low molecular weight secondary metabolites produced by filamentous fungi in food and feed, pose serious global threat to both human health and world economy. Due to their mutagenic, teratogenic, carcinogenic and immunosuppressive effects, the International Agency for Research on Cancer has classified various MYTs under Group 1 to 3 category with aflatoxins being designated under Group 1 category (carcinogenic to humans). Also, the presence of MYTs in trace amounts in diverse food matrices necessitates exploration of highly sensitive methods for onsite analysis. Although conventional chromatographic methods are highly sensitive, they are expensive, tedious and cannot be applied for rapid onsite analysis. In recent years the application of nanomaterials especially carbon-based nanomaterials (CNMs) in the fabrication of low-cost and miniaturized electrochemical and optical sensors has enabled rapid onsite analysis of MYTs with high sensitivity and specificity. Moreover, the CNMs are employed as effective solid phase extraction (SPE) adsorbents possessing high specific surface area for effective enrichment of MYTs to improve the sensitivity of chromatographic methods for MYT analysis in food. This article aims to overview the recent trends in the application of CNMs as SPE adsorbents for sample pretreatment in chromatographic methods as well as in the fabrication of highly sensitive electrochemical and optical sensors for rapid analysis of MYTs in food. Initially, the efficiency of various functionalized CNMs developed recently as adsorbent in packed SPE cartridges and dispersive SPE adsorbent/purification powder is discussed. Then, their application in the development of various electrochemical immunosensors involving functionalized carbon nanotubes/nanofibers, graphene oxide, reduced graphene oxide and graphene quantum dots is summarized. In addition, the recent trends in the use of CNMs for fabrication of electrochemical and fluorescence aptasensors as well as some other colorimetry, fluorometry, surface-enhanced Raman spectroscopy and electrochemical based sensors are compared and tabulated. Collectively, this review article can provide a research update on analysis of MYTs by carbon-based nanomaterials paving a way for identifying future perspectives.
... The principal genera of mycotoxin-producing fungi are Penicillium, Aspergillus and Fusarium, and mycotoxins may remain even after fungal destruction. [1][2][3][4] Aflatoxins are more widely studied mycotoxins. Chemically, they are substituted coumarins, among which the most important are aflatoxin B1 (AFB1). ...
The present work describes a facile synthesis of a nanocomposite based on gold nanoparticles (NPAu) and graphene quantum dots (GQD) and its use as an electrode modifier for a non-biological alternative of Aflatoxin B1 (AFB1) voltammetric determination. TEM and SEM techniques showed a uniform and well-distributed dispersion of gold nanoparticles (19 ± 6 nm of average size) and GQD, which was used as a reducing and stabilizing particle formation. Modified screen-printed electrode (NPAuGQD-SPE) was characterized by electrochemical experiments, as linear voltammetry and electrochemical impedance spectroscopy. A significative electrocatalytic effect was observed towards AFB1 oxidation using the proposed device, shifting the peak potential to less positive values and improving the voltammetric response. EIS experiments showed lower RCT values for the modified electrodes, calculated as 117 Ω, 14.9 kΩ, and 40.3 kΩ for NPAuGQD-SPE, GQD-SPE, and SPE, respectively. Under optimized conditions, an analytical curve with linear region of 1.0–50.0 nmol L⁻¹ was obtained, reaching detection and quantification limits of 0.47 and 1.5 nmol L⁻¹, respectively. Samples of malted barley were fortified based on the maximum residue limit (MRL) allowed by Brazilian legislation, and recoveries in the range of 76–103% were obtained, indicating that there is no significant matrix effect, considering the low concentration values used and simple sample treatment.
... In normal conditions, graphene is hydrophobic, impermeable to liquids and gases. However, graphene derivatives, including graphene oxide and reduced graphene oxide, can be dispersed in aqueous media, and capable of covalently immobilizing antibodies due to their rich oxygen-containing functional groups such as carboxyl group (Pérez-López and Merkoçi 2011;Chen and Inbaraj 2016;Wang and others 2016). ...
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The presence of mycotoxins in foodstuff causes serious health problems to consumers and economically affects the food industry. Among the mycotoxins, aflatoxins are very toxic and highly carcinogenic contaminants which affect the safety of many foods, and therefore endanger human health. Aflatoxin M1 (AFM1) found in milk results from the biotransformation of aflatoxin B1. Many efforts have been made to control the source of AFM1 from farmers to dairy product companies. However, AFM1 escapes ordinary methods of food treatment such as cooking, sterilization, and freezing, hence it appears in milk and dairy products. The presence of high levels of AFM1 constitutes an alarming threat as milk and dairy products contain essential nutrients for human health, especially for infants and children. For this reason, there is a pressing need for developing a fast and reliable screening method for detecting trace aflatoxins in food. Several analytical methods based on high-performance liquid chromatography (HPLC) and mass spectroscopy have been used for aflatoxin detection; however, they are expensive, time-consuming, and require many skills. Recently, immunoassay methods, including enzyme-linked immunosorbent assay (ELISA), immunosensors, and lateral flow immunoassay (LFIA), have been preferred for food analysis because of their improved qualities such as high sensitivity, simplicity, and capability of onsite monitoring. This paper reviews the new developments and applications of immunoassays for the rapid detection of AFM1 in milk.
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The evolution of nanotechnology has made an appreciable contribution in the domain of food science from the selection of appropriate procedures for tracking and tracing of contaminants to the incorporation of various nanomaterials as health supplements and antimicrobial agents in food products. The technique has also taken into account the safety and quality aspects of the food products by preventing them from the proximity of physical, chemical, and biological hazards. Nanotechnology has a great interest in assessing food safety by detecting pathogens, pesticides, viruses, toxic residues, and several other contaminants found in food products by the action of nanobiosensors. Different kinds of communication methods, including internal and external indicators monitor the interaction between food, packaging material, and the external environment. One of the important sectors where nanotechnology can be looked upon as a boon is traceability where various nano-based communication devices such as radio-frequency identification (RFID) tags and barcodes are integrated with packaging materials, building a revolutionary force in the field of food packaging technology. Furthermore, the use of these devices ensures the authenticity, anti-counterfeiting, anti-theft of the food products. In this chapter, various nanotechnology applications would be studied concerning their applications in traceability throughout the food chain.
Xanthomegnin, a known fungal toxin, secondary metabolite, and pigment diffuses from the dermatophytes has gained attention as local virulence factor because of the mutagenicity, toxicity, cytocidal, and immunosuppressive properties. Not only as a dermatophyte in skin related disorders, the production of xanthomegnin is implicated as a powerful diagnostic marker in patients suffering from ocular mycoses. Incidentally also attributed to death in livestock's majorly by exposing themselves to food-borne fungi like Aspergillus and Penicillium. The production of xanthomegnin in dermetophytic species Trichophyton rubrum, found commonly in infected skin and nails. In this study nickel/nickel hydroxide nanoparticles decorated reduced graphene oxide (Ni/Ni(OH)2-rGO) modified glassy carbon electrode has been successfully used for non-enzymatic detection of xanthomegnin. The Ni/Ni(OH)2-rGO composites were synthesized through a simple microwave assisted technique with less harmful reducing agent. The UV-visible spectroscopy (UV-vis), X-ray photoelectron spectroscopy (XPS), Scanning electron microscopy - energy dispersive X-ray spectroscopy (SEM-EDS), and electrochemical investigations demonstrated the robust formation of the sensor. The sensor exhibited improved electrochemical properties with enhanced electrochemical active area and excellent electrochemical behavior towards xanthomegnin detection with a limit of detection of 0.12 μM. The selectivity, stability, and analytical recovery studies proved the potential use of the sensor for the detection of xanthomegnin in real samples. Further, the sensor successfully detected xanthomegnin produced by the Trichophyton rubrum, the most common superficial fungus, accounting for at least 60% of all superficial fungal infections in humans. Validation studies showed satisfiable and quantifiable amount of xanthomegnin in comparison with common bench mark UV-Vis studies meant for fungal mycotoxin detection.
The term “emerging mycotoxins” was coined in 2008 for lesser-known Fusarium metabolites, fusaproliferin, enniatins, beauvericin, and moniliformin. The emerging mycotoxins and their derivatives can occur in high concentrations and high frequency in cereals, cereal-based products, fruits, processed and unprocessed/raw food, animal feed, and feeding stuffs. Some of them are carried over in animal-derived products. Notwithstanding the high prevalence, determination and legislative regulations of emerging mycotoxins are confined to Europe and the Mediterranean. Since many emerging mycotoxins are more or less toxic to the consumers, their importance to food safety is not addressed properly. In view of this, the present chapter focuses on some emerging or minor mycotoxins produced by Aspergillus spp., Penicillium spp., Fusarium spp., and Alternaria spp.
For the management and prevention of many chronic and acute diseases, the rapid quantification of toxicity in food and feed products have become a significant concern. Technology advancements in the area of biosensors, bioelectronics, miniaturization techniques, and microfluidics have shown a significant impact than conventional methods which have given a boost to improve the sensing performance towards food analyte detection. In this article, recent literature of Aflatoxin B1 (AFB1), worldwide permissible limits, major outbreaks and severe impact on healthy life have been discussed. An improvement achieved in detection range, limit of detection, shelf-life of the biosensor by integrated dimensional nanomaterials such as zero-dimension, one-dimension and two-dimension for AFB1 detection using electrical and optical transduction mechanism has been summarized. A critical overview of the latest trends using paper-based and micro-spotted array integrated with the anisotropic shape of nanomaterials, portable microfluidic devices have also been described together with future perspectives for further advancements.
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Owing to the potential health hazards and the contamination of agricultural products, the presence of mycotoxins in food and feed products appears to be a real problem, which poses a serious threat to the country's economy and international trade and have detrimental effects on the lives of humans, domestic animals, and livestocks. Recently, nanobiotechnology, especially in the area of biosensors, has emerged as an innovative strategy to design a simple, facile, and reliable detection platform for mycotoxin analysis in a broad range of matrices and also served as a promising alternative to the conventional methodologies to fulfill the requirements of limit of detection and maximum permissible limit set by the legislation. In this contribution, different types of mycotoxins such as aflatoxins, ochratoxins, citrinin, patulin, and fusarium are briefly introduced. In addition, we provided a detailed overview about different types of biosensors (DNA‐based biosensor, electrochemical, carbon nanotubes, quartz crystal microbalance, etc.) employed for the specific and sensitive detection of different fungal toxins and apprise recent progress made in the development of biosensors for mycotoxin detection in food stuff. Undoubtedly, biosensor technology has enabled swift, low‐cost, high‐throughput, portable, and ultrasensitive determination of mycotoxins with an additional feature of multiplexing, allowing to assess the food safety on site.
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This paper describes a thin‐layer chromatographic (TLC) method, which allows the determination of sterigmatocystin at a level of 2 µg/kg in various cereal grains of interest. Sterigmatocystin is extracted from the food matrix and further purified by phenyl‐bond solid‐phase extraction (SPE). The separation and identification is performed on an amino‐derivatised high‐performance TLC (HP‐TLC) plate. The derivatisation for densitometric measurement and visual inspection is achieved reagent free by heating the plate. Sterigmatocystin results in highly fluorescent spots. The method has shown good recovery values for the various cereals analysed (e.g., around 80% for wheat). The method was applied for monitoring the sterigmatocystin content in 85 cereal samples, purchased from the local (Italian) market in 2002. However, none of the samples was found to be positive, indicating that this mycotoxin was not a problematic contaminant in products of this particular region and in this specific year.
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Mycotoxin analysis and detection in food and drinks is vital for ensuring food quality and safety, eliminating and controlling the risk of consuming contaminated foods, and complying with the legislative limits set by food authorities worldwide. Most analysis of these toxins is still conducted using conventional methods; however, biosensor methods are currently being developed as screening tools for use in field analysis. Biosensors have demonstrated their ability to provide rapid, sensitive, robust and cost-effective quantitative methods for on-site testing. The development of biosensor devices for different mycotoxins has attracted much research interest in recent years with a range of devices being designed and reported in the scientific literature. However, with the advent of nanotechnology and its impact on the evolution of ultrasensitive devices, mycotoxin analysis is also benefiting from the advances taking place in applying nanomaterials in sensors development. This paper reviews the developments in the area of biosensors and their applications for mycotoxin analysis, as well as the development of micro/nanoarray transducers and nanoparticles and their use in the development of new rapid devices.
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There is an increased interest toward the development of bioelectronic devices for food toxin (mycotoxins) detection. Mycotoxins are highly toxic secondary metabolites produced by fungi like Fusarium, Aspergillus, and Penicillium that are frequently found in crops or during storage of food including cereals, nuts, fruits, etc. The contamination of food by mycotoxins has become a matter of increasing concern. High levels of mycotoxins in the diet can cause adverse, acute, and chronic effects on human health and a variety of animal species. Side effects may particularly affect the liver, kidney, nervous system, endocrine system, and immune system. Among 300 mycotoxins known till date, there are a few that are considered to play an important part in food safety, and for these, a range of analytical methods have been developed. Some of the important mycotoxins include aflatoxins, ochratoxins, fumonisins, citreoviridin, patulin, citrinin, and zearalenon. The conventional methods of analysis of mycotoxins normally require sophisticated instrumentation, e.g., liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. Hence, new analysis tools are necessary to attain more sensitive, specific, rapid, and reliable information about the desired toxin. For the last about two decades, the research and development of simpler and faster analytical procedures based on affinity biosensors has aroused much interest due to their simplicity and sensitivity. The nanomaterials have recently had a great impact on the development of biosensors. The functionalized nanomaterials are used as catalytic tools, immobilization platforms, or as optical or electroactive labels to improve the biosensing performance to obtain higher sensitivity, stability, and selectivity. Nanomaterials, such as carbon nanomaterials (carbon nanotubes and graphene), metal nanoparticles, nanowires, nanocomposites, and nanostructured metal oxide nanoparticles are playing an increasing role in the design of sensing and biosensing systems for mycotoxin determination. Furthermore, these nanobiosystems are also bringing advantages in terms of the design of novel food toxin detection strategies. We will focus on some of the recent results related to fabrication of nanomaterial-based biosensors for food toxin detection obtained in our laboratories.
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Ochratoxin A (OTA) is a nephrotoxic mycotoxin with carcinogenic properties. Its presence was detected in various foodstuffs all over the world but with significantly higher frequency and concentrations in areas with endemic nephropathy (EN). Even though food is often contaminated with more than one mycotoxin, earlier studies focused on the occurrence and toxicology of only OTA. Only a limited number of surveys showed that OTA co-occurs in food with mycotoxins (citrinin-CIT, penicilic acid, fumonisin B1-FB1, aflatoxins-AF) which exert nephrotoxic, carcinogenic or carcinogen-promoting activity. This review summarises the findings on OTA and its co-occurrence with the mentioned mycotoxins in food as well as experimental data on their combined toxicity. Most of the tested mycotoxin mixtures involving OTA produced additive or synergistic effects in experimental models suggesting that these combinations represent a significant health hazard. Special attention should be given to mixtures that include carcinogenic and cancer-promoting mycotoxins.
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We report a sensitive electrochemical immunosensor for Ochratoxin A (OTA), which is a frequent mycotoxin contaminant in cereals and other kinds of agricultural commodities, based on a biotinylated monoclonal antibody against OTA (mAbOTA–bi) and the avidin–biotin coupling of the tracer extravidin–horseradish peroxidase (ea–HRP). The analytical performance has been improved with respect to a previously developed immunosensor based on a polyclonal antibody against OTA and a secondary aIgG antibody labeled with alkaline phosphatase as tracer. The immunosensor relies on indirect competitive assay format after the passive physical adsorption of the antigen conjugated to bovine serum albumin (OTA–BSA) or bound to gold nanoparticles (OTA–BSA–AuNPs), and the screen-printed technology for voltammetric (DPV) measurements. The new design is simpler (only one capture probe), requires less time (only one incubation time with antibody), and shows an increased slope at the linear range of the calibration plot due to higher affinity of the monoclonal antibody compared to the polyclonal one. The newly designed immunosensor has a linear dynamic range of 0.15 to 9.94 ng mL−1 of OTA (R ≥ 0.9900), lower detection limit (0.10 ng mL−1 OTA), and a variability between assays of about 10%. The immunosensor was validated with certified wheat samples after extraction of OTA in acetonitrile:water (6/4) (v/v), and allows the determination of OTA in concentration levels well below those permitted in cereals under European Union recommendations (3 ng g−1).
A one-step simultaneous immunchromatographic (OS-ICG) assay using colloidal gold-monoclonal antibody (gold-MAb) conjugates was developed for the rapid multianalysis of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in feed samples. Visual detection limits for AFB1 and OTA were 0.5 and 2.5 ng/mL, respectively, and the results were obtained within 15 min. Matrix interference from the feed extracts was efficiently reduced by appropriate dilution with buffer. Cut-off values of the OS-ICG assay for the feed spiked with AFB1/OTA mixtures (5/5, 10/10, 25/25, 50/55, 100/100 μg/kg) were 10 and 50 μg/kg for AFB1 and OTA. The comparative analyses of 65 feed samples by OS-ICG, enzyme-linked immunosorbent assay (ELISA), and high performance liquid chromatography (HPLC) showed good agreement. In this study, we confirmed that simultaneous analysis based on immunoassay is possible and it can be used as an on-site multianalysis of AFB1 and OTA in feed, food, and agricultural products.
The mycotoxins reviewed in this section comprise a family of closely related sesquiterpenoids produced by various species of fungi such as Fusarium, Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium and Stachybotrys,and are classified as trichothecenes after trichothecin, the first member of the group to be discovered (Freeman and Morrison, 1949). In the early stage of trichothecene studies, the first isolate, trichothecin, was discovered as a result of screening of antifungal metabolites in a culture of Trichothecium roseum. The following survey demonstrated that Myrothecium roridum and M. verrucaria produced potent antifungal metabolites, which were designated as verrucarins A through J and rondins A through E (Härri et al.,1962). Structural studies on these compounds demonstrated that they are macrocyclic trichothecenes (Böhner et al., 1965).
A novel electrochemical aptasensor, based on disposable screen-printed electrodes was developed for the sensitive detection of the mycotoxin ochratoxin A (OTA). Two strategies were investigated by using an indirect and a direct competitive assay, based on the use of superparamagnetic nanoparticles. The performance of the optimized aptasensors in terms of reproducibility, stability, sensitivity, and analysis time was studied. The best strategy was found to be the direct competitive format. In this assay, free OTA competed with labeled alkaline phosphatase–OTA for the binding to the DNA aptamer immobilized on magnetic beads. The electrochemical detection was thus achieved through a suitable substrate for the enzyme ALP, by Differential Pulse Voltammetry. The aptasensor obtained using this novel approach allowed detection limit of 0.11ng/mL, and was also validated for real sample analysis.