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Highly sensitive toxin microarray assay for aflatoxin B1 detection
in cereals
Azadeh Beizaei
a
,
b
,
*
, Sara L. O' Kane
a
, Abolfazl Kamkar
b
, Ali Misaghi
b
, Gary Henehan
c
,
Dolores J. Cahill
a
,
**
a
Conway Institute of Biomolecular &Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
b
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, University of Tehran, Qareeb Street, Azadi Av. P.O. Box: 14155-6453,
Tehran, Iran
c
Dublin Institute of Technology, Campus Planning Office, Grangegorman Lower, Dublin 7, Ireland
article info
Article history:
Received 11 November 2014
Received in revised form
9 March 2015
Accepted 17 March 2015
Available online 25 April 2015
Keywords:
Toxin microarray
Aflatoxin B
1
Competitive immunoassay
Cereal samples
abstract
We report a rapid, highly sensitive microarray method for quantitative aflatoxin B
1
(AFB
1
) detection in
cereal samples. Following optimisation using an indirect competitive immunoassay, optimised amounts
of AFB
1
-bovine serum albumin (AFB
1
-BSA)-conjugate were contact-printed onto 16 isolated sub-arrays
on multi-pad nitrocellulose coated slides subsequently used in competitive binding assays.
The toxin microarray working range for AFB
1
was established in the range of 15 pg g
1
to 3.04 ng g
1
,
with a detection limit of 1 pg g
1
. To determine assay sensitivity in contaminated food models, wheat
flour and barley grains samples were spiked with AFB
1
standard dilutions. Following extraction, the
working ranges of 0.11e4.15 and 0.18e4.31 ng g
1
were determined, with detection limits of 30 and
90 pg g
1
, respectively. The sensitivity of the developed assay is below the European commission limit
set for AFB
1
detection and the assay procedure was completed in 3 h time. Good recoveries (98% ±11%)
obtained demonstrate the suitability of the proposed method for rapid and sensitive quantification of
AFB
1
in contaminated cereal samples.
©2015 Elsevier Ltd. All rights reserved.
1. Introduction
Aflatoxins are extremely toxic and carcinogenic secondary me-
tabolites produced by some Aspergillus species namely A. flavus,
A. parasiticus and the rare A. nomius contaminate a wide range of
agricultural products (Zhang et al., 2009). Although more than 20
aflatoxins have been identified, only aflatoxin B
1
(AFB
1
), aflatoxin B
2
(AFB
2
), aflatoxin G
1
(AFG
1
), and aflatoxin G
2
(AFG
2
) are classified as
human's carcinogens (Chun, Kim, Ok, Hwang, &Chung, 2007). AFB1
presents the highest toxic potential (CAST, 2003); being
hepatotoxic and carcinogenic in human and animals (Nogueira
et al., 2009; Pitt, 2000) and listed as a Group I carcinogen by the
International Agency for Research on Cancer (IARC) (IARC, 2002). It
is a potent carcinogen, teratogen and mutagen (Speijers &Speijers,
2004). Although Aflatoxins contaminate a wide range of agricul-
tural products such as cereals, nuts, peanuts, fruits, oilseeds and
dried fruits both in the field and during storage (Zhang et al., 2009)
however, AFB1 is a predominant form in cereal and oil seed con-
taminations (CAST, 2003). They enter the food chain mainly by
ingestion via the dietary route in humans and animals. (Piemarini,
Micheli, Ammida, Palleschi, &Moscone, 2007). In farm and labo-
ratory animals, chronic exposure to aflatoxins compromises im-
munity and interferes with protein metabolism and several
micronutrients that are critical to health. Aflatoxin exposure and
their toxic effects on immunity and nutrition combine to negatively
affect health that account for 40% of the burden of disease in
developing countries (Williams et al., 2004).
Regard to impact of Aflatoxins on human an animals, legal
limits for aflatoxin intake via food and feed products have been
*Corresponding author. Conway Institute of Biomolecular &Biomedical
Research, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: þ353 87 940
0321.
** Corresponding author. School of Medicine and Medical science, Conway Insti-
tute of Biomolecular and Biomedical Science, University College Dublin, Belfield,
Dublin 4, Ireland. Tel.: þ353 861725572; fax: þ353 1 716 6701.
E-mail addresses: a.beizaee@gmail.com (A. Beizaei), slokane1@gmail.com
(S.L. O' Kane), akamkar@ut.ac.ir (A. Kamkar), a_misaghi@hotmail.com
(A. Misaghi), Gary.Henehan@dit.ie (G. Henehan), Dolores.cahill@ucd.ie (D.J. Cahill).
Contents lists available at ScienceDirect
Food Control
journal homepage: www.elsevier.com/locate/foodcont
http://dx.doi.org/10.1016/j.foodcont.2015.03.039
0956-7135/©2015 Elsevier Ltd. All rights reserved.
Food Control 57 (2015) 210e215
established in many countries. For example, the current maximum
allowable levels set by the European Commission are 2 ng g
1
for
AFB
1
and 4 ng g
1
for total aflatoxins (B
1
þB
2
þG
1
þG
2
)in
groundnuts, nuts, dried fruits, and cereals (Kolosova, Shim, Yang,
Eremin, &Chung, 2006). There are well-established methodolo-
gies for analysing aflatoxins in different foodstuffs. These include
methods such as fourier transform near-infrared spectroscopy
(FTIR) (Tripathi and Mishra, 2009), high-performance liquid
chromatography (HPLC) (Ghali et al., 2009; Khayoon, Saad, Lee, &
Sallah, 2012), liquid chromatographyetandem mass spectrometry
(LCeMS/MS) (Cervino, Asam, Knopp, Rychlik &Niessner, 2008;
Rubert., Soler, &Manes, 2012), immunochromatographic assay
(ICA) (Zhang, Li, Yang et al., 2011; Zhang, Li, Zhang, &Zhang, 2011)
ion mobility spectrometry (Sheibani, Tabrizchi, &Ghaziaskar,
2008) and enzyme-linked immunosorbent assay (ELISA) (Li
et al., 2009; Wang et al., 2012). Although chromatic techniques
have considerable advantages providing accurate quantification of
analytes, they require expensive equipment, extensive samples
preparation and skilled operators which make them unsuitable for
routine screening. Therefore, it is required to establish a rapid, low
cost and sensitive method as routine screening method for
monitoring purpose (He et al., 2012). Nowadays, immunochemical
assays such as lateral flow strips and ELISA are widely used for
mycotoxin detection as routine screening methods. Moreover,
microarray based immunoassay technology has offered the pos-
sibility to quantitatively measure multiple samples simultaneously
for multiple analytes and has a great potential as monitoring
systems for the rapid assessment of water or food samples
(Ngundi et al., 2005). Despite the fact that total aflatoxin detection
is a legislative requirement, there is however the need for further
improvement of current methods for highly sensitive detection of
aflatoxin B1 e.g. as low as 0.1
m
gkg
1
set by EU commission for
cereal based and dietary food products for infants and young
children. Therefore, the aim of our study was to generate a very
sensitive assay for AFB1 detection in microarray platform and
evaluate the produced sensitivity with ELISA. Here, we report the
development of a toxin microarray for rapid and sensitive detec-
tion of AFB
1
in cereal samples. The efficacy of this toxin microarray
was evaluated in cereal samples using spiked wheat flour and
barley.
2. Material and method
2.1. Materials, reagents and instruments
Maxisorp polystyrene microtiter plates were purchased from
NUNC. 384-well microtiter plates were obtained from Molecular
Devices. 16-pad nitrocellulose coated FAST slides and incubation
chambers (6 mm 6 mm pad) were purchased from Whatman Int.
Ltd. AFB
1
, AFB2, AFG1, AFG2, AFB
1
-bovine serum albumin (AFB
1
-
BSA), monoclonal anti-AFB
1
antibody, sheep anti-mouse IgG eCy3
(IgG-Cy3), 3,3
0
,5,5
0
-Tetramethylbenzidine (TMB) liquid substrate
system (peroxidase substrate) solution were purchased from Sig-
maeAldrich. Goat anti-mouse HRP conjugate IgG was purchased
from Abcam. Alexa Fluor
®
647 Goat anti-Mouse IgG (H þL), highly
cross-adsorbed antibody were purchased from Invitrogen. All other
chemicals were of analytical grade (A.R.) and purchased from Sig-
maeAldrich (Dublin, Ireland). Q-Array System (Genetix) and 1
blunt-ended stainless steel print pin with a tip diameter of 150
m
m
was used to generate the toxin microarrays. The arrays were
scanned using a confocal microarray reader (Genepix 4000B).
Washing steps for ELISA were performed by Fluido 2 microplate
washer (Anthos). The experimental data for ELISA were obtained by
Spectra MaxM2 microplate reader (Molecular device).
2.2. Competitive ELISA and cross-reactivity
Maxisorp plates coated with 50
m
L/well of AFB
1
-BSA (1
m
gml
1
)
in bicarbonate buffer (100 mM Na
2
CO
3
NaHCO
3
pH 9.6) were
incubated at 4
C for overnight. After two washes with TBST
(150 mM NaCl, 10 mM Tris-HCL pH 7.5, 0.1% v/v Tween 20), plates
were blocked with skim milk powder 5% (v/v) in TBST (40 0
m
l/well)
for 1 h at 37
C. Following three washes (50
m
l/well), pre-incubated
AFB
1
in different concentrations with monoclonal antibody were
incubated for 30 min at 37
C. After three washes, HRP conjugated
goat anti-mouse (0.2
m
gml
1
in skim milk powder 5% (v/v) in TBST,
50
m
l/well) were added for 1 h at 37
C. Followed by three washes,
TMB solution (50
m
l/well) were added and plate were incubated for
30 min at 37
C. The final absorbance was measured at 650 nm. To
assess the specificity of developed ELISA, cross reactivity of Mab
were constructed by using AFB1, AFB2, AFG1 and AFG2 as
competitor under optimised condition. Four serial diluted standard
curves were performed in the ELISA and the competition curves
were graphed and fitted by a four-parameter logistic equation. The
50% inhibition concentration (IC50) values of different aflatoxins
were determined and the cross reactivity was gained by comparing
the IC50 values of analytes with the following formula: cross-
reactivity (%) ¼[IC50 (AFB1)/IC50 (analyte)] 100 ( Jiang et al.,
2012).
2.3. Contact printing and immobilization of toxin microarray
16-pad nitrocellulose coated FAST slides were placed in Q-Array
spotting chamber with relative humidity of 60%e75%. Each sub-
array consisted of 32 replicates of AFB
1
-BSA, 8 replicates of mouse
IgG-Cy3 (printing control), 4 replicates of monoclonal anti- AFB
1
antibody (positive control) and 4 replicates of BSA 2% in PBS
(negative control). The average size of each generated spot was
around 200
m
m. After printing, the microarray slides were stored at
4
C for at least 24 h before use.
2.4. Toxin microarray procedure
An indirect competitive immunoassay was performed on each
sub array after fixing spotting chambers on slides. Then slides were
blocked with 100
m
l of BSA 2% in PBS for 1 h and washed two times
with PBST (0.01 M phosphate buffer contains 0.0027 M KCl and
0.137 M NaCl, pH 7.4, at 25
C, 0.01% (v/v) Tween 20). 50
m
l pre-
incubated mixture of AFB
1
standard solutions at different concen-
trations with monoclonal anti-AFB
1
(0.19
m
g/ml) in BSA 1% PBST
were added and the slides incubated for 30 min at 37
C. Followed
by three washes, 50
m
l of Alexa Fluor
®
647 anti-mouse IgG
(1 mg ml
1
) 1:5000 (v/v) in BSA 1%- PBST was added for 45 min in
37
C. Then slides were centrifuged at 3000 rpm for 3 min at 4
C
after last three washes. Dried slides were scanned at 532 nm and
635 nm wavelength with a scan resolution of 10
m
m. Total proce-
dure completed in 3 h duration.
2.5. Food sample preparation
Barley grains were ground in a householder blender at high
speed for 5 min. Then barley and wheat flour were artificially
contaminated by adding 100
m
l of AFB
1
standard solutions (0, 0.1, 1
and 10
m
g/ml) to 5 g of sample. The extraction method used was a
modification of that used by Strachan and Garden (Garden &
Strachan, 2001). For this, 15 ml methanol-water (80: 20) was
added to 5 g of sample. The suspensions were vortexed for 1 min
and then centrifuged at 4000 g for 15 min. The aqueous layer was
diluted 1 in 10 for the assays. The concentration of AFB
1
in diluted
sample extracts was measured by reference to a calibration curve
A. Beizaei et al. / Food Control 57 (2015) 210e215 211
and used to estimate the concentration in the original sample
(Table 2).
2.6. Data extraction and analysis
Toxin microarray data was extracted using Genepix Pro 5.1
software (Axon Instruments). The value used for analysis generated
by “mean foreground minus mean background”intensity for each
spot. IgG-Cy
3
value were applied for normalisation in each sub
array. Standard curves were produced using standard solutions of
AFB
1
(0, 0.05, 0.1, 0.2, 0.5, 1, 5 and 10 ng ml
1
) and were generated
from one chip in parallel, and repeated two times. Calibration
curves for wheat flour and barley, produced by using artificially
contaminated samples as described in section 2.5. Recovery esti-
mations were measured on two different days. (Table 2).
3. Results and discussion
3.1. ELISA optimisation and cross reactivity test
A spectrophotometric ELISA used to asses immunoassay sensi-
tivity and specificity of Mab. The developed ELISA protocol was
similar to that applied by Ammida et al. (Ammida, Micheli, &
Palleschi, 2004), with minor modifications. Standard curves were
produced using standard solutions of AFB
1
(0, 5, 10, 20 and
50 ng ml
1
). To achieve the optimal condition for assay, Mab
antibody gradually titrated from 1: 10 000 to 1:35 000 (v/v) dilu-
tion. In the Fig. 1 generated calibration curves has been shown.
Detection limit of optimised ELISA defined as the concentration
corresponding to the f(x) value obtained by subtracting three times
the standard deviation of the zero standard measurement from
standard curve (Ammida et al., 2004). Using this formula sensitivity
of developed ELISA determined as 0.7 ng g
1
. The specificity of
ELISA was evaluated by determining the cross-reactivity of Mab
with AFB1, AFB2, AFG1 and AFG2. The IC50 was obtained from the
standard curves. The calculated cross-reactivity measured as 100%,
72%, 35% and 32% for AFB1, AFB2, AFG1 and AFG2, respectively. High
cross-reactivity obtained from AFB2 showing that Mab should be
re-consider for selective detection of AFB1.
3.2. Optimization on toxin microarray
Utilizing the dot blot technique, the concentration of
100
m
gml
1
of AFB
1
-BSA and the concentrations of 0.33 mg ml
1
of
monoclonal antibody and 0.01 mg ml
1
of IgG-Cy3 (positive con-
trols) were chosen for printing on FAST nitrocellulose slides. To
establish the working range in the microarray format, further ti-
trations of monoclonal antibody were performed. The four final
optimised dilution of monoclonal antibody applied on the sub ar-
rays were 1:150 000, 1:160 00, 1:170 000 and 1:180 000 (v/v) and
were evaluated by generating standard competition curves for
AFB
1
. Comparison of the calibration curves generated using the
toxin microarray indicated that the dilution of 1: 170 000 (v/v)
(equal to the concentration of 0.19
m
gml
1
) can be used for
quantitative detection of AFB
1
(Fig. 2). The minimum antibody
dilution used for ELISA was 1: 35 000 (v/v) (0.94
m
gml
1
) indicates
that the toxin microarray has the advantage of a reduction of
analytical usage and consumable costs by requiring near 5 fold less
of expensive Mab. Several microarrays were screened without
competitors in order to evaluate the experimental variation in spot
intensities among each array. Relative standard deviation no higher
than 10% was observed. The variation across the slide is affected by
the distance of the slide from microtitre plate, and the exact loca-
tion on the slide.
3.3. Calibration curves
The result of calibration curves, the equations for estimation of
the LOD values (equivalent to IC
10
) and the working ranges are
shown in Table 1. Each concentration of AFB
1
had 32 replicates in a
sub array and each sub array is represented two times. The logistic
correlation coefficient (R
2
) which was above 0.98 indicated the
excellent analytical performance of this optimised toxin microarray
assay method. The data show that this microarray assay can detect
the pure toxin at a level of 1 pg g
1
which is 1.4 10
3
fold more
sensitive than developed immunoassay using ELISA. Since LOD
reflects the sensitivity (Wang et al., 2012), the proposed method
could achieve much lower LOD than developed ELISA. Not using the
enzymatic step in microarray format helped to reduce the assay
time from almost 5 h with ELISA to 3 h with toxin microarray. The
limits of detection of AFB
1
in wheat and barley samples were 30 and
90 pg g
1
, respectively. The decreased sensitivity in real samples
indicated that the matrix of food affects the performance of the
toxin microarray assay therefore further research should be done to
address the issue. Nonetheless, it achieves adequate sensitivity
require for applications in cereal samples.
3.4. Recovery in food samples
The recovery results of artificially contaminated wheat flour and
barley samples were measured. Good recoveries (98% ±11%) were
obtained, demonstrating the suitability of the proposed assay for
accurate determination of AFB
1
concentration in wheat and barley
samples. The recovery values were the mean of two extraction
procedure repeated on two different days. Each extraction value
was the average of 16 measurements. The precision was estimated
by calculating the relative standard deviation (% R.S.D) for replicate
measurements (see Table 2).
4. Conclusion
In recent years the antibody-based microarrays have provided a
powerful tool that can be used to generate rapid and detailed
expression profiles of a defined set of analytes in complex samples
and are potentially useful for generating rapid immunological as-
says of food contaminants (Ngundi et al., 2005). There are very few
studies that effectively employ this promising technology for spe-
cific detection of mycotoxins. In a preliminary study conducted by
Lamberti et al., (Lamberti, Tanzarella, Solinas, Padula, &Mosiello,
2009) an antibody-based microarray assay for simultaneous
detection of pure AFB
1
and FB
1
in standard solutions were devel-
oped by using glass slides coated with a copolymer of
Table 1
Limit of Detection measured for AFB1 detection by using toxin microarray assay.
Matrix Standard curve LOD (ng/g) Working range (ng/g)
Buffer y ¼2.1386 þ1.2023/(1þ(x/0.3708^2.1386))1.2023,R2 ¼0.98 0.001 0.015e3.04
Wheat y ¼4.0836 þ0.3898/(1þ(x/1.0089^0.7033))0.3898,R2 ¼1 0.03 0.11e4.15
Barley y ¼3.2549 0.3609/(1þ(x/0.6139^1.1255))þ0.3609,R2 ¼1 0.09 0.18e4.31
A. Beizaei et al. / Food Control 57 (2015) 210e215212
N,Ndimethylacrylamide, N,N-acryloyloxysuccinimide and [3-
(methacryloyl-oxy)-propyl] trimethoxysilyl (DMANAS- MAPS). The
LOD obtained with this method was 3 and 43 ng ml
1
for AFB
1
and
FB
1
, respectively. With the advantage of using fluorescence for
detection, recently an immunochip was developed to detect six
different mycotoxins, namely, AFB
1
, AFM
1
, DON, OTA, T-2, and ZEN
in drinking water with LODs ranging between 10 pg and 16 ng ml
1
(Wang et al., 2012). In all studies analysing mycotoxin levels, the
LOD is an important parameter e.g., the LOD of AFB
1
was
3.00 ng ml
1
using surface plasmon resonance (Daly et al., 2000),
12.5 ng g
1
by ELISA in food stuffs (Saha, Acharya, Roy, &Dhar,
2007)0.16ngml
1
by HPLC (Ghali et al., 2009) and 1.00 ng ml
1
using LC/APPI-MS/MS (Capriotti et al., 2010); 1 ng ml
1
by novel
selective immunochromatographic assa (Zhang, Li, Yang et al.,
2011); 0.03 ng ml
1
by an ultra-sensitive immunochromato-
graphic assay (Zhang, Li, Zhang et al., 2011) and 1 mg kg
1
by lateral
flow immunoassay (Anfossi et al., 2011).
The LOD of our developed method was as low as 1 pg g
1
which is 10 fold more sensitive than the recent report of myco-
toxin assays using immunochip technology (Wang et al., 2012).
The toxin microarray procedure was completed within 3 h indi-
cating rapid detection of this proposed method. The performance
of the microarray assay in commodities was evaluated using
spiked wheat flour and barley (whole grain) samples. The sensi-
tivity of this method was very high detecting between 30 and
90 pg g
1
in wheat and barley samples, respectively. Good re-
coveries (98% ±11%) demonstrate the suitability of the proposed
assay for determination of AFB
1
in contaminated cereal samples.
Although the high cross reactivity with AFB
2
(72%) suggests that
Mab should be reconsider for selective detection of AFB
1
. The
detection of toxins in wheat and barley compares very favourably
with the detection LOD of pure toxin in buffer of 1 pg g
1
,
although more research should be done to eliminate the matrices
effect. Using this method, small quantities of reagents and samples
are required and parallel assays can be performed for multiple
samples. This work was an initial step toward adapting high
throughput screening platform for improved AFB1 detection.
Future work would focus on evaluating different antibodies to
improve the performance and extending the platform for simul-
taneous detection of total aflatoxins. Moreover using certified
cereal samples to fully optimize our detection method and
extending the application in different matrices such as nuts (al-
monds, pistachios, hazelnuts) and animal feeds would be valuable.
In addition, this method could be extended to detect other food-
borne hazards (such, as food borne pathogens, bacterial toxins,
chemicals, antibiotics residues etc.) on a single chip format.
Table 2
Estimation of AFB1 in spiked wheat and barley samples by using toxin microarray assay.
Sample AFB1 spiked (ng/g) AFB1 measured after extraction (ng/g) (Mean ±S.D.) R.S.D.(%) Recovery (%)
First extraction Second extraction
Wheat 2 2.01 1.79 1.9 ±0.15 7.87 95.3
20 16.02 17.99 17.01 ±1.39 8.19 85.06
200 206.28 199.8 203.04 ±4.58 2.25 101.52
Barley 2 2.85 1.8 2.32 ±0.74 32.12 116.45
20 17.75 17.99 17.87 ±0.17 0.98 89.37
200 199.8 199.8 199.8 ±006 0 99.9
Each extraction value is the average of 16 measurements. Each measurement performed on two separate days.
Fig. 1. Standard curves for AFB1 detection generated by competitive ELISA. Effect of decreasing concentrations of Mab on standard curve has been shown. Mab concentrations were
3.3, 1.65, 1.32, 1.1 and 0.94
m
gml
1
, respectively.
A. Beizaei et al. / Food Control 57 (2015) 210e215 213
Acknowledgements
This study was supported by the Ministry of Science, Research
and Technology of Iran; the Research Council of University of
Tehran, Iran; the School of Food Science and Environmental Health,
Dublin Institute of technology, Dublin, Ireland; with the facilities of
Conway Institute of Biomedical and Bimolecular Research, Univer-
sity College Dublin, Dublin, Ireland.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.foodcont.2015.03.039.
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Fig. 2. Detection of AFB1 using toxin microarray (A) Image of generated toxin array. The concentrations of AFB
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m
gml
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