Article

pubs.acs.org/ac

Multiplex Lateral Flow Immunoassay for Mycotoxin Determination

Suquan Song,†,∥ Na Liu,†,∥ Zhiyong Zhao,† Emmanuel Njumbe Ediage,‡ Songling Wu,§ Changpo Sun,§
Sarah De Saeger,‡ and Aibo Wu*,†
†
Institute for Agro-food Standards and Testing Technology, Laboratory of Quality & Safety Risk Assessment for Agro-products
(Shanghai), Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403, China
‡
Laboratory of Food Analysis, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
§
Academy of State Administration of Grain P.R.C, No. 11 Baiwanzhuang Avenue, Xicheng District, Beijing 100037, China
*
S Supporting Information

ABSTRACT: A new lateral flow immunoassay (LFA) is proposed for qualitative and/or
semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxy-
nivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the
mycotoxin specific antibody was class specific and there was no cross reactivity to other
groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6,
and 10 μg/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection
(cLOD) were 0.05, 1, and 3 μg/kg, respectively. Meanwhile the cutoff values were
achieved at 1, 50, and 60 μg/kg for AFB1, ZEA and DON, respectively. Recoveries ranged
from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three
mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated
maize samples resulted in a good agreement between the multiplex LFA and LC−MS/MS
(100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further
explained the general overestimation of LFA compared to chromatographic methods for
quantification of mycotoxins.

M ycotoxins are secondary metabolites produced by many

invading species of filamentous fungi, which contami-
nate various agricultural commodities under favorable temper-
ature and humidity conditions.1 Cereals, such as corn, wheat,
and rice, are examples of plant-derived products especially
susceptible to fungal infestation. About 25% of the world’s food
crops are contaminated by mycotoxins, resulting in an
estimated annual loss of 1 billion metric tons of food products
equivalent to about 5 billion dollars per year.2 Besides,
mycotoxins are carcinogenic, nephrotoxic, hepatotoxic, neuro-
toxic, mutagenic, estrogenic, and immunosuppressive agents.
They may usually pose great threat to human and livestock
when they enter the food chain through contaminated cereals
or feedstuffs.1 Owning to their wide contamination with high
frequency, mycotoxins have become a major concern in global
food safety issues.3 Among the mycotoxins that have been
found in cereals and derived products, aflatoxins (AFs),
zearalenone (ZEA), and related compounds (ZEAs) as well
as deoxynivalenol (DON) and related compounds (DONs)
have been reported as the most frequently occurring toxins.4−7
AFs are commonly produced by Aspergillus molds while ZEAs
and DONs are mainly produced by Fusarium species. ZEA and
its analogues are suspected to be triggering factors for central
precocious puberty observed in adolescent females in the
United States8 and have also been reported to be carcinogenic
with symptoms of reproductive toxicity.9 Ingestion of high dose
of DON is reported to cause acute gastroenteritis with vomiting
effects, while low dose impairs growth as well as an altered
immune function.10 The AFs are of great concern due to their
highest toxicity and are potent cancer-promoting agents,
causing liver cirrhosis or primary liver carcinomas.11 Because
of the serious threat posed by these mycotoxins and to
guarantee food safety, most countries have established
regulations to control mycotoxins contamination in food and
feed. Typically, the European Commission has set the
maximum levels (MLs) for the sum of AFs (AFB1, AFB2,
AFG1, and AFG2) at 4 μg/kg and that for AFB1 at 2 μg/kg in
cereals and products derived from cereals. Likewise, the MLs
for DON and ZEA in unprocessed maize are set at 1750 μg/kg
and 200 μg/kg, respectively.12,13
The increasing awareness about mycotoxins as well as the
intensifying legislative framework worldwide has aroused the
need for fast and efficient analytical methods for monitoring
mycotoxins in food and feed.14,15 Chromatographic-based
methods such as liquid chromatography tandem mass
spectrometry (LC−MS/MS) have been extensively used as a
confirmatory method in recent years.4,16−18 In spite of the
numerous advantages that such techniques offer, they are time-
consuming, expensive, and labor-intensive with extensive
sample preparation. They require skilled technicians for
operation and are unsuitable for field applications.19 Immu-
nochemical techniques such as enzyme-linked immunosorbent
Received: February 8, 2014
Accepted: April 18, 2014
Published: April 18, 2014

© 2014 American Chemical Society 4995 dx.doi.org/10.1021/ac500540z | Anal. Chem. 2014, 86, 4995−5001
Analytical Chemistry
Figure 1. (A) Scheme of the multiplex lateral flow immunoassay (LFA) for multiplex myxotoxins. (B1) Semiquantitative analysis platform for LFA.
(B2) Qualitative analysis platform for LFA. Strips 1 to 9 are the schematic illustrations of detection results. (1) AFs (−), ZEAs (−), DONs (−); (2)
AFs (+), ZEAs (−), DONs (−); (3) AFs (−), ZEAs (+), DONs (−); (4) AFs (−), ZEAs (−), DONs (+); (5) AFs (−), ZEAs (+), DONs (+); (6)
AFs (+), ZEAs (−), DONs (+); (7) AFs (+), ZEAs (+), DONs (−); (8) AFs (+), ZEAs (+), DONs (+); (9) invalid result.
assay (ELISA) have been widely used to screen mycotoxins.20,21
However, conventional ELISA methods require some labo-
ratory operations which are time-consuming. Lateral flow
immunoassay (LFA) has been widely developed for rapid
detection (qualitative) of single or multiple analytes,3,22−24 with
the possibility to inaccurate results due to individual
subjectivity. Recently quantitative immuno-chromatographic
assays have been developed for single analyte detection.24−27
However, considering co-occurrence of mycotoxins in cereal
grains efforts to design simultaneous detection of multi-
mycotoxins were made in a pioneering study.28,29 In spite of
these developments, there are still some theoretical and
technological gaps, unanswered questions, and continuous
demands from end-users which call for more research with the
existing technology especially in the area of multiplex
mycotoxin determination. Plant metabolites can conjugate
with mycotoxins to form the masked mycotoxins, which stay in
the plant vacuoles and cell wall and escape routine monitoring.
Several studies have highlighted their potential threat to human
and animal health as some of these masked forms can undergo
partial or total cleavage reverting to the native compound and
hence are able to exert the same toxic effect as the native
compounds.30,31 Realistically the coexistence of the parent
mycotoxins and their masked forms can always lead to
underestimation of the total mycotoxin content in the sample
and therefore underestimation of the exposure of consumers at
doses exceeding the MLs.32 A possible remedy is to broaden
the current analytical methods for mycotoxins by integrating
conjugates and other mycotoxin metabolites in the routine
monitoring process.
With the availability of three class specific monoclonal
antibodies, a rapid immunoassay platform for simultaneous
multiclass qualification or semiquantification of deoxynivalenol,
zearalenone, and aflatoxins and their main conjugates or
analogues was successfully established. The developed assay
was applied for the determination of mycotoxins in spiked and
naturally contaminated cereal samples. To our knowledge, this
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is the first report on multiclass analysis of mycotoxins and their
analogues by LFA with three monoclonal antibodies.
■
EXPERIMENTAL SECTION
Experimental Section. The mycotoxin standards aflatoxin
B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1),
aflatoxin G2 (AFG2), zearalenone (ZEA), zearalanone
(ZAN), α-zearalenol (α-ZOL), β-zearalanol (β-ZOL), deoxy-
nivalenol (DON), 3-acetyldeoxynivalenol (3-AC-DON), 15-
acetyldeoxynivalenol (15-AC-DON), deoxynivalenol-3-gluco-
side (D3G), T-2, fumonisin B1 (FB1), fumonisin B2 (FB2),
and ochratoxin A (OTA) were provided by Sigma-Aldrich (St.
Louis). The monoclonal antibodies (mAbs) against AFB1,
ZEA, and DON together with the conjugates AFB1-bovine
serum albumin (BSA), ZEA-BSA, as well as DON-BSA were
developed in our lab. Hydrogen tetrachloroaurate (III) hydrate
(HAuCl4·3H2O), sodium citrate (C6H5Na3O7·2H2O), and goat
antimouse immunoglobulin (IgG) were purchased from Sigma-
Aldrich (St. Louis). Sample pad (spun bonded polyester, 6613),
gold conjugate pad (borosilicate glass fiber with PVA binder,
8964), as well as nitrocellulose (NC) membranes (vivid 170)
were purchased from PALL Corporation (NY). Absorbing pad
was purchased from Shanghai Goldbio Tech Co., Ltd.
(Shanghai, China). Water was purified with a Milli-Q system
from Millipore (Bedford, MA). All the organic solvents in the
study were of analytical reagent grade.
Apparatus. HGS510 dispenser and sprayer platform and
HGS201 cutter (Hangzhou Autokun Technology Co., Ltd.,
Hangzhou, China) were used to prepare test strips. The
CHR100 strip reader was purchased from KAIWOOD
Technology Co., Ltd. (Taiwan, China). A Shimadzu LC/MS-
8030 triple quadrupole mass spectrometer (LC/MS/MS)
equipped with an electrospray ionization interface (Shimadzu,
Kyoto, Japan) was used in the study.
Samples. Wheat and maize samples were from local
markets in China, Shanghai province. Because certified blank
samples were not available, samples with undetected levels of
the analytes after LC−MS/MS screening were chosen as
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Analytical Chemistry
“blank” and used in spiking and recovery experiments. For
sample preparation, 5 g of samples was extracted with 25 mL of
methanol/water (70:30, v/v) for 15 min. After centrifugation at
4000g for 10 min, 5 mL of supernatant was dried and the
residue was reconstituted with the same (5 mL) volume of PBS
(0.01 M, pH 7.4).
Preparation of LFA Strip. Colloidal gold (CG) with a
mean diameter of 25 nm and anti-AFB1-CG conjugates were
prepared by the method described by Liu et al.33 Furthermore,
the anti-DON-CG and anti-ZEA-CG conjugates were prepared
with the same procedure. Before preparing the LFA strips,
parameters such as the pH value, the proportion of different
constituents in the blocking buffer, the ratio of CG labeled
mAb, the speed of the dispensing platform, and the volume of
liquid were optimized. The LFA strips consisted of four parts as
follows: sample pad, conjugate pad, NC membrane, and
absorbent pad (Figure 1). The absorbent pads were employed
without any pretreatment. The sample pads and conjugate pads
were first blocked with blocking buffer (0.1 M PBS (pH 7.4),
containing 10% (w/v) sucrose, 1% (w/v) BSA, and 0.5% (w/v)
trehalose) followed by overnight drying at 37 °C. For preparing
the conjugate pads, the three CG labeled mAbs (CG-mAbs)
were mixed at equal ratio and then diluted 10-fold with 0.1 M
PBS. Afterward, the mixture was dispensed onto the glass fiber
at a speed of 2 μL/cm and dried. For preparing the test zones,
the three capture reagents (DON-BSA, ZEA-BSA, and AFB1-
BSA) and goat antimouse immunoglobulin (0.25 mg/mL) were
separately spotted onto the NC membrane in turn at a jetting
rate of 0.7 μL/cm to generate three test lines and one control
line. The four lines were positioned at a 3.8 mm interval. Finally
the sample pad, conjugated pad, NC membrane, and absorbent
pad were laminated onto a plastic backing and divided into
strips, which were installed in the shell and stored with
desiccators at room temperature until use.
Qualitative or Semiquantitative Immunoassay Proce-
dure. For qualitative assay, 60 μL of mycotoxin standard or
extracted sample solution were loaded onto the sample pad of
the LFA strip. Driven by capillary forces, the liquid migrated to
the absorbent pad. The CG-mAbs, immobilized on the
conjugate pad, were then redissolved in the solution and
reacted with the mycotoxins (if present) while the whole
complex migrated along the membrane. Upon reaching the test
line, CG-mAbs were captured by the corresponding capture
reagents (mycotoxin-BSA) resulting in the appearance of a pink
line. The color intensity (CI) of the test line was inversely
correlated with the mycotoxin concentration in the sample. In
the absence of target mycotoxin, the largest amounts of CG-
mAbs were trapped by the competitive antigen and the most
intensive red color band developed on the corresponding test
line. However, if there were enough target analytes in the
sample, all the CG-mAbs were occupied. Therefore, no mAbs
could react with the mycotoxin-BSA reagents immobilized on
the NC membrane and no visible band appeared in the test
line. The control line should always be visible because of the
reaction between CG-mAbs and goat antimouse IgG, which
was considered to be an indicator of the good functionality of
the test.
For semiquantitative analysis, the intensity of the test lines
was determined after 15 min using the strip reader and the data
were expressed as relative optical density (ROD). ROD is the
ratio of the optical densities of the positive (B) to the negative
(B0) sample. The concentration of the three analytes in the
samples were quantified from a calibration curve (B/B0 × 100%
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versus the concentration of each analyte), which was run
simultaneously.
Calibration, Sensitivity, and Specificity of the LFA. The
standard curve for each analyte was constructed in matrix by
spiking at different concentrations for the different analytes.
The concentration range was 0.1 to 100 μg/kg for DON and
ZEA and 0.025 to 10 μg/kg for AFB1 (Figure 2). The standard
curve was fitted using the four parameter logistic equation by
SigmaPlot (version 12.0)
Figure 2. Calibration curves for multiplex mycotoxins (AFB1, ZEA,
and DON) detection with the developed LFA. B/B0 is the ratio of the
optical densities of the positive sample to the negative sample. The
inset shows the visual detection limits of three mycotoxin standards.
AFB1/ZEA/DON concentrations were as follows (from left to right):
0/0/0, 0.01/0.4/2.5, 0.02/0.8/5, 0.03/1.6/10, 0.06/3/20, 0.12/6/30,
0.3/12/40, 0.5/25/50, 1/50/60 μg/kg. Error bars represent standard
error with n = 6.
The assay sensitivity was evaluated by analyzing a series of
concentrations of the mycotoxin mixture. The visual limit of
detection (vLOD) for qualitative evaluation was defined as the
minimum concentration that gave very weak color intensity in
the test line visibly different from that of the negative control
line (very intense coloration). The cutoff value was the
concentration that gave a complete disappearance of the visible
band. For semiquantitative evaluation, the calculated limit of
detection (cLOD) was defined as the concentration at which
B/B0 equals to 80% (thus 20% inhibition of the signal, IC20),
and the IC80 was used to evaluate the maximum detection
ability of the LFA in this study.
The specificity, expressed as cross reactivity (CR), was
evaluated by assessing the recognition of the specific analyte-
mAb toward other mycotoxins or analogues. The CR was
expressed as the percentage of the concentration at 50%
inhibition (IC50) of the analogues to the corresponding target
analyte.
LC−MS/MS Analysis. The samples were first extracted with
25 mL of acetonitrile/water (84:16, v/v) for 60 min. Before
analysis, 1 mL of supernatant was diluted by an equal volume of
a methanol/water mixture (20:80, v/v). The analytical column
was an Agilent poroshell 120 EC-C18 (100 mm × 3 mm, 2.7
μm). A mobile phase consisting of ammonium acetate (5 mM)
was used at a flow rate of 0.3 mL/min. The gradient elution
program applied was as follows: 0−1 min, 20% methanol; 1−2
min, 20−90% methanol; 2−6 min, 90% methanol; 6−6.2 min,
dx.doi.org/10.1021/ac500540z | Anal. Chem. 2014, 86, 4995−5001
Analytical Chemistry Article

90−20% methanol; 6.2−8 min, 20% methanol. The injection Table 1. Cross Reactivity (CR) of Analytes with Monoclonal
volume was 5.0 μL. The MS conditions were thoroughly Antibody Detected by LFAa
optimized for each mycotoxin. The optimal parameters were
class analytes antibody IC50 (μg/kg) CR (%)
listed in Table S-1 in the Supporting Information. Quantifica-
tion of mycotoxins was performed by measuring peak areas in AFs AFB1 Anti-AFB1 mAb 0.2 100
the MRM mode. AFB2 0.2 124
Safety Precautions. AFB1 is a known liver carcinogen, AFG1 0.3 66
therefore direct exposure and laboratory contamination should AFG2 0.2 96
be avoided. All experiments were carried out in the fume hood, ZEAs ZEA Anti-ZEA mAb 4 100
and researchers should wear laboratory coat, safety glasses, ZAN 1 251
gloves, mouth-muffle, and face mask. α-ZOL 2 193

■
RESULTS AND DISCUSSION
Optimization of the LFA Strips. A variety of labels have
been used for signal generation in immunoassays, such as
enzyme, quantum dots, dye-loaded liposomes, carbon nano-
particles, or magnetic beads.34 Because of its ease of
conjugation with antibodies, CG has been widely used in
LFA. A 25 nm CG was chosen since it has been reported35,36 to
show good stability and sensitivity compared to other
commercially available sizes. Hence, a 25 nm CG labeled
antibody was adopted as a signal molecule.
For convenience, three different conjugates of CG-mAbs
were mixed together and dispensed onto a single conjugate pad
(Figure 1) instead of coating them on three overlapping
conjugate pads. With the use of the single conjugate pad, the
pink colored lines developed within 5 min. However, 15 min
was recommended for the development of a more stable
intensity with the reason that, after this time point, the B/B0
ratio of the samples exhibited the lowest value which implied
good assay sensitivity. Furthermore, 60 μL of sample extract
was determined sufficient to dissolve the CG-mAbs and
develop a satisfactory consequence in the end-results. The
performance of different types of NC membrane (PALL vivid,
Whatman-AE99 and Sartorius CN 140) were also evaluated.
PALL vivid 170 was chosen for its good functionality when
compared with the other NC membranes (Whatman-AE99 and
Sartorius CN140). Other parameters also optimized included
the concentration of the coated antigens (0.5 mg/mL) and goat
antimouse IgG (0.25 mg/mL), while the antibody concen-
tration was 10 μg/mL for the CG conjugate of anti-DON, anti-
ZEA, and anti-AFB1.
Determination of Limits of Detection and Inhibition
Concentrations for the Different Test Lines. The
inhibition curves obtained with the strip reader for the different
analytes are shown in Figure 2. The calculated LOD (cLOD)
for AFB1, ZEA, and DON were 0.05, 1, and 3 μg/kg. The visual
cutoff was 1 μg/kg, 50 μg/kg, and 60 μg/kg for AFB1, ZEA,
and DON, which were far below the MLs established in the
EU.12,13 The IC50 values were calculated to be 0.2 μg/kg, 4 μg/
kg, and 10 μg/kg for AFB1, ZEA, and DON, respectively
(Figure 2 and Table 1). The vLOD of the LFA for qualitative
analysis was determined with different mycotoxin concen-
trations spiked in a blank sample. As shown in the inset of
Figure 2, the intensity of the test lines decreased with increasing
mycotoxin concentration. At the following concentrations, 0.03
μg/kg for AFB1, 1.6 μg/kg for ZEA, and 10 μg/kg for DON,
the intensity of the test line showed an obvious difference from
the control line. Hence these concentrations were set as the
vLODs. By performing background subtraction and normal-
ization, the CI was within uniform standard, which significantly
improved the detection capability and greatly reduced the
uncertainty of measurements near the cutoff value.
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β-ZOL 2 153
DONs DON Anti-DON mAb 10 100
3-AC-DON 8 127
15-AC-DON 200 5
D3G 20 49
a
The analysis was performed in standard solution (n = 4).
Cross Reactivity. For the simultaneous detection of three
mycotoxins by the LFA, the corresponding capture antigens
(AFB1-BSA, DON-BSA, or ZEA-BSA) were immobilized at
different sites on the strip constituting different test zones. It
was shown that, when a single CG-mAb specific (anti-AFB1
mAb or anti-ZEA mAb or anti-DON mAb) was loaded directly
onto the NC membrane, only the corresponding test line
appeared (AFB1-BSA/ZEA-BSA/DON-BSA) with no pink line
for the other mycotoxins. This implies each of the three
antibodies were each specific to the corresponding mycotox-
in(s) and no cross reactivity with any other of the two
mycotoxin test zones.
The cross reactivity toward other mycotoxins was also
evaluated. The results are illustrated in Table 1. Most of the
antibodies exhibited high cross reactivity to the class specific
analogues. For ZEA and its analogues, high CRs were observed
with ZAN (251%), α-ZOL (193%), and β-ZOL (153%). For
DON and its analogues, high CRs were also observed with 3-
AC-DON (127%) and D3G (49%), with the exception of 15-
AC-DON (5%). For the AFs, high CRs for AFB2 (124%),
AFG1 (66%), and AFG2 (96%) were observed. For the
mycotoxins not belonging to the same class, the cross reactivity
was below 0.1% with IC50 higher than 1000 μg/kg. Hence the
antibodies used in the development of the LFA were class
specific. With the LFA developed in this work, the masked
mycotoxins could also be determined to some extent (D3G).
Hence this multicomponent LFA could also be used for
multimycotoxin determination.
Optimization of the Sample Preparation and Loading
Step. Two approaches were evaluated. In the first approach,
the extract was diluted 3.5 times in PBS after extraction and
then loaded onto the LFA. While in the second approach, the
sample extract was preconcentrated (through evaporation) and
reconstituted in injection solvent. Results are shown in Table S-
2 in the Supporting Information. The IC80 and IC50 for DON
and AFB1, respectively, were far higher than their respective
ML of 1750 μg/kg and 9 μg/kg. Faced with these problems, we
opted for a second approach where the sample extract was first
evaporated to dryness and then equal volumes of PBS were
used to reconstitute the sample before loading onto the strip.
With the second approach, the sensitivity of the LFA toward
the three mycotoxins improved significantly. Hence the IC50 for
all these analytes were far below the MLs.
Validation with Cereal Samples. The LFA was validated
for maize and wheat samples. The parameters were obtained by
dx.doi.org/10.1021/ac500540z | Anal. Chem. 2014, 86, 4995−5001
Analytical Chemistry Article

Table 2. Recoveries (R) of AFB1, ZEA, and DON in Spiked Maize and Wheat (n = 6)
LFA LC−MS/MS
sample analytes spiked concentration (μg/kg) mean ± SD (μg/kg) R (%) RSD (%) mean ± SD (μg/kg) R (%) RSD (%)
AFB1 0.5 0.5 ± 0.1 106 17 0.6 ± 0.1 128 19
1 1 ± 0.2 98 19 0.9 ± 0.1 86 17
2 2 ± 0.2 87 13 1.7 ± 0.1 83 4
ZEA 10 10 ± 0.5 96 5 8.0 ± 1.6 80 21
maize 25 22 ± 1 88 7 25.6 ± 3.4 102 13
50 44 ± 3 87 7 59.1 ± 2.0 118 3
DON 40 32 ± 6 80 19 30.0 ± 4.7 75 16
80 66 ± 8 82 12 70.2 ± 6.5 88 9
150 169 ± 19 112 11 107.5 ± 2.7 72 3
AFB1 0.5 0.6 ± 0.1 122 5 0.4 ± 0.1 70 7
1 1 ± 0.2 95 18 0.9 ± 0.2 93 6
2 2 ± 0.1 85 8 1.9 ± 0.2 97 10
ZEA 10 9 ± 1.3 92 13 8.6 ± 1.5 86 18
wheat 25 30 ± 5 120 20 26.6 ± 1.0 106 4
50 52 ± 9 103 17 57.3 ± 2.9 115 5
DON 40 35 ± 2 88 7 32.6 ± 2.5 82 8
80 70 ± 5 88 7 84.7 ± 10.9 106 13
150 129 ± 9 86 7 116.9 ± 9.9 78 8

Table 3. Comparison of the Analysis Results for AFs, ZEAs, and DONs in Naturally Contaminated Samples by the Developed
LFA and LC−MS/MS (n = 4)a
LFA LC−MS/MS
maize sample AFB1 (μg/kg) ZEA (ZAN, α-ZOL, β-ZOL)b (μg/kg) DON (3-Ac-DON, D3G)c (μg/kg) AFB1 (μg/kg) ZEA (μg/kg) DON (μg/kg)
1 n/d 113 (45, 59, 74) P n/d 81.2 1730.5
2 n/d P P n/d 103.8 1267.9
3 n/d n/d 104 (82, 215) n/d 6.7 83.8
4 n/d 29 (11, 15, 19) 69 (54, 141) n/d 17.6 255.5
5 n/d 116 (46, 60, 75) P n/d 82.3 645.5
6 n/d 61 (25, 32, 40) P n/d 49.8 1256.7
7 n/d 16 (7, 8, 11) 169 (132, 347) n/d 18.5 151.7
8 n/d P P n/d 447.7 6149.0
9 n/d P P n/d 381.4 4607.7
10 n/d P P n/d 149.7 960.5
11 n/d P P n/d 999.3 9821.7
12 n/d 128 (51, 66, 84) P n/d 85.5 1829.0
13 n/d 32 (13, 16, 21) P n/d 20.0 574.0
14 n/d P 237 (186, 487) n/d 276.4 260.2
15 n/d n/d 66 (52,135) n/d 7.2 113.9
16 n/d 20 (8, 10, 13) 206 (162, 425) n/d 11.6 258.2
17 n/d 66 (26, 34, 43) 403 (317, 830) n/d 46.3 520.9
18 n/d n/d 193 (152, 397) n/d 8.6 221.2
19 n/d n/d 119 (93, 244) n/d 7.0 131.2
20 n/d 25 (10, 13, 16) 358 (281, 736) n/d 17.5 391.5
21 n/d n/d 124 (97, 255) n/d 7.5 177.7
a
n/d = not detected. P = above cutoff. bThe calculated concentration of each mycotoxin compound according to the concentration of ZEA. cThe
calculated concentration of each mycotoxin compound according to the concentration of DON.

spiking the samples at six concentration levels and quantified by
use of matrix-matched calibration curves. The results are shown
in Table S-3 in the Supporting Information. The coefficients of
determination (R2) for the different analytes were higher than
0.97, which indicated good linearity of the analytical ranges.
The parameters of cLOD, IC50, cutoff value, vLOD, and
working range of the qualitative and semiquantitative LFA are
also listed in Table S-3 in the Supporting Information. Both the
vLOD and cLOD for three mycotoxins in the two cereal
matrixes were lower than the MLs; therefore, the method could
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be applied for simultaneous quantification of mycotoxins in real
samples.
To evaluate the recoveries of the sample preparation, spiked
blank maize and wheat were investigated. The parameters were
obtained by spiking the cereal samples at three concentration
levels. The spiked samples were also analyzed in-parallel by
LC−MS/MS. The results (Table 2) demonstrated good
agreement between the measured values and the fortified
concentration in both LFA and LC−MS/MS, with recoveries
ranging from 80% to 122% for LFA and from 70% to 128% for
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Analytical Chemistry
LC−MS/MS. Moreover, acceptable RSDs ranging from 5% to
20% were also achieved with LFA.
Results of Naturally Contaminated Samples. Analysis
of 21 naturally contaminated maize samples was performed
using the developed LFA followed by LC−MS/MS for
confirmation (Table 3). With the LC−MS/MS analysis, all
21 samples were found positive with DON and ZEA and no
AFB1. Of the 21 samples, 4 were contaminated above the MLs
of 1750 μg/kg for DON and 200 μg/kg for ZEA. Using the
LFA, all 21 samples were contaminated with DONs (100%
agreement) whereas only 17/21 (81%) samples were found
contaminated with ZEAs. As listed in Table S-3 in the
Supporting Information, the cLOD of the LFA for ZEA was 9
μg/kg, which was higher than the concentrations confirmed by
LC−MS/MS in the selected five samples.
For concentrations above the cutoff of the LFA, a value “P”
(positive) was marked, which meant the concentration was
above the calculated range of the LFA. However, for samples 2
and 10, the concentration of ZEA confirmed by LC−MS/MS
was 104 μg/kg and 150 μg/kg, which was within the detection
range of the LFA (9 μg/kg to 186 μg/kg for ZEA), but the data
was still marked as “P”. Such overestimation of ZEA could be
the result of an actual contamination (cocontamination) of
“masked-ZEAs” in the sample. Also for sample 13, an
overestimation for DON was detected. This result explains
the general overestimation of LFA compared to chromato-
graphic methods for quantification of mycotoxins.37,38
■
CONCLUSION
In the present study, a multiplex LFA platform was constructed
and validated which could simultaneously qualify or semi-
quantify AFs, ZEAs, and DONs within a total time of 15 min.
The technical platform offers multiple advantages for simplicity,
rapidity, sensitivity, cost-effectiveness, and time-efficiency. The
validation results of the method in the spiked samples showed
that the developed approach was reliable and could be
employed for multiplex mycotoxin detection in cereals. Because
the monoclonal antibodies adopted were class specific, the LFA
strip could simultaneously detect three groups of mycotoxins in
a single assay. In real sample analysis, the vLOD and cLOD for
three mycotoxins were lower than the EU maximum levels. The
proposed method was successfully applied to naturally
contaminated maize samples. The results were in good
agreement with those obtained using confirmatory LC−MS/
MS. The overestimation of some mycotoxins was related to the
recognition capability of the class specific antibodies utilized in
the LFA.
■
ASSOCIATED CONTENT
*
S Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-21-37196975. Fax: +86-21-62203612. E-mail:
wuaibo@saas.sh.cn.
Author Contributions
∥ C.; Giraudi, G. Food Addit. Contam., Part A: Chem. Anal. Control Expo.
S.S. and N.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
5000
■
Article
ACKNOWLEDGMENTS
The authors thank the funds from the National Basic Research
Program of China (Grant 2013CB127801), Pujiang Plan of
Shanghai (Grant No. 13PJ1407200), Shanghai Municipal
Commission for Science and Technology (Grant
13231202800), and the Chinese-Belgian Joint Project of
BELSPO, MOST, China (Grant S2012GR0 016) and Belgium
(Grant BL/02/C58).
■
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