Multiplex Lateral Flow Immunoassay for Mycotoxin DeterminationSuquan 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/orsemiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxy-nivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of themycotoxin specific antibody was class specific and there was no cross reactivity to othergroups 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 wereachieved at 1, 50, and 60 μg/kg for AFB1, ZEA and DON, respectively. Recoveries rangedfrom 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the threemycotoxins were lower than the EU maximum levels. Analysis of naturally contaminatedmaize 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 furtherexplained the general overestimation of LFA compared to chromatographic methods forquantification of mycotoxins.

Mycotoxins are secondary metabolites produced by manyinvading 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 especiallysusceptible to fungal infestation. About 25% of the world’s foodcrops are contaminated by mycotoxins, resulting in anestimated annual loss of 1 billion metric tons of food productsequivalent 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 livestockwhen they enter the food chain through contaminated cerealsor feedstuffs.1 Owning to their wide contamination with highfrequency, mycotoxins have become a major concern in globalfood safety issues.3 Among the mycotoxins that have beenfound in cereals and derived products, aflatoxins (AFs),zearalenone (ZEA), and related compounds (ZEAs) as wellas 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 ZEAsand DONs are mainly produced by Fusarium species. ZEA andits analogues are suspected to be triggering factors for centralprecocious puberty observed in adolescent females in theUnited States8 and have also been reported to be carcinogenicwith symptoms of reproductive toxicity.9 Ingestion of high doseof DON is reported to cause acute gastroenteritis with vomitingeffects, while low dose impairs growth as well as an altered

immune function.10 The AFs are of great concern due to theirhighest toxicity and are potent cancer-promoting agents,causing liver cirrhosis or primary liver carcinomas.11 Becauseof the serious threat posed by these mycotoxins and toguarantee food safety, most countries have establishedregulations to control mycotoxins contamination in food andfeed. Typically, the European Commission has set themaximum levels (MLs) for the sum of AFs (AFB1, AFB2,AFG1, and AFG2) at 4 μg/kg and that for AFB1 at 2 μg/kg incereals and products derived from cereals. Likewise, the MLsfor DON and ZEA in unprocessed maize are set at 1750 μg/kgand 200 μg/kg, respectively.12,13

The increasing awareness about mycotoxins as well as theintensifying legislative framework worldwide has aroused theneed for fast and efficient analytical methods for monitoringmycotoxins in food and feed.14,15 Chromatographic-basedmethods such as liquid chromatography tandem massspectrometry (LC−MS/MS) have been extensively used as aconfirmatory method in recent years.4,16−18 In spite of thenumerous advantages that such techniques offer, they are time-consuming, expensive, and labor-intensive with extensivesample preparation. They require skilled technicians foroperation and are unsuitable for field applications.19 Immu-nochemical techniques such as enzyme-linked immunosorbent

Received: February 8, 2014Accepted: April 18, 2014Published: April 18, 2014

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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 flowimmunoassay (LFA) has been widely developed for rapiddetection (qualitative) of single or multiple analytes,3,22−24 withthe possibility to inaccurate results due to individualsubjectivity. Recently quantitative immuno-chromatographicassays have been developed for single analyte detection.24−27

However, considering co-occurrence of mycotoxins in cerealgrains efforts to design simultaneous detection of multi-mycotoxins were made in a pioneering study.28,29 In spite ofthese developments, there are still some theoretical andtechnological gaps, unanswered questions, and continuousdemands from end-users which call for more research with theexisting technology especially in the area of multiplexmycotoxin determination. Plant metabolites can conjugatewith mycotoxins to form the masked mycotoxins, which stay inthe plant vacuoles and cell wall and escape routine monitoring.Several studies have highlighted their potential threat to humanand animal health as some of these masked forms can undergopartial or total cleavage reverting to the native compound andhence are able to exert the same toxic effect as the nativecompounds.30,31 Realistically the coexistence of the parentmycotoxins and their masked forms can always lead tounderestimation of the total mycotoxin content in the sampleand therefore underestimation of the exposure of consumers atdoses exceeding the MLs.32 A possible remedy is to broadenthe current analytical methods for mycotoxins by integratingconjugates and other mycotoxin metabolites in the routinemonitoring process.With the availability of three class specific monoclonal

antibodies, a rapid immunoassay platform for simultaneousmulticlass qualification or semiquantification of deoxynivalenol,zearalenone, and aflatoxins and their main conjugates oranalogues was successfully established. The developed assaywas applied for the determination of mycotoxins in spiked andnaturally contaminated cereal samples. To our knowledge, this

is the first report on multiclass analysis of mycotoxins and theiranalogues by LFA with three monoclonal antibodies.

■ EXPERIMENTAL SECTION

Experimental Section. The mycotoxin standards aflatoxinB1 (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-bovineserum albumin (BSA), ZEA-BSA, as well as DON-BSA weredeveloped in our lab. Hydrogen tetrachloroaurate (III) hydrate(HAuCl4·3H2O), sodium citrate (C6H5Na3O7·2H2O), and goatantimouse 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 padwas purchased from Shanghai Goldbio Tech Co., Ltd.(Shanghai, China). Water was purified with a Milli-Q systemfrom Millipore (Bedford, MA). All the organic solvents in thestudy were of analytical reagent grade.

Apparatus. HGS510 dispenser and sprayer platform andHGS201 cutter (Hangzhou Autokun Technology Co., Ltd.,Hangzhou, China) were used to prepare test strips. TheCHR100 strip reader was purchased from KAIWOODTechnology 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 localmarkets in China, Shanghai province. Because certified blanksamples were not available, samples with undetected levels ofthe analytes after LC−MS/MS screening were chosen as

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.

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“blank” and used in spiking and recovery experiments. Forsample preparation, 5 g of samples was extracted with 25 mL ofmethanol/water (70:30, v/v) for 15 min. After centrifugation at4000g for 10 min, 5 mL of supernatant was dried and theresidue 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 wereprepared by the method described by Liu et al.33 Furthermore,the anti-DON-CG and anti-ZEA-CG conjugates were preparedwith the same procedure. Before preparing the LFA strips,parameters such as the pH value, the proportion of differentconstituents in the blocking buffer, the ratio of CG labeledmAb, the speed of the dispensing platform, and the volume ofliquid were optimized. The LFA strips consisted of four parts asfollows: sample pad, conjugate pad, NC membrane, andabsorbent pad (Figure 1). The absorbent pads were employedwithout any pretreatment. The sample pads and conjugate padswere 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 preparingthe conjugate pads, the three CG labeled mAbs (CG-mAbs)were mixed at equal ratio and then diluted 10-fold with 0.1 MPBS. Afterward, the mixture was dispensed onto the glass fiberat 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) wereseparately spotted onto the NC membrane in turn at a jettingrate of 0.7 μL/cm to generate three test lines and one controlline. The four lines were positioned at a 3.8 mm interval. Finallythe sample pad, conjugated pad, NC membrane, and absorbentpad were laminated onto a plastic backing and divided intostrips, which were installed in the shell and stored withdesiccators at room temperature until use.Qualitative or Semiquantitative Immunoassay Proce-

dure. For qualitative assay, 60 μL of mycotoxin standard orextracted sample solution were loaded onto the sample pad ofthe LFA strip. Driven by capillary forces, the liquid migrated tothe absorbent pad. The CG-mAbs, immobilized on theconjugate pad, were then redissolved in the solution andreacted with the mycotoxins (if present) while the wholecomplex migrated along the membrane. Upon reaching the testline, CG-mAbs were captured by the corresponding capturereagents (mycotoxin-BSA) resulting in the appearance of a pinkline. The color intensity (CI) of the test line was inverselycorrelated with the mycotoxin concentration in the sample. Inthe absence of target mycotoxin, the largest amounts of CG-mAbs were trapped by the competitive antigen and the mostintensive red color band developed on the corresponding testline. However, if there were enough target analytes in thesample, all the CG-mAbs were occupied. Therefore, no mAbscould react with the mycotoxin-BSA reagents immobilized onthe NC membrane and no visible band appeared in the testline. The control line should always be visible because of thereaction between CG-mAbs and goat antimouse IgG, whichwas considered to be an indicator of the good functionality ofthe test.For semiquantitative analysis, the intensity of the test lines

was determined after 15 min using the strip reader and the datawere expressed as relative optical density (ROD). ROD is theratio of the optical densities of the positive (B) to the negative(B0) sample. The concentration of the three analytes in thesamples were quantified from a calibration curve (B/B0 × 100%

versus the concentration of each analyte), which was runsimultaneously.

Calibration, Sensitivity, and Specificity of the LFA. Thestandard curve for each analyte was constructed in matrix byspiking at different concentrations for the different analytes.The concentration range was 0.1 to 100 μg/kg for DON andZEA and 0.025 to 10 μg/kg for AFB1 (Figure 2). The standardcurve was fitted using the four parameter logistic equation bySigmaPlot (version 12.0)

The assay sensitivity was evaluated by analyzing a series ofconcentrations of the mycotoxin mixture. The visual limit ofdetection (vLOD) for qualitative evaluation was defined as theminimum concentration that gave very weak color intensity inthe test line visibly different from that of the negative controlline (very intense coloration). The cutoff value was theconcentration that gave a complete disappearance of the visibleband. For semiquantitative evaluation, the calculated limit ofdetection (cLOD) was defined as the concentration at whichB/B0 equals to 80% (thus 20% inhibition of the signal, IC20),and the IC80 was used to evaluate the maximum detectionability 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 wasexpressed as the percentage of the concentration at 50%inhibition (IC50) of the analogues to the corresponding targetanalyte.

LC−MS/MS Analysis. The samples were first extracted with25 mL of acetonitrile/water (84:16, v/v) for 60 min. Beforeanalysis, 1 mL of supernatant was diluted by an equal volume ofa methanol/water mixture (20:80, v/v). The analytical columnwas an Agilent poroshell 120 EC-C18 (100 mm × 3 mm, 2.7μm). A mobile phase consisting of ammonium acetate (5 mM)was used at a flow rate of 0.3 mL/min. The gradient elutionprogram applied was as follows: 0−1 min, 20% methanol; 1−2min, 20−90% methanol; 2−6 min, 90% methanol; 6−6.2 min,

Figure 2. Calibration curves for multiplex mycotoxins (AFB1, ZEA,and DON) detection with the developed LFA. B/B0 is the ratio of theoptical densities of the positive sample to the negative sample. Theinset 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 standarderror with n = 6.

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90−20% methanol; 6.2−8 min, 20% methanol. The injectionvolume was 5.0 μL. The MS conditions were thoroughlyoptimized for each mycotoxin. The optimal parameters werelisted in Table S-1 in the Supporting Information. Quantifica-tion of mycotoxins was performed by measuring peak areas inthe MRM mode.Safety Precautions. AFB1 is a known liver carcinogen,

therefore direct exposure and laboratory contamination shouldbe avoided. All experiments were carried out in the fume hood,and researchers should wear laboratory coat, safety glasses,gloves, mouth-muffle, and face mask.

■ RESULTS AND DISCUSSIONOptimization of the LFA Strips. A variety of labels have

been used for signal generation in immunoassays, such asenzyme, quantum dots, dye-loaded liposomes, carbon nano-particles, or magnetic beads.34 Because of its ease ofconjugation with antibodies, CG has been widely used inLFA. A 25 nm CG was chosen since it has been reported35,36 toshow good stability and sensitivity compared to othercommercially available sizes. Hence, a 25 nm CG labeledantibody 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 overlappingconjugate pads. With the use of the single conjugate pad, thepink colored lines developed within 5 min. However, 15 minwas recommended for the development of a more stableintensity with the reason that, after this time point, the B/B0ratio of the samples exhibited the lowest value which impliedgood assay sensitivity. Furthermore, 60 μL of sample extractwas determined sufficient to dissolve the CG-mAbs anddevelop a satisfactory consequence in the end-results. Theperformance 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 whencompared with the other NC membranes (Whatman-AE99 andSartorius CN140). Other parameters also optimized includedthe concentration of the coated antigens (0.5 mg/mL) and goatantimouse 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. Theinhibition curves obtained with the strip reader for the differentanalytes are shown in Figure 2. The calculated LOD (cLOD)for AFB1, ZEA, and DON were 0.05, 1, and 3 μg/kg. The visualcutoff was 1 μg/kg, 50 μg/kg, and 60 μg/kg for AFB1, ZEA,and DON, which were far below the MLs established in theEU.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 qualitativeanalysis was determined with different mycotoxin concen-trations spiked in a blank sample. As shown in the inset ofFigure 2, the intensity of the test lines decreased with increasingmycotoxin concentration. At the following concentrations, 0.03μg/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 fromthe control line. Hence these concentrations were set as thevLODs. By performing background subtraction and normal-ization, the CI was within uniform standard, which significantlyimproved the detection capability and greatly reduced theuncertainty of measurements near the cutoff value.

Cross Reactivity. For the simultaneous detection of threemycotoxins by the LFA, the corresponding capture antigens(AFB1-BSA, DON-BSA, or ZEA-BSA) were immobilized atdifferent sites on the strip constituting different test zones. Itwas shown that, when a single CG-mAb specific (anti-AFB1mAb or anti-ZEA mAb or anti-DON mAb) was loaded directlyonto the NC membrane, only the corresponding test lineappeared (AFB1-BSA/ZEA-BSA/DON-BSA) with no pink linefor the other mycotoxins. This implies each of the threeantibodies were each specific to the corresponding mycotox-in(s) and no cross reactivity with any other of the twomycotoxin test zones.The cross reactivity toward other mycotoxins was also

evaluated. The results are illustrated in Table 1. Most of theantibodies exhibited high cross reactivity to the class specificanalogues. For ZEA and its analogues, high CRs were observedwith ZAN (251%), α-ZOL (193%), and β-ZOL (153%). ForDON 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 themycotoxins not belonging to the same class, the cross reactivitywas below 0.1% with IC50 higher than 1000 μg/kg. Hence theantibodies used in the development of the LFA were classspecific. With the LFA developed in this work, the maskedmycotoxins could also be determined to some extent (D3G).Hence this multicomponent LFA could also be used formultimycotoxin determination.

Optimization of the Sample Preparation and LoadingStep. Two approaches were evaluated. In the first approach,the extract was diluted 3.5 times in PBS after extraction andthen loaded onto the LFA. While in the second approach, thesample extract was preconcentrated (through evaporation) andreconstituted in injection solvent. Results are shown in Table S-2 in the Supporting Information. The IC80 and IC50 for DONand AFB1, respectively, were far higher than their respectiveML of 1750 μg/kg and 9 μg/kg. Faced with these problems, weopted for a second approach where the sample extract was firstevaporated to dryness and then equal volumes of PBS wereused to reconstitute the sample before loading onto the strip.With the second approach, the sensitivity of the LFA towardthe three mycotoxins improved significantly. Hence the IC50 forall these analytes were far below the MLs.

Validation with Cereal Samples. The LFA was validatedfor maize and wheat samples. The parameters were obtained by

Table 1. Cross Reactivity (CR) of Analytes with MonoclonalAntibody Detected by LFAa

class analytes antibody IC50 (μg/kg) CR (%)

AFs AFB1 Anti-AFB1 mAb 0.2 100AFB2 0.2 124AFG1 0.3 66AFG2 0.2 96

ZEAs ZEA Anti-ZEA mAb 4 100ZAN 1 251α-ZOL 2 193β-ZOL 2 153

DONs DON Anti-DON mAb 10 1003-AC-DON 8 12715-AC-DON 200 5D3G 20 49

aThe analysis was performed in standard solution (n = 4).

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spiking the samples at six concentration levels and quantified byuse of matrix-matched calibration curves. The results are shownin Table S-3 in the Supporting Information. The coefficients ofdetermination (R2) for the different analytes were higher than0.97, which indicated good linearity of the analytical ranges.The parameters of cLOD, IC50, cutoff value, vLOD, andworking range of the qualitative and semiquantitative LFA arealso listed in Table S-3 in the Supporting Information. Both thevLOD and cLOD for three mycotoxins in the two cerealmatrixes were lower than the MLs; therefore, the method could

be applied for simultaneous quantification of mycotoxins in realsamples.To evaluate the recoveries of the sample preparation, spiked

blank maize and wheat were investigated. The parameters wereobtained by spiking the cereal samples at three concentrationlevels. The spiked samples were also analyzed in-parallel byLC−MS/MS. The results (Table 2) demonstrated goodagreement between the measured values and the fortifiedconcentration in both LFA and LC−MS/MS, with recoveriesranging from 80% to 122% for LFA and from 70% to 128% for

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 (%)

maize

AFB1 0.5 0.5 ± 0.1 106 17 0.6 ± 0.1 128 191 1 ± 0.2 98 19 0.9 ± 0.1 86 172 2 ± 0.2 87 13 1.7 ± 0.1 83 4

ZEA 10 10 ± 0.5 96 5 8.0 ± 1.6 80 2125 22 ± 1 88 7 25.6 ± 3.4 102 1350 44 ± 3 87 7 59.1 ± 2.0 118 3

DON 40 32 ± 6 80 19 30.0 ± 4.7 75 1680 66 ± 8 82 12 70.2 ± 6.5 88 9150 169 ± 19 112 11 107.5 ± 2.7 72 3

wheat

AFB1 0.5 0.6 ± 0.1 122 5 0.4 ± 0.1 70 71 1 ± 0.2 95 18 0.9 ± 0.2 93 62 2 ± 0.1 85 8 1.9 ± 0.2 97 10

ZEA 10 9 ± 1.3 92 13 8.6 ± 1.5 86 1825 30 ± 5 120 20 26.6 ± 1.0 106 450 52 ± 9 103 17 57.3 ± 2.9 115 5

DON 40 35 ± 2 88 7 32.6 ± 2.5 82 880 70 ± 5 88 7 84.7 ± 10.9 106 13150 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 DevelopedLFA 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 1730.52 n/d P P n/d 103.8 1267.93 n/d n/d 104 (82, 215) n/d 6.7 83.84 n/d 29 (11, 15, 19) 69 (54, 141) n/d 17.6 255.55 n/d 116 (46, 60, 75) P n/d 82.3 645.56 n/d 61 (25, 32, 40) P n/d 49.8 1256.77 n/d 16 (7, 8, 11) 169 (132, 347) n/d 18.5 151.78 n/d P P n/d 447.7 6149.09 n/d P P n/d 381.4 4607.710 n/d P P n/d 149.7 960.511 n/d P P n/d 999.3 9821.712 n/d 128 (51, 66, 84) P n/d 85.5 1829.013 n/d 32 (13, 16, 21) P n/d 20.0 574.014 n/d P 237 (186, 487) n/d 276.4 260.215 n/d n/d 66 (52,135) n/d 7.2 113.916 n/d 20 (8, 10, 13) 206 (162, 425) n/d 11.6 258.217 n/d 66 (26, 34, 43) 403 (317, 830) n/d 46.3 520.918 n/d n/d 193 (152, 397) n/d 8.6 221.219 n/d n/d 119 (93, 244) n/d 7.0 131.220 n/d 25 (10, 13, 16) 358 (281, 736) n/d 17.5 391.521 n/d n/d 124 (97, 255) n/d 7.5 177.7

an/d = not detected. P = above cutoff. bThe calculated concentration of each mycotoxin compound according to the concentration of ZEA. cThecalculated concentration of each mycotoxin compound according to the concentration of DON.

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LC−MS/MS. Moreover, acceptable RSDs ranging from 5% to20% were also achieved with LFA.Results of Naturally Contaminated Samples. Analysis

of 21 naturally contaminated maize samples was performedusing the developed LFA followed by LC−MS/MS forconfirmation (Table 3). With the LC−MS/MS analysis, all21 samples were found positive with DON and ZEA and noAFB1. Of the 21 samples, 4 were contaminated above the MLsof 1750 μg/kg for DON and 200 μg/kg for ZEA. Using theLFA, all 21 samples were contaminated with DONs (100%agreement) whereas only 17/21 (81%) samples were foundcontaminated with ZEAs. As listed in Table S-3 in theSupporting Information, the cLOD of the LFA for ZEA was 9μg/kg, which was higher than the concentrations confirmed byLC−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 wasabove the calculated range of the LFA. However, for samples 2and 10, the concentration of ZEA confirmed by LC−MS/MSwas 104 μg/kg and 150 μg/kg, which was within the detectionrange of the LFA (9 μg/kg to 186 μg/kg for ZEA), but the datawas still marked as “P”. Such overestimation of ZEA could bethe result of an actual contamination (cocontamination) of“masked-ZEAs” in the sample. Also for sample 13, anoverestimation for DON was detected. This result explainsthe 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 constructedand 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. Thevalidation results of the method in the spiked samples showedthat the developed approach was reliable and could beemployed for multiplex mycotoxin detection in cereals. Becausethe monoclonal antibodies adopted were class specific, the LFAstrip could simultaneously detect three groups of mycotoxins ina single assay. In real sample analysis, the vLOD and cLOD forthree mycotoxins were lower than the EU maximum levels. Theproposed method was successfully applied to naturallycontaminated maize samples. The results were in goodagreement with those obtained using confirmatory LC−MS/MS. The overestimation of some mycotoxins was related to therecognition capability of the class specific antibodies utilized inthe LFA.

■ ASSOCIATED CONTENT

*S Supporting InformationAdditional information as noted in text. This material isavailable 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∥S.S. and N.L. contributed equally to this work.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors thank the funds from the National Basic ResearchProgram of China (Grant 2013CB127801), Pujiang Plan ofShanghai (Grant No. 13PJ1407200), Shanghai MunicipalCommission for Science and Technology (Grant13231202800), and the Chinese-Belgian Joint Project ofBELSPO, MOST, China (Grant S2012GR0 016) and Belgium(Grant BL/02/C58).

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