Development of a Simultaneous Lateral Flow Strip Test for the Rapid and Simple Detection of Deoxynivalenol and Zearalenone Kyeong-Yeol Kim, Won-Bo Shim, Jeong-Sook Kim, and Duck-Hwa Chung
The objective of this study was to develop a 1-step simultaneous lateral flow strip test for the rapid and simple detection of deoxynivalenol (DON) and zearalenone (ZEA) in grains. Two monoclonal antibodies (MAbs) against DON and ZEA were respectively conjugated with gold nanoparticles and used to develop a lateral flow strip test for a single toxin and multiple toxins. First, individual lateral flow strips for a single toxin were optimized, and their conditions were used to develop a simultaneous lateral flow strip for multiple toxins. Limits of detection of both lateral flow strip tests for DON and ZEA were the same (DON: 50 ng/mL, ZEA: 1 ng/mL). Both methods showed cross-reactivity for α-zearalenol and β-zearalenol, but no cross-reaction to other mycotoxins. The results can be completed obtained within 15 min. The cut-off values of the simultaneous lateral flow strip for the spiked rice and corn were 500 and 10 ng/g for DON and ZEA, respectively. The results demonstrated that the developed simultaneous lateral flow strip test offers a rapid, easy-to-use, and portable analytical system and can be used as a convenient qualitative tool for the on-site detection of DON and ZEA in food and agricultural commodities.
Keywords: deoxynivalenol, lateral strip test, simultaneous detection, zearalenone
M: Food Microbiology & Safety
Simultaneous lateral strip test is useful for a rapid detection of DON and ZEA at a time in food
and grain samples.
Introduction A variety of Fusarium fungi produce a number of mycotoxins of the trichothecene class, such as deoxynivalenol (DON), zearalenone (ZEA), fumonisin, nivalenol, and T-2 and HT-2 toxins (Kamimura 1993; Sobrova and others 2010). Among them, DON and ZEA are found worldwide in a number of cereal crops, such as maize, rice, oats, and wheat (Kuiper-Goodman and others 1987; Tanaka and others 1988; Ok and others 2014). Since both mycotoxins were produced by Fusarium fungi, the simultaneous occurrence of DON and ZEA is potential and frequently reported (Kolosova and others 2007). Recently, DON and ZEA are classified as a Group 3 (not classifiable as to its carcinogenicity to humans) by the international agency for research on cancer (IARC 2014). Adverse effects of DON include dose-dependent induction of feed refusal, diarrhea and emesis in livestock, and ZEA causes infertility, abortion or other breeding problems, especially in swine (Agag 2004; Kolosova and others 2007). Therefore, the presence of these mycotoxins in food items may cause a serious damage to humans. In order to prevent the importation and distribution of foods and grains contaminated with both mycotoxins, maximum tolerated levels by the European Commission for cereals (intended for direct human consumption) have been MS 20140322 Submitted 2/27/2014, Accepted 8/8/2014. Authors Kyeong-Yeol Kim and Chung are with Div. of Applied Life Science, Graduate School, Gyeongsang National Univ., Jinju, Gyeongnam 660-701, Republic of Korea. Authors Shim is with School of Physics and Chemistry, Gwangju Inst. of Science and Technology, Gwangju 500-712, Republic of Korea. Authors Jeong-Sook Kim and Chung are with Inst. of Agriculture and Life Science, Gyeongsang Natl. Univ., Jinju, Gyeongnam 660-701, Republic of Korea. Direct inquiries to author Won-Bo Shim (E-mail: [email protected]
Journal of Food Science r Vol. 79, Nr. 10, 2014
set as 750 ng/g for DON and 75 ng/g for ZEA (EC 2006). In South Korea, maximum tolerated levels are 500 to 1000 ng/g for DON and 50 to 200 ng/g for ZEA in unprocessed cereals (MFDS 2013). Analyses of DON and ZEA have been usually performed by gas chromatography (GC) (Schwadorf and Muller 1992; Eke and others 2004), gas chromatography-mass spectrometry (GC-MS) (Tanaka and others 2000; Zhao and others 2013), high performance liquid chromatography (HPLC) (Saeger and others 2003; Omurtag and Beyo˘glu 2007) and immunoassays (Kuzdrali´nski and others 2013). However, chromatographic methods are unsuitable for the routine screening of large number of samples because GC and HPLC require a complicated clean-up, enrichment step prior to determination, sophisticated technical equipment, and highly skilled personnel (Kolosova and others 2006). On the other hand, most traditional immunoassays including ELISA have often been limited to laboratories equipped with tools and devices and could not detect multianalytes in an analytical cycle. Recently, on-site and multiple detection technologies have received a great deal of attention in the development of analytical methods for mycotoxins determination as well as diagnosing human diseases and hazards in environmental and food samples (Cella and others 2010; Wang and others 2011). Therefore, there are many studies that have been conducted for the development of methods based on chromatographic (Garrido and others 2013; Li and others 2014) and biosensor (Czeh and others 2013; Wang and others 2013) techniques for multiple mycotoxins detection. A lateral flow strip, often called immunochromatography, is representative of on-site detection technologies and can reduce time and cost for analysis. The method also allows for simpler assay R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12647 Further reproduction without permission is prohibited
Simultaneous detection of DON and ZEA . . .
Materials and Methods Chemicals and materials DON was purchased from Wako Pure Chemical Industries (Osaka, Japan). ZEA and related mycotoxins (α-zearalenol, βzearalenol, α-zearalanol, and β-zearalanol) and other mycotoxins (nivalenol, 3-acetyl deoxynivalenol, T-2 toxin, aflatoxin B1 , ochratoxin A, patulin, citrinin), bovine serum albumin (BSA), ovalbumin (OVA), soybean trypsin inhibitor (STI), tetrachloroauric acid, sodium citrate, Tween 20, sucrose, dextran, and goat anti-mouse IgG (whole molecule) were obtained from SigmaAldrich (St. Louis, Mo., U.S.A.). All other chemicals and organic solvents were of analytical grade or higher. Mycotoxinprotein conjugates were prepared in our laboratory. DON–protein conjugates (DON–BSA, DON–OVA, and DON–STI) were synthesized according to the method described in a previous report (Maragos and McCormick 2000). ZEA protein conjugates (ZEA–BSA, ZEA–OVA, and ZEA–STI) were synthesized by the activated ester method described in a previous report (Thongrussamee and others 2008). The protein concentration of the mycotoxin-protein conjugates was determined by by Quick Start Bradford Protein Assay (Bio-Rad, Hercules, Calif., U.S.A.) The mycotoxin-protein conjugates were used as capture reagents immobilized at the test lines on membranes in the lateral flow strip system. Protein G-agarose for the purification of a monoclonal antibody (MAb) was purchased from Bioprogen (Daejeon, Korea). A nitrocellulose membrane (HiFlow Plus HFB18004) was purchased from Millipore Corporation (Bedford, Mass., U.S.A.). The sample, absorbent and conjugate pads were purchased from Pall Co.
(Port Washington, N.Y., U.S.A.). Semi-gride polyethylene sheets were obtained from a local market.
Production of monoclonal antibodies (MAbs) to DON and ZEA A MAb specific to DON was newly developed in this study by cell fusion of spleen cells obtained from mice immunized with DON–BSA conjugate. A monoclonal hybridoma cell line named with DON 3 was developed and confirmed to produce produced an MAb specific to DON. The anti-DON monoclonal antibody also possessed the cross-reactivity to 3-acetyl deoxynivalenol (23%). Another MAb (ZEA 2C5) against ZEA has already been confirmed to be highly specific to ZEA and showed the cross-reactivity to other related mycotoxins such as αzearalenol (121.5%), β-zearalenol (65.3%), α-zearalanol (21.5%), and β-zearalanol (18.9) by DC-ELISA in a previous report (Thongrussamee and others 2008). However, both MAbs did not react to other mycotoxins including aflatoxin B1, ochratoxin A, patulin, citrinin, and T-2 toxin. Both hybridoma cells (DON 3 and ZEA 2C5) were expended in 10% fetal bovine serum/bulbecco modified eagle medium and intraperitoneally injected into BALB/c mice that had been pretreated with an intraperitoneal injection of 0.5 mL of pristine. Ascite fluid was taken from the mice and purified by precipitation with saturated ammonium sulfate followed with affinity chromatography on a protein G agarose. Protein concentration of the purified MAbs was determined with a protein assay kit (Bio-Rad Laboratories, Richmond, Calif., U.S.A.). Preparation of MAb–gold nanoparticles conjugate Colloidal gold nanoparticles (diameter 40 nm) generally used as a marker in lateral flow strips were produced in our laboratory according to the method of Frens (1973) and respectively conjugated with DON 3 MAb and ZEA 2C5 MAb by the method of Roth (1982). Due to the surface charge of typical gold nanoparticles is negative, the electronic properties of gold nanoparticles become neutral when excess cations are added to the solution of gold nanoparticles. This state causes nanoparticles to aggregate resulting in the color change of the solution from red to blue. However, the aggregation can be prevented by coating or labeling gold nanoparticles with biological recognition molecules such as antibody and single strand DNA. Therefore, gold nanoparticles conjugated with appropriate amount of antibody will not be aggregated by excess cations (stabilized state). Before the conjugation of the MAb and gold nanoparticles, the minimal MAb concentration needed for the stabilization of the gold nanoparticle was determined. Briefly, 0.1 mg of lyophilized MAb was dissolved in 1 mL of 2 mM borate buffer (pH 7.2), and the gold nanoparticle solution was adjusted to pH 9.0 with 0.1 M K2 CO3 . One milliliter of colloidal gold was distributed into each of a series of 1.5 mL tubes. Both MAbs (0, 10, 20 to 90 μL) were separately added to each tube and adjusted to the same volume (total volume: 1.09 mL) with 2 mM borax buffer (pH 9.4). The tubes were shaken for 1 min and then incubated for 5 min at room temperature. Fifty microliter of 10% NaCl was added to all tubes and agitated for 1 min, and then the solution was measured with a spectrophotometer at 540 nm. If a tube containing a minimum amount of the MAb for stabilization of gold nanoparticle, the color did not change from red wine to blue. Five microgram of DON 3 MAb and 3 μg of ZEA 2C5 MAb per were determined as minimal amount to stabilize 1 mL gold nanoparticle. Vol. 79, Nr. 10, 2014 r Journal of Food Science M2049
M: Food Microbiology & Safety
and decreases the sample volume required. This assay has been widely applied for the detection of mycotoxins, such as aflatoxin B1 (Zhang and others 2011), ochratoxin A (Lai and others 2009), ZEA (Shim and others 2009a,b,c), and fumonisin B1 (Shiu and others 2010) since this method provides several benefits including a user friendly format, short assay time, long-term stability over a wide range of climates, and cost-effectiveness. These characteristics make it ideally suited for on-site screening by people who are not skilled analysts (Shim and others 2006). Recently, it has been effectively used to detect multiple mycotoxins with one single test, such as aflatoxin B1 and ochratoxin A (Shim and others 2009a,b,c), ochratoxin A and zearalenone (Shim and others 2009a,b,c), and DON and ZEA (Kolosova and others 2007). Especially, Kolosova and others (2007) reported a lateral-flow immunoassay for the rapid simultaneous detection of ZEA and DON that has cut-off levels of 1500 ng/g for DON and 100 ng/g for ZEA in grain samples. However, the method was not suitable for determination of DON in unprocessed cereals (other than durum wheat, oats, and maize) (1250 ng/g for DON and 100 ng/g for ZEA), cereals intended for direct human consumption (750 ng/g for DON and 75 ng/g for ZEA), and processed cereal-based foods and baby foods for infants and young children (200 ng/g for DON and 20 ng/g for ZEA) set by EU (EC 2006). Therefore, a more sensitive lateral flow strip for simultaneous determination of DON and ZEA is strongly demanded in terms of rapid on-site analysis. In this paper, we report a simultaneous lateral flow strip with higher sensitivity compared to that reported by Kolosova and others (2007) for the rapid, simple, and on-site detection of DON and ZEA and its application to rice and corn samples artificially spiked with DON and ZEA.
Simultaneous detection of DON and ZEA . . . For conjugation, 5.5 μg of DON 4 MAb and 3.3 μg of ZEA 2C5 MAb per 1 mL gold nanoparticle were respectively added and incubated for 1 h at room temperature. One-hundred and fifty microliter of 10% (w/v) BSA solution was added and stirred for 1 h. The mixture was centrifuged at 15000 rpm at 4 °C for 15 min, and the supernatants were removed. The pellets were washed 3 times with 2 mM borate buffer (pH 7.2), and the final pellets were resuspended with 1 mL of 2 mM borate buffer (pH 7.2) containing 1% BSA, 1% sucrose, and 0.05% sodium azide and kept at 4 °C before use.
M: Food Microbiology & Safety
A lateral flow strip test for a single mycotoxin Before developing a simultaneous lateral flow strip test for DON and ZEA, lateral flow strip for a single toxin were optimized. The lateral flow strip test for a single toxin consists of 3 pads (sample, 1.5 × 0.5 cm; conjugate, 1.5 × 0.5 cm; absorbent pads, 1.5 × 0.5 cm) and a nitrocellulose membrane (2.5 × 0.5 cm). In our previous study (Shim and others 2009a,b,c), we recognized that mycotoxins were often absorbed to a sample pad. Thus, in order to prevent the absorption of mycotoxin, a sample pad was soaked with 50-mM borate buffer (containing 1% BSA, 0.5% Tween 20, 5% sucrose, 5% dextran, and 0.05% sodium azide, pH 7.4) and dried at 60 °C for 1 h. A conjugate pad was treated with DON 3 MAb–gold nanoparticle diluted 50-fold (DON) or ZEA 2C5 MAb–gold nanoparticle (5 μL) diluted 40-fold (ZEA) in 20 mM borate buffer (pH 7.4) containing 0.5% BSA, 5% sucrose and 0.5% Tween 20 and dried at 37 °C for 30 min. The absorbent pads were not treated. The nitrocellulose membrane was treated with ZEA– STI (200 ng) or DON–BSA (40 ng) conjugate in 1 μL of PBS and goat antimouse antibody (1 μg in 1 μL of PBS) for test and control lines by using a dispenser (DCI100; Zeta Corporation, Kyunggi-do, South Korea) and dried for 30 min at 37 °C. The distance between test and control lines was 5 mm. The pad and nitrocellulose membrane were attached on semirigid polythylene sheet. The membrane was placed at the center of the sheet, and the absorbent pad was pasted on the top of the sheet with 3 mm overlap to the membrane. The conjugate pad was attached by overlapping 2 mm with the other side of the membrane, and the absorbent pad was placed on the bottom of the sheet with 3 mm overlap to the conjugate pad. A simultaneous lateral flow strip test for multiple toxins detection A format of a simultaneous lateral flow strip for rapid and simple detection of both mycotoxins (DON and ZEA) was shown in Figure 1. Nitrocellulose membranes were treated with 2 test lines (DON and ZEA tests) and 1 control line by the dispenser as described above. DON–BSA (40 ng) and ZEA–STI (200 ng) conjugates were placed at 2 discrete test lines, and 1 μL of goat antimouse IgG (1.0 mg/mL in PBS) was immobilized at the control line. The distance of each line was 5 mm. To determine nonspecific binding between the DON–protein (or ZEA–protein) conjugate and ZEA 2C5 MAb–gold (or DON 3 MAb–gold) conjugate, conjugate pads were respectively prepared by spraying with DON 3 MAb–gold (5 μL), ZEA 2C5 MAb–gold (5 μL), and the sum of both MAb–gold conjugates (10 μL; 5 μL DON 3 MAb–gold and 5 μL ZEA 2C5 MAb–gold). All pads and membrane were treated as described above, dried at 37 °C for 30 min, and attached to their positions on the semirigid polyethylene sheet. Mycotoxinfree standard solution (200 μL 10% MeOH/PBS) was placed in the wells of microtiter plates, and the simultaneous lateral flow M2050 Journal of Food Science r Vol. 79, Nr. 10, 2014
strip assembled was dipped into the well at the sample pad side. The results were determined by naked eyes.
Sensitivity and specificity of the simultaneous lateral flow strip The detection limits of the simultaneous lateral flow strip for DON and ZEA were tested with the individual mycotoxin standard (0, 1, 10, 50, and 100 ng/mL for DON and 0, 0.1, 0.5, 1, and 5 ng/mL for ZEA) and the mixture of DON/ZEA standards (DON/ZEA; 0/0, 0.5/0.5, 1/1, 5/5, 10/10, 50/50, and 100/100 ng/mL). On the other hand, the specificity of the method was subjected with other mycotoxin standards such as nivalenol, 3-acetyl dexoynivalenol, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, T-2 toxin, aflatoxin B1 , ochratoxin A, patulin, and citrinin. The mycotoxins standard solutions (200 μL) were placed into wells of a microtiter plate, and the side of sample pad of the lateral flow strip was dipped into the wells. The results of the method were visually evaluated as described above within 15 min. Sample preparation Mycotoxin-free and positive rice and corn samples were prepared by spiking mycotoxin mixture solutions at 0/0, 10/10, 50/50, 100/100, 500/500 and 1000/1000 ng/g of DON/ZEA. The spiked samples (5 g) were dried for 1 h at room temperature in a darkroom and extracted with 10 mL of 60% methanol containing 4% NaCl for 30 min by a vortexer (Science Industries Inc. Bohemia, N.Y., U.S.A.). After centrifuging at 3000 rpm for 10 min, the supernatant was filtered through filter paper (Advantec NO. 1), diluted with PBST to minimize matrix interferences and methanol concentration, and immediately applied to the simultaneous lateral flow strip. The results of the simultaneous lateral flow strip were compared with those by HPLC performed as described in previous report (Thongrussamee and others 2008; Ok and other 2014). For ZEA analysis, ground samples (20 g) of corn and rice with 2-g NaCl were mixed with 100 mL of acetonitrile:water (90:10, v/v) and blended at high speed for 3 min. The mixture was collected and filtered through Whatman no.1 filters, and 10 mL of the filtrate was diluted with 40 mL of distilled water. The diluted extract was slowly passed through an immuno-affinity column (ZearalaTest, Vicam IAC, Milford, Mass., U.S.A.) with a flow rate about 1.5 mL/min. The column was washed with 10-mL distilled water, and the ZEA bound to the column was eluted with 1.5-mL HPLC grade methanol and the elute was immediately used in HPLC analysis. For HPLC, a reverse phase SUPELCOSILTM LC-18 (25 × 4.6 mm, 5-μm particle size) was used as an analytical column. The column was maintained at 30 ˚C and equilibrated with water:acetonitrile:methanol (43:35:22, v/v/v) at flow rate at 1.5 mL/min. Fifty microliters of the eluted samples were injected into HPLC system (Agilent 1100 series, Waldbronn, Germany). The detection of ZEA was carried out at wavelength of 274 nm excitation and 440 nm emission (Fluorescence Spectrofotometer Detector, model 1046A, Agilent). For DON analysis, 20 g of corn and rice were added to 100 mL of distilled water and blended at high speed for 5 min. The mixture was centrifuged for 15 min at 4000 rpm and filtered through Whatman no.1 filters. Four milliliters of filtrate was slowly passed through an immuno-affinity column (DON test, Vicam) with a flow rate about 1.5 mL/min. The column was washed with 10-mL distilled water. The DON bound to the column was eluted with 3-mL HPLC grade acetonitrile, and immediately evaporated to dryness under nitrogen gas at 50 °C and dissolved in 1 mL of water:acetonitrile (83:17, v/v). Fifty microliter was injected into
Agilent 1100 series HPLC system. The detection of DON was carried out with ultraviolet light detector at 220 nm. In addition, rice and corn samples spiked with DON/ZEA mixture at 0/0, 0/10, 500/0, and 500/10 ng/g were prepared on different days (3 d) and analyzed 3 times for each day by the simultaneous lateral flow strips to validate the reproducibility, precision, and accurate of the method.
sensitivity compared to that obtained by the lateral flow strip with ZEA–BSA conjugate. Consequentially, the combinations of ZEA 2C5 MAb–gold nanoparticles and ZEA–STI and DON 3 MAb–gold nanoparticles and DON–BSA were chosen to develop lateral flow strip tests for single toxin and multiple toxins (data not shown).
Results and Discussion
Optimization and characterization of the lateral flow strip for a single mycotoxin The simultaneous lateral flow strip was based on the competition reaction of the MAb–gold nanoparticles between mycotoxin and mycotoxin–protein conjugate. In typical competitive immunoassay for small molecules as well as mycotoxins, increasing concentration of immunoreagents decreases the sensitivity of immunoassays. Therefore, the determination of appropriate amount of detector (MAb–gold nanoparticles) and capture (mycotoxin– protein conjugate) reagents is key factor to develop a highly sensitive lateral flow strip. For the optimization of the lateral flow strip for a mycotoxin, amount of the detector and capture reagents were investigated. In case of DON lateral flow strip, checkerboard titration with different volumes (3, 5, and 7 μL) of DON 3 MAb–gold nanoparticles diluted 1/50 and amounts (30, 35, and 40 ng in 1 μL) of DON–BSA conjugate were investigated. The optimal concentrations of the detector and capture reagents exhibiting the highest different of red band intensities between DON negative (0 ng/mL) and positive (100 ng/mL) test were 5 μL of DON 3 MAb–gold nanoparticles and 40 ng of DON–BSA
Colloidal gold–monoclonal antibody We synthesized MAb–gold nanoparticles with 5.5 μg of DON 3 MAb and 3.3 μg of ZEA 2C5 MAb responding to 1 mL of gold nanoparticles. For the determination of the suitability of MAb– gold nanoparticles to develop a lateral flow strip, DON–BSA, DON–OVA, and DON–STI conjugates were treated at the test line on a membrane and tested with DON negative and positive tests. DON 3 MAb–gold nanoparticles did not bind to DON– OVA and DON–STI conjugates (no red line at the test zone by the negative test). However, the lateral flow strip test performed with DON–BSA appeared clear different results at the test line between positive (no line) and negative (red line) tests. In case of ZEA, ZEA–OVA conjugate was unsuitable to develop a lateral flow strip. Whereas, ZEA–STI and ZEA–BSA conjugates were suitable as capture reagents in the development of a lateral flow strip, we finally chose ZEA–STI conjugate as a capture reagent to develop a lateral flow strip for ZEA analysis since the results of the lateral flow strip treated ZEA–STI conjugate showed the highest
Figure 1–Schematic description of a simultaneous lateral flow strip for DON and ZEA detection (A), and expected results of the method (B). C, DT, and ZT on figures mean control line, DON test line, and ZEA test line, respectively. Vol. 79, Nr. 10, 2014 r Journal of Food Science M2051
M: Food Microbiology & Safety
Simultaneous detection of DON and ZEA . . .
Simultaneous detection of DON and ZEA . . .
M: Food Microbiology & Safety
conjugate, respectively. In order to optimize the lateral flow strip for ZEA, checkerboard titration with 2C5 MAb–gold nanoparticles and ZEA–STI conjugate was determined as same as DON test. ZEA–STI of 200 ng and 5 μL of 2C5 MAb–gold nanoparticles diluted 40-fold per strip were finally chosen as appropriate amounts resulting in the highest different color intensity between ZEA negative and positive tests (data not shown). With these conditions described above, the individual lateral flow strips for a mycotoxin, DON and ZEA were developed. A lateral flow strip has been widely used for a qualitative and on-site detection of target at threshold levels without any instruments and complicated experimental steps. Therefore, results between negative and positive tests are needed to be easily distinguished by the naked eyes. The detection limits of both lateral flow strips optimized for a single mycotoxin were 50 ng/mL for DON and 1 ng/mL for ZEA. In our previous report (Shim and others 2009a,b,c), we reported the development of lateral flow strip with the ZEA 2C5 MAb–gold nanoparticles and ZEA–BSA conjugate for ZEA determination, and its detection limit was 5 ng/mL of ZEA. The lateral flow strip with ZEA–STI conjugate developed in this study provided the enhanced sensitivity. The lateral flow strip for DON was confirmed to be specific to DON, but ZEA lateral flow strip showed cross-reactivities for α-zearalenol and β-zearalenol. Both single toxin lateral flow strips for DON and ZEA require just sample application (dipping the strip into sample extracts) and could be completed in 15 min after sample application (data not shown).
disappeared at ࣙ1 ng/mL for ZEA and ࣙ50 ng/mL for DON, respectively. The visual detection limits of the simultaneous lateral flow strip for DON and ZEA was the same with those of the single toxin lateral flow strips. The cross-reactivities of the simultaneous lateral flow strip also investigated with other mycotoxin as same as that of the single mycotoxin lateral flow strip. The simultaneous lateral flow strip was confirmed to be specific to DON and ZEA and showed cross-reaction to α-zearalenol and β-zearalenol (Table 1). Comparison to the cross-reactivity, both single and mupltiple mycotoxin lateral flow strips possessed the same cross-reactivity (data not shown).
Assessment of the matrix effect from rice and corn The simultaneous lateral flow strip for multiple mycotoxins analysis developed in this study was validated by analyzing rice and corn samples artificially spiked with known concentrations of both mycotoxins. Before the analysis, the tolerance of the simultaneous lateral flow strip to methanol (0% to 60% in deionized water; v/v) was investigated, since both mycotoxins (ZEA and DON) contaminated in real samples such as grains and food were generally extracted by 60% methanol (methanol:water = 60:40). The simultaneous lateral flow strip tolerated to 40% methanol. However, more than 50% methanol caused decreasing color intensities at control and test lines. Although both methods kept their performance up to 40% methanol, we choose 30% methanol in PBST as a working solution to assure the performance of the methods. From the extraction step with 60% methanol, coextracted substances from rice and corn samples resulting in matrix effects on Development of a simultaneous lateral flow strip for DON immunoassays were presented in the extracts. The matrix effect can be effectively removed by the dilution with an appropriate buffer and ZEA analysis The key advantage of the utilization of membranes is that im- In this study, the mycotoxin-free rice and corn samples (5 g) were munoreagents such as antibody and target-protein conjugate could extracted with 10 mL of 60% (v/v) methanol containing 4% (w/v) be immobilized at different places on membranes. Therefore, if the interaction between one mycotoxin and the corresponding MAb–gold nanoparticle is not affected by the other toxin and the other MAb–gold nanoparticles, multiple detection is possible on the lateral flow membranes. In practice, several lateral flow assays based on membranes for multiple toxins analysis have been reported (Kolosova and other 2007; Shim and others 2009a,b,c; Li and others 2013; Wang and others 2013). As shown in Figure 2, 2 test lines and a control line were treated with ZEA–STI, DON– BSA, and antimouse IgG at different sites on the nitrocellulose membrane. Once both MAb–gold nanoparticles were applied to the simultaneous lateral flow strip, color development for both test lines and control line was observed. Whereas, when the DON 3 MAb–gold conjugate (or ZEA 2C5 MAb–gold conjugate) was added to the conjugation pad, the color at the DON test line (or ZEA test line) and control line was observed, but no color was observed at ZEA test line (or DON test line). It meant that the interaction between DON–BSA (or ZEA–STI) and DON 3 MAb–gold nanogoldparticle (or ZEA 2C5 MAb–gold nanoparticle) was not affected by the present of the other toxin and the other corresponding MAb–gold nanoparticles. Therefore, the simultaneous lateral flow strip test could be developed by using the optimized conditions of the lateral flow strips for a single toxin. The simultaneous lateral flow strip was assembled using the optimized conditions of single toxin lateral flow strip without Figure 2–Confirmation of a specific interaction between MAb–gold conany change and subjected to determine its sensitivity. Figure 3 jugates on conjugate pad and corresponding mycotoxin-protein conjushows the sensitivities of the simultaneous lateral flow strip for the gates at each test line. (A) Conjugate pad was treated with BSA– nanoparticles conjugate, (B) conjugate pad was treated with DON 3 MAb– presence of a single mycotoxin and both mycotoxins. ZEA and nanoparticles conjugate, (C) conjugate pad was treated with ZEA 2C5 DON test lines of the strips performed with the DON/ZEA mix- MAb–nanoparticles conjugate, (D) conjugate pad was treated with both ture (0/0, 0.5/0.5, 1/1, 5/5, 10/10, 50/50, and 100/100 ng/mL) DON 3 MAb–gold nanoparticles and ZEA 2C5 MAb–gold nanoparticles. M2052 Journal of Food Science r Vol. 79, Nr. 10, 2014
Simultaneous detection of DON and ZEA . . . toxins. As shown in Table 2, red color development appeared at both test lines corresponding to each mycotoxin when the blank sample was applied to the assay. However, color development at DON and ZEA test lines decreased as the concentrations of DON and ZEA increased, and completely disappeared at ࣙ500 ng/g of DON and 10 ng/g of ZEA. This confirmed that the cut-off values of the method in real rice and corn samples are 500 and 10 ng/g for DON and ZEA, respectively. The results of DON and ZEA determination by the simultaneous lateral flow strip were compared with HPLC results and shown in Table 2. The results obtained by both analytical methods (lateral flow strip and HPLC) showed very good agreement. Kolosova and others (2007) reported the development of a colloidal gold-based lateral flow immunoassay for the rapid simultaneous detection of ZEA and DON. The detection limits of the method were 1500 ng/g for DON and 100 ng/g for ZEA. The simultaneous lateral flow strip developed
M: Food Microbiology & Safety
NaCl, and undiluted and diluted extracts (1/3, 1/5, and 1/7) with PBST. Up to a 3-fold dilution of the extracts, strong matrix effect was still observed. However, matrix effect was effectively reduced by ࣙ5-fold dilution with PBST, and we selected 5-fold dilution with PBST for the preparation of samples. This means that the initial concentration of both mycotoxins in rice and barley would be diluted 10-fold after extraction by 60% methanol and 5-fold dilution with PBST. Therefore, we expected that the lateral flow strip for multiple toxins analysis exhibiting the detection limits of 50 ng/mL for DON and 1 ng/mL for ZEA can simultaneously detect DON and ZEA contaminated at ࣙ500 ng/g and ࣙ10 ng/g in real samples, respectively. The mycotoxin-positive rice and corn samples (n = 3) were prepared by spiking known concentration of both mycotoxins (ZEA/DON; 0/0, 10/10, 50/50, 100/100, 500/500, and 1000/1000 ng/g), prepared as previously described, and analyzed by the simultaneous lateral flow strip for multiple
Figure 3–Visible detection limits of the lateral flow strips for a single mycotoxin (upper) and both mycotoxins (bottom). The labels on the figures indicate the concentrations of respective and mixed concentration DON and ZEA. VDL means a visible detection limit of the simultaneous lateral flow strip. Vol. 79, Nr. 10, 2014 r Journal of Food Science M2053
Simultaneous detection of DON and ZEA . . . Table 1–Cross-reactivity of the simultaneous lateral flow strips for deoxynivalenol and zearalenone determination. Mycotoxin concentrations (ng/mL) Mycotoxinsa (n
DON ZEA DON ZEA DON ZEA DON ZEA DON ZEA DON ZEA DON ZEA DON ZEA DON ZEA
3-Acetyl deoxynivalenol Nivalenol Zearalenone α-zearalenol β-zearalenol α-zearalanol β-zearalanol T-2 toxin
+b + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + − + + + + + + + + + +
+ + + + + + + − + − + + + + + + + +
−c + + + + + + − + − + ±d + + + + + +
− + + + + + + − + − + − + + + + + +
Note Red bands at the control line were observed from the all lateral flow strip tested. a Other mycotoxins such as aflatoxin B1, ochratoxin A, citrinin, and patulin were also tested and determined to be negative by the simultaneous lateral flow strip. b +: an obvious red band was observed, negative result. c −: no band was observed, positive result. d ±: a faint band was observed, negative result.
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Table 2–Analyses of DON and ZEA in spiked rice and corn samples by the simultaneous lateral flow strip. Results of analyses Simultaneous lateral flow strip Test lines Samples
Spiked DON/ZEA (ng/g)
0/0 10/10 50/50 100/100 500/500 1000/1000 0/0 10/10 50/50 100/100 500/500 1000/1000
+,+,+ −c ,−,− −,−,− −,−,− −,−,− −,−,− +,+,+ −,−,− −,−,− −,−,− −,−,− −,−,−
ND 9.3 ± 0.5 49.1 ± 6.2 92.2 ± 5.8 452.9 ± 32.7 974.5 ± 53.1 ND 8.6 ± 0.4 52.1 ± 3.4 105.2 ± 7.2 476.8 ± 43.1 921.6 ± 92.3
Rice (n = 3)
Corn (n = 3)
a +: an obvious red band b ND: not detected. c
+,+,+ +,+,+ +,+,+ −,−,− −,−,− +,+,+ +,+,+ +,+,+ +,+,+ −,−,− −,−,−
8.2 ± 0.21 44.2 ± 1.4 100.8 ± 9.1 482.1 ± 26.5 942.1 ± 38 ND 8.7 ± 0.7 48.1 ± 4.6 93.5 ± 6.3 464.3 ± 36.9 952.5 ± 72.2
−: no band was observed.
in this study possesses a higher sensitivity than that developed by them. According to the maximum tolerated levels set by EU and South Korea for cereals (intended for direct human consumption), the limits of detection for DON and ZEA of the method in this study were sufficient to be useful tool for the rapid, multiple, and on-site detection of both mycotoxins, DON and ZEA, in various food and grain samples. In addition, we prepared DON (500 ng/g) positive corn and rice samples and ZEA positive (10 ng/g) corn and rice samples, and DON/ZEA-free and DON/ZEA positive (500/10 ng/g) corn and rice samples on different 3 d (triplicate per each day) and analyzed the samples to determine the ability of the method to detect negative samples as negative and positive samples as positive. As shown in Table 3, even if a faint red band at ZEA test line was obtained from 8th test with ZEA positive corn and 5th with DON/ZEA positive corn, the method could specifically de-
M2054 Journal of Food Science r Vol. 79, Nr. 10, 2014
tect negative and positive samples as negative and positive results, respectively. The demonstrated the suitability of the method for the on-site, simple, and rapid detection of multiple mycotoxins, DON and ZEA in grains. In conclusion, multiple detection technology has been received a great attention in analytical method development as well as onsite detection technology. The aim of this study is to develop the simultaneous lateral flow strip for rapid, on-site, and multiple detection of DON and ZEA in grain. A lateral flow strip for a single toxin (DON or ZEA) was first optimized, and its optimized conditions were used to develop the simultaneous lateral flow strip for DON and ZEA detection. The limits of detection of the simultaneous method were 50 ng/mL for DON and 1 ng/mL for ZEA, and cut-off values for rice and corn samples artificially spiked with known concentration of DON and ZEA were 500 ng/g for DON and 10 ng/g for ZEA. Results of the simultaneous
Simultaneous detection of DON and ZEA . . . Table 3–Repeatability and reproducibility tests of the simultaneous lateral flow strip test by using spiked rice and corn samples. Spiked DON/ZEA (ng/g) Samples
Rice (n = 9)
DON ZEA DON ZEA
Corn (n = 9)
+,+,+/+,+,+/+,+,+ +,+,+/+,+,+/+,+,+ +,+,+/+,+,+/+,+,+ +,+,+/+,+,+/+,+,+
+,+,+/+,+,+/+,+,+ −,−,−/−,−,−/−,−,− +,+,+/+,+,+/+,+,+ −,−,−/−,−,−/−,±,−
−,−,−/−,−,−/−,−,− +,+,+/+,+,+/+,+,+ −,−,−/−,−,−/−,−,− +,+,+/+,+,+/+,+,+
−,−,−/−,−,−/−,−,− −,−,−/−,−,−/−,−,− −,−,−/−,−,−/−,−,− −,−,−/−,±,−/−,−,−
lateral flow strip for the spiked samples were a good agreement with those of HPLC. The simultaneous assay does not require time-consuming, expensive equipment, and laborious for DON and ZEA analysis and can be accomplished within 15 min with an experimental step (sample application). The results obtained in this study demonstrate that the lateral flow strip for multiple mycotoxins detection has sufficient sensitivity for screening of DON and ZEA in grain samples (intended for direct human consumption) within maximum tolerated limits set by EU and South Korea and could be a useful screening method to qualitative detection of DON and ZEA.
Acknowledgments This work was supported by grants from Advanced Production Technology Development Program, Korean Ministry of Agriculture, Food and Rural Affairs and from Fishery Commercialization Technology Development Program, Korean Ministry of Oceans and Fisheries.
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Vol. 79, Nr. 10, 2014 r Journal of Food Science M2055
M: Food Microbiology & Safety
Note The spiked samples were prepared on different days (3 d). Triplicate spiked samples were prepared and analyzed in a day.