Article pubs.acs.org/JAFC
Europium Nanospheres-Based Time-Resolved Fluorescence for Rapid and Ultrasensitive Determination of Total Aflatoxin in Feed Du Wang,†,§,# Zhaowei Zhang,*,†,# Peiwu Li,*,†,‡,§ Qi Zhang,†,§,# Xiaoxia Ding,†,§ and Wen Zhang†,⊥ †
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China Key Laboratory of Biology and Genetic Improvement of Oil Crops, §Laboratory of Quality & Safety Risk Assessment for Oilseed Products (Wuhan), #Key Laboratory of Detection for Mycotoxins, and ⊥Quality Inspection & Test Center for Oilseed Products, Ministry of Agriculture, Wuhan 430062, China
‡
ABSTRACT: Immunochromatographic (IC) assays are considered suitable diagnostic tools for the determination of mycotoxins. A europium nanospheres-based time-resolved fluorescence immunoassay (Eu-Nano-TRFIA), based on a monoclonal antibody and a portable TRFIA reader, was developed to determine total aflatoxin (including aflatoxins B1, B2, G1, and G2) levels in feed samples. Under optimized conditions, the Eu-Nano-TRFIA method detected total aflatoxin within 12 min. It showed good linearity (R2 > 0.985), LOD of 0.16 μg/kg, a wide dynamic range of 0.48−30.0 μg/kg, recovery rates of 83.9−113.9%, and coefficients of variation (CVs) of 3.5−8.8%. In the 397 samples from company and livestock farms throughout China, the detection rate was 78.3%, concentrations were 0.50−145.30 μg/kg, the highest total aflatoxin content was found in cottonseed meal, and corn was found to be the most commonly contaminated feed. This method could be a powerful alternative for the rapid and ultrasensitive determination of total aflatoxin in quality control and meet the required Chinese maximum residue limits. KEYWORDS: Eu-Nano-TRFIA, europium label, nanospheres, total aflatoxin, feed
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INTRODUCTION Aflatoxins (AF), including AFB1, AFB2, AFG1, and AFG2, are mainly produced by Aspergillus flavus and Aspergillus parasiticus (Figure 1).1 They are commonly present in the food supply
avoid human exposure to aflatoxins, rigorous regulations have been set up by various international government agencies to monitor aflatoxin levels in feed.4,10 A number of methods have been employed for the determination of aflatoxins. IC assays are new, rapid detection technologies with several advantages, including simple and rapid operation, immediate results, and low cost.11−13 Labeled immunoassays, including fluorescence, bioluminescence, chemiluminescence, organic dyes, colloidal gold, magnetic beads, etc.,14 are widely used to detect binding events in many biomedical and diagnostic analyses. Although several efforts have been made toward the determination of aflatoxins using a test strip rapid methods are commercially available,15 they are not ultrasensitive and cannot quantitatively determine aflatoxins. Therefore, a rapid, quantitative determination method for aflatoxins sensing is needed. The time-resolved fluoroimmunoassay (TRFIA) was introduced in the early 1980s. This method uses lanthanide and fluorescent marker complexes, which have rapidly developed to become a new milestone in the development of labeled immunoassays for clinical medicine.16−19 The lanthanide and fluorescent complexes (e.g., Eu3+, Tb3+) provide longer relaxation times and reduced background signals with nanoto microsecond-long fluorescence,20,21 but their shortcoming is that the luminescence is in low quantum yields in comparison with those of organic fluorescence dyes. Due to the complex nature of feed matrices,22 enhancing sensitivity by improving
Figure 1. Chemical structures of the studied aflatoxins B1, B2, G1, and G2.
chain, such as in production, storage, transport, and consumables.2,3 Aflatoxins are regarded as one of the most harmful contaminants in feed, due to their carcinogenic, hepatotoxic, teratogenic, and mutagenic adverse effects toward animals,4,5 and have been shown to be related to the hepatic and extrahepatic carcinogenesis of humans and livestock.6,7 The order of toxicity, AFB1 > AFG1 > AFB2 > AFG2, indicates that the terminal furan moiety of AFB1 is the critical point for determining the degree of biological activity of these mycotoxins.5 Lactating animals can also be infected by aflatoxins by consuming contaminated feed, whereby metabolites of aflatoxins M1 and M2 are then excreted in milk.8,9 To © XXXX American Chemical Society
Received: July 30, 2015 Revised: November 11, 2015 Accepted: November 12, 2015
A
DOI: 10.1021/acs.jafc.5b03746 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Diagrammatic description for principle of competitive assay in test strip format. lamp served as the excitation source at 365 ± 10 nm, and signal acquisition was obtained at 613 ± 10 nm using a photomultiplier tube (PMT). The light source and PMT tube were set at a 120° angle. When the exciting light was irradiated on the strip test area, the fluorescence signal was recorded by the PMT after a 400 μs delay. Meanwhile, the fluorescence with short relaxation time decayed rapidly, reducing the background noise and achieving a high signal-tonoise ratio. The fluorescence signals on test line (T line) and control line (C line) peak were processed using data processing software for quantitative analysis. Preparation of Eu-Nanospheres. The Eu-nanospheres were developed via a modified method.26,27 The carboxylated polystyrene nanospheres were diluted with a 10.0 mL mixture of DI water/acetone (1:1, v/v) to a final density of 1 × 1014 nanospheres in 10.0 mL. Then, a solution containing EuCl3 (100 μL, 0.1 mol/L), stearoyl benzoyl methane (400 μL, 0.1 M), phenanthroline (100 μL), and trioctylphosphine oxide (300 μL) was added and stirred for 10 h at 60 °C and subsequently for 2 h at room temperature. The resulting solution underwent vacuum distillation to remove the organic solvents and was dialyzed for 5 days. The dialysate was collected and stored at 4 °C with 0.05% NaN3. Coupling Antibody−Aflatoxins to Eu-Nanospheres. Two hundred microliters of Eu-nanospheres was added to 800 μL of boric acid buffer solutions (pH 8.2) and ultrasonicated (30 kHz frequency, 20% amplitude, 0.6 intermittent frequencies) at 20 °C for 10 min. Then, 40 μL of EDC (15 mg/mL) was added and stirred for 15 min. The suspension was separated by centrifugation at 17000g for 10 min. The precipitate was resuspended in 1.0 mL of boric acid buffer (pH 8.2) by ultrasonication for 2 min. After 1.0 ng/μL monoclonal anti-aflatoxins antibody (15, 25, 35, 45, and 55 μL, respectively) was added, the mixed solution was shaken for 12 h before pelleting at 17000g for 10 min. The residue was resuspended in 1.0 mL of boric acid buffer (0.5% BSA). The reaction was continued for another 2 h under agitation at 20 °C, and the solution was centrifuged at 17000g, resuspended in 1 mL of boric acid buffer (pH 8.2), and stored at 4 °C for further experiment. Fabrication of IC Strip. The IC strip is a single-step test based on a competitive immunoassay format (Figure 2). An IC strip contained a sample pad, an NC membrane, and an absorbent pad. The AFB1−BSA and rabbit anti-mouse IgG antibody were coated on the NC membrane as a T line and a C line by using Bio Dot XYZ Platform at an appropriate jetting rate. The distance between the T line and the C line was 10 mm. The coated membrane was dried at 37 °C for 2 h. The concentrations of AFB1−BSA and rabbit anti-mouse IgG antibody were optimized by a checkerboard method. The sample pad, NC membrane, and absorbent pad were then assembled on a plastic scale board. The whole assembled scale board was divided into 4.5 mm × 70 mm strips with a guillotine cutter (CM 4000) and stored at 4 °C. Optimization of the Eu-Nano-TRFIA Method. An indirect competitive immunoassay was performed on the strip. The antiaflatoxins−mAb Eu-nanospheres were diluted by 50, 100, 200, 300, and 500 times with protective reagents (6.0% sucrose, 4.0% bovine serum albumin, and 1.0% mannitol in 0.05 mol/L PBS buffer at pH 7.4). A 150 μL dilution was introduced in glass penicillin bottles before lyophilization. The concentrations of AFB1−BSA and rabbit anti-
either fluorescence or detection power for the purpose of rapid on-site sensing is required. One major signal amplification method is to pack the luminescence into a nanospheres capsule, thus enhancing its fluorescence up to thousands of times. After doping, the Eu of nanospheric particles and COOH-activated Eu-nanospheres surfaces were conjugated with monoclonal antibody against aflatoxins, to form an immunoreagent. This immunoreagent was further employed in the determination of aflatoxins by an IC strip via a homemade portable TRFIA reader. Herein, a Eu-Nano-TRFIA method is described, combining the advantages of the long fluorescent lifetime properties of lanthanide elements (Eu), signal amplifications of nanospheres, and IC assays.23,24 This protocol could dramatically enhance the sensitivity and linear range of the immunoreagent and could be employed in complex feed matrices.
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MATERIALS AND METHODS
Reagents and Apparatus. Standard solutions of aflatoxins (AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), zearalenone (ZEN), T2 toxin (T2), deoxynivalenol (DON), AFB1-conjugated BSA (AFB1− BSA), and rabbit anti-mouse IgG were purchased from Sigma-Aldrich (Urbana, IL, USA). Mannitol, sucrose, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), bovine serum albumin (BSA), and Tween 20 were purchased from Roche Applied Science (Roche, Indianapolis, IN, USA). The MycoSep 226 solid phase extraction (SPE) matrix was purchased from Romer Laboratories (Romer, Tulln, Austria). Deionized water (DI) was prepared using a Milli-Q quality water system (Bedford, MA, USA). All other inorganic chemicals and organic solvents were of analytical reagent grade or better. Anti-aflatoxin mAb4F12 was produced in our laboratory and further purified with a protein G immunoaffinity column.6 The cross-reactivity with four major aflatoxins was 100% of AFB1, 90.1% of AFB2, 84.6% of AFG1, and 20.7% of AFG2. Carboxylated polystyrene nanospheres were obtained from Shanghai Uni Biotech Ltd. (Shanghai, China), and polystyrene nanospheres (190 ± 10 nm particle size, 170−200 μequiv/ g surface charge, 25−35.7 sq.A/grp surface area) were obtained. Nitrocellulose (NC) membranes were obtained from Millipore Corp. (Millipore, Bedford, MA, USA). A 3050 dispensing platform and a CM 4000 Cutter (Bio Dot, Irvine, CA, USA) were used to prepare test strips. The HPLC apparatus consisted of an Agilent 1100 HPLC system (Palo Alto, CA, USA) including an isocratic pump (Agilent G1310A) connected to an automatic sampler (Agilent G1313A) and a fluorescence detector (Agilent G1321A). Postcolumn derivatization was carried out with electrochemically generated bromine in Cobra cells (Coring System Diagnostics GmbH, Gernsheim, Germany) using a reaction tube of 340 × 0.5 mm i.d. PTFE to enhance the fluorescence intensity. The analytical column Symmetry Alltima C18 (150 × 4.6 mm i.d., 5 μm; Agilent), kept at 25 °C, was used for aflatoxin determination. This system was piloted by Chemstation 3D software. TRFIA Apparatus. The portable TRFIA reader was homemade, and used for the quantitative determination of aflatoxins. A mercury B
DOI: 10.1021/acs.jafc.5b03746 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry mouse IgG were used for the test and control lines, respectively, and prepared with serial dilutions of 0.1−1.0 mg/mL. In line dispensing, the dispensed rate was evaluated from 4 to 10 μL/s, whereas the moving rate of the horizontal platform was optimized as 100 mm/s. Concentrations of immunoreagent were similarly screened as a checkerboard titration in an ELISA. An aflatoxin standard solution of 0.25 μg/kg (AFB1, AFG1, AFB2, and AFG2, 0.0625 μg/kg) was prepared using a blank feed sample (aflatoxin-free as determined by HPLC). Inhibitory rate values were recorded to determine the optimum concentrations. Aflatoxin-free feed sample extracts spiked with aflatoxins (0, 0.5, 1.5, 4.5, 9.0, 13.5, 22.5, and 30.0 μg/kg) were presented to the test strip to estimate the sensitivity and linear range of the method. A homemade TRFIA reader was used to measure the T line and C line peak areas. A calibration curve was fitted and expressed as T/C versus the natural logarithm of the aflatoxins concentration:
average light density (OD value) of a 10000 times diluted Eunanospheres sample was 0.356 at 340 nm. According to a multifunctional microplate reader, the average lifetime was 740 μs. To estimate the loading capacity of Eu on the nanospheres, the fluorescent intensities from Eu alone and Eu-nanospheres were measured.29 According to the linear relationship between Eu and the Eu-nanospheres, the Eu-nanospheres contained 150,000−160,000 Eu particles. Optimization of Preparation of Eu-Nanospheres Antibody−Aflatoxins. Carboxylated Eu-nanospheres served as carriers for the conjugation of mAbs against aflatoxins. Typical EDC conjugation methods were used because mild conditions maintained the antibody activity. After optimization, 40 μL (15 mg/mL) of EDC was used to activate a 1.0 mL Eunanospheres solution (containing ∼1012 particles). The mAb was then conjugated onto the Eu-nanospheres surfaces. After optimization, a 40 ng mAb against aflatoxins was conjugated with 1.0 mL of Eu-nanospheres, the solution was added to the strip, and the strip had the strongest fluorescence intensity at the same dilution. It was observed that with a lower density of mAb on the Eu-nanospheres, the coefficients of variation (CVs) of the T line increased; however, a higher mAb density could decrease the sensitivity. To obtain a minimum required dosage of Eu-nanospheres antibody and a clear T line, serial concentrations of 50, 100, 200, 300, and 500 times diluted 1.0 mL were used. The 200 times dilution was selected for further experimentation. Optimization of the IC Strip. Before fabricating the IC strip, nonspecific protein adsorption was prevented by using a blocking buffer on the sample pad. An optimized blocking buffer was used and contained 0.01 mol/L, pH 7.4, PBS, 1.0% BSA, 2.5% sucrose, 2.5% Tween 20, and 0.02% NaN3. The BSA prevented nonspecific protein adsorption, and sucrose aided the adsorption and desorption of immunoreagent for a more uniform chromatographic process; Tween 20 increased the specificity by maintaining an effective interaction between the mAb and the antigen, and NaN3 provided a preservation function. Using a checkerboard method, immunoreagent consumption was optimized at 7.5 μL/s of AFB1−BSA (0.5 mg/mL) and 6.0 μL/s of rabbit anti-mouse IgG (0.25 mg/mL) on the IC strip, whereas the moving rate was optimized as 100 mm/s, respectively. Optimization of Eu-Nano-TRFIA Method. Lyophilized Eu-nanospheres antibody−aflatoxins were pipetted into each glass penicillin bottle, and the extracting solution was then added. After mixing, an IC strip was inserted into the glass penicillin bottle and incubated at 37 °C for 8 min. For negative controls, the Eu-nanospheres antibody−aflatoxins present in the solution flowed laterally along the strip toward the T line, where the Eu-nanospheres antibody−aflatoxins are captured by the immobilized AFB1−BSA on the T line. Excess Eunanospheres antibody−aflatoxins flowed toward the C line and were captured by the rabbit anti-mouse IgG on the C line. The T line and C line were then inspected under UV exposure (Figure 3). For the positive feed sample, aflatoxins-conjugated Eu-nanospheres antibody−aflatoxins did not interact with the immobilized AFB1−BSA on the T line, but reached the C line, as indicated by a faint red color (i.e., a positive sample with 10.0 μg/kg of the aflatoxins) or no color (i.e., a positive sample with 30.0 μg/kg of the aflatoxins) on the T line under UV exposure (Figure 3). The ratio of the fluorescent intensities on the T line and C line was inversely proportional to the aflatoxins
Y (value T /C) = aX (ln Caflatoxins) + b Twenty blank samples were analyzed using standard curves to calculate the LOD according to previous reports25 with the homemade TRFIA reader. The LOQ was calculated as the 3 times the LOD. The recovery was studied to evaluate the accuracy of the Eu-NanoTRFIA method. Random aflatoxin concentration in samples was further determined by both Eu-Nano-TRFIA and HPLC methods, to calculate their recovery. Intra-batch repeatability was studied using five strips from one batch. Inter-batch accuracy was measured over five batches by calculating the mean value of five batches. The recovery was calibrated according to previous reports.26 Specificity was evaluated by investigating the cross-reactivity of the Eu-Nano-TRFIA method with mycotoxins in 30.0 μg/kg in blank feed sample, respectively. Sample Preparation for Eu-Nano-TRFIA Method. Feed samples were finely ground with a laboratory mill and sieved through a 20-mesh screen. A 20 g sample in 100 mL of 70% methanol (v/v) was homogenized for 2 min. Then, a 5 mL suspension was centrifuged at 8000g for 2 min. The extracted solutions were purified by MycoSep 226 SPE column, in which 1 mL of supernatant was diluted with 7 mL of 0.4% Tween 20 solution before measurement by the Eu-NanoTRFIA method. Comparison between Eu-Nano-TRFIA and HPLC Method. To validate the Eu-Nano-TRFIA method, the results between Eu-NanoTRFIA and HPLC methods were compared. Samples with random aflatoxin concentrations were compared. The HPLC method was according to the national standard.12 Extracts were filtered through double-layered filter paper. A 10.0 mL sample of the extract was diluted with 20.0 mL of water. The resulting solution was filtered via a 0.45 μm filter membrane before loading onto a homemade immunoaffinity chromatography column packed with anti-aflatoxins mAb. Aflatoxins were eluted using 1.0 mL of methanol and collected in a tube, followed by injection into an HPLC equipped with a fluorescence detector (λex = 360 nm, λem = 440 nm). Aflatoxin analysis was performed with an isocratic elution mode using a mixture of water/methanol (45:55, v/v) at a flow rate of 1.0 mL/min (run time of 18 min). The injection volume of suspended sample was 10.0 μL.28 Application in Real Feed Sample. A total of 397 feed samples were purchased from feed companies and livestock farms throughout China. The optimized method was used to analyze various feed samples, such as corn, wheat bran, soybean meal, peanut meal, cottonseed meal, distillers dried grains with soluble (DDGS), alfalfa forage, silage, and swine and poultry feed.
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RESULTS AND DISCUSSION Characterization of Eu-Nanospheres. The solid content of Eu-nanospheres was 0.99%, as determined by weighing blank nanospheres and Eu-nanosphere complexes. The morphology of the Eu-nanospheres was obtained by transmission electron microscopy. The Eu-nanospheres were uniform and had a welldistributed size, with an average diameter of 204 nm. The C
DOI: 10.1021/acs.jafc.5b03746 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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(Figure 4), which showed that the Eu-Nano-TRFIA method has better specificity. Sample Preparation. Because aflatoxins were in trace amounts in feed, and there is an external restriction factor of uneven distribution and complex matrix nature of the feed, sample pretreatment must ensure representativeness and remove interference. To ensure the sample representativeness, the point-quartering method to take samples for extraction was applied. The extracted solutions were purified by MycoSep 226 SPE, by pushing the column into a test tube containing the sample extract, forcing the extract to filter upward through the packing material of the column. Interferences such as pigments, salt, and other proteins adhere to the chemical packing in the column, and the purified extract, containing the analytes of interest, passes through the column and is detected by the EuNano-TRFIA method.32 To improve the reaction efficiency of extracted solutions and Eu-Nano-TRFIA, 0.4% Tween 20 was used to dilute the extract solution. Tween 20 can increase the identification ability of EuNano-TRFIA, as well as reduce interactions between proteins. The results of comparison of the Eu-Nano-TRFIA and HPLC methods are shown in Table 3. The average recovery was 96.3%, ranging from 82.1 to 115.4%, with a mean CV of 6.1% (3.8−7.9%). Determination and Evaluation of Real Feed Samples. A total of 397 samples from feed companies and livestock farms were collected. These feed samples were categorized as feedstuffs and feed products. Feedstuffs included cottonseed meal, DDGS, corn, peanut meal, soybean meal, and wheat bran. Feed products included swine feed, poultry feed, alfalfa forage, and silage. As shown in Table 4, a total of 311 samples (78.3% of 397 samples) were found to contain aflatoxins having a concentration of 0.50−145.30 μg/kg. Furthermore, 254 samples (64.0% of the 397 samples) have aflatoxin concentrations 50 μg/kg. Aflatoxins were found in 93.3% of 45 corn samples, 59.4% of 32 wheat bran samples, 83.0% of 47 peanut meal samples, 34.9% of 43 soybean meal samples, 89.7% of 29 cottonseed meal samples, and 90.0% of 30 DDGS samples, respectively. The highest aflatoxin concentrations were found in cottonseed meal samples.33 Corn samples held the highest detection rates among the feedstuffs. Aflatoxin contamination was found in 45.7% of 45 alfalfa forage samples, 69.0% of 29 silage samples, 100% of 52 swine feed samples, and 100% of 55 poultry feed samples. Lower concentrations of aflatoxins in the alfalfa forage were likely due to higher moisture content, which may prevent
Figure 3. Gradient concentration of the aflatoxin-spiked feed sample under UV light.
concentration. On the basis of this relationship, the levels of aflatoxins were determined in the feed sample. To establish a standard curve, the aflatoxins-spiked feed sample was employed. By plotting the ratio of T line value to C line value versus the natural logarithm of the concentration (ln c), dependent on the different feed matrices, as seen in Table 1, a linear correlation was found between the T line and C line value ratio and the aflatoxins concentration. Figure 3 shows the gradient concentration of the aflatoxins-spiked feed sample under UV light. For the LOD evaluation, the mean value of five repeated experiments was recorded using 20 blank feed samples. On the basis of the calibration curve, the aflatoxin concentration was further calculated and defined as the LOD. The LOQ was calculated at 3 times the LODs. For different feed matrices, LODs were recorded from 0.16 to 0.25 μg/kg, whereas LOQs were found to be 0.48−0.75 μg/kg. The LOQs of a 0.5 μg/kg strip are shown in Figure 3. Linear wide dynamic ranges of 0.48−30.0 μg/kg were obtained, and a positive sample with 30.0 μg/kg aflatoxins showed no color on the T line.30 This established the Eu-Nano-TRFIA method had a 3 times lower LOD when compared with the ELISA method for broiler feed (1.25 μg/kg).31 For recovery, random feed samples were first measured using HPLC. Average recoveries for inter-batch reproducibility of 101.3% ranged from 83.9 to 113.9%, with a mean CV of 5.3% (3.5−7.9%). For inter-batch reproducibility, the test strips of five different batches were used for replicate testing of samples, with average recoveries of 96.0%, ranging from 89.7 to 99.6%, and mean CVs of 6.0% (3.9−8.8%). Results are shown in Table 2. This demonstrated that the Eu-Nano-TRFIA method was able to meet the regulatory standards in feed sample. To evaluate the method’s specificity, AFB1, AFB2, AFG1, AFG2, ZEN, OTA, DON, and T2 were tested for crossreactivity at spiked concentrations of 30.0 μg/kg in blank feed sample, respectively. Each toxin was measured by UV exposure Table 1. Standard Curves for Feed Sample Matrices sample corn wheat bran soybean meal peanut meal cottonseed meal DDGS alfalfa forage silage swine feed
standard curve Y Y Y Y Y Y Y Y Y
= = = = = = = = =
−0.341X −0.364X −0.347X −0.338X −0.403X −0.345X −0.338X −0.354X −0.391X
+ + + + + + + + +
1.264 1.291 1.211 1.173 1.414 1.287 1.329 1.305 1.328
LOD (μg/kg)
LOQ (μg/kg)
linear range (μg/kg)
R2
0.16 0.20 0.21 0.18 0.25 0.19 0.17 0.24 0.22
0.48 0.60 0.63 0.54 0.75 0.57 0.51 0.72 0.66
0.48−30.0 0.60−30.0 0.63−30.0 0.54−30.0 0.75−30.0 0.57−30.0 0.51−30.0 0.72−30.0 0.66−30.0
0.985 0.992 0.987 0.991 0.985 0.990 0.995 0.986 0.987
D
DOI: 10.1021/acs.jafc.5b03746 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 2. Recovery and CVs for the Eu-Nano-TRFIA Methoda intra-batch (n = 5)
a
inter-batch (n = 5)
sample
HPLC found aflatoxins (μg/kg)
av found (μg/kg)
av CV (%)
recovery (%)
av found (μg/kg)
av CV (%)
recovery (%)
corn wheat bran peanut meal soybean meal cottonseed meal DDGS alfalfa forage silage swine feed poultry feed
18.80 6.50 14.25 3.22 22.65 20.63 1.16 1.87 10.24 8.92
18.22 6.21 13.87 2.70 22.60 21.08 1.31 2.13 10.97 9.24
4.2 5.1 4.6 6.8 3.5 4.1 7.2 7.9 4.9 5.1
96.9 95.5 97.3 83.9 99.8 102.2 112.9 113.9 107.1 103.6
18.64 6.42 14.19 2.97 21.98 20.10 1.04 1.78 9.65 8.60
4.8 5.8 5.2 7.5 3.9 4.5 8.2 8.8 5.5 5.7
99.1 98.8 99.6 92.2 97.0 97.4 89.7 95.2 94.2 96.4
CV, coefficient of variation.
aflatoxin contamination.34 Because corn is the raw material for the preparation of DDGS, the DDGS sample was heavily contaminated with aflatoxins. These observations were in agreement with results reported by Paterson and Grazina.35,36 Higher concentrations of contaminants were found in feedstuffs than in feed products. This Eu-Nano-TRFIA method can be widely applied as a rapid, ultrasensitive determination method for aflatoxins in feed samples. Figure 4. Cross-reactivities with AFB1, AFB2 AFG1, AFG2, ZEN, OTA, DON, and T2 at the concentration of 30.0 μg/kg in feed sample.
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Table 3. Comparison of the Eu-Nano-TRFIA and HPLC Methods for Aflatoxins in Feed Samplesa
*(ZW.Z.) Phone: +86 27 86812943. Fax: +86 27 86812862; Email: [email protected]. *(PW.L.) E-mail: [email protected].
Corresponding Authors
Funding
aflatoxins found (μg/kg) sample
HPLC
Eu-Nano-TRFIA
RSD (%)
recovery (%)
corn wheat bran peanut meal soybean meal cottonseed meal DDGS alfalfa forage silage swine feed poultry feed
15.31 8.76 11.64 3.24 2.78 0.64 nd nd 19.57 3.64
15.13 9.30 10.94 2.66 2.46 0.56 nd nd 19.11 4.20
4.1 6.3 6.0 7.5 6.9 7.9
98.8 106.2 94.0 82.1 88.5 87.5
3.8 6.1
97.6 115.4
6.1
96.3
av a
AUTHOR INFORMATION
This project was supported by the Special Fund for Agroscientific Research in the Public Interest (201203094-4), Special Fund for Grain-scientific Research in the Public Interest (201513006-02), and National Natural Science Foundation of China (31171702, 31401601). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Thevenot, D. R.; Toth, K.; Durst, R. A.; Wilson, G. S. Electrochemical biosensors: recommended definitions and classification. Biosens. Bioelectron. 2001, 16, 121−131. (2) Loock, H. P.; Wentzell, P. D. Detection limits of chemical sensors: applications and misapplications. Sens. Actuators, B 2012, 173, 157−163.
nd, not detected.
Table 4. Sample Detection Data Distribution by the Eu-Nano-TRFIA Methoda
a
sample
N
av (μg/kg)
scope (μg/kg)
detection rate (%)
50 μg/kg (%)
corn wheat bran peanut meal soybean meal cottonseed meal DDGS alfalfa forage silage swine feed poultry feed
45 32 47 43 29 30 35 29 52 55
9.21 1.46 6.51 3.21 9.81 8.66 0.82 2.61 7.59 8.66
0.65−81.21 0.78−15.25 0.91−103.21 0.70−43.21 0.90−145.30 0.92−102.24 0.70−12.51 1.00−19.82 0.50−81.69 0.80−67.08
93.3 59.4 83.0 34.9 89.7 90.0 45.7 69.0 100.0 100.0
84.4 59.4 68.1 27.9 69.0 56.7 45.7 69.0 75.0 74.6
6.7 0.0 10.6 7.0 13.8 23.3 0.0 0.0 15.4 16.4
2.2 0.0 4.3 0.0 6.9 10.0 0.0 0.0 9.6 9.0
Detection rate is the sample contained total aflatoxin. E
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Journal of Agricultural and Food Chemistry (3) Li, X.; Li, P. W.; Zhang, Q.; Li, R.; Zhang, W.; Zhang, Z. W.; Ding, X. X.; Tang, X. Q. Multi-component immunochromatographic assay for simultaneous detection of aflatoxin B1, ochratoxin A and zearalenone in agro-food. Biosens. Bioelectron. 2013, 49, 426−432. (4) Salwa, A. A.; Anwer, W. Effect of naturally contaminated feed with aflatoxins on performance of laying hen and the carryover of aflatoxin B1 residues in table eggs. Pakistan J. Nutr. 2009, 8, 181−186. (5) Han, Z.; Ren, Y. P.; Zhu, J. F.; Cai, Z. X.; Chen, Y.; Luan, L. J. Multianalysis of 35 mycotoxins in traditional Chinese medicines by ultra-high-performance liquid chromatography-tandem mass spectrometry coupled with accelerated solvent extraction. J. Agric. Food Chem. 2012, 60, 8233−8247. (6) Zhang, D. H.; Li, P. W.; Zhang, Q.; Zhang, W.; Huang, Y. L.; Ding, X. X.; Jiang, J. Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure. Anal. Chim. Acta 2009, 636, 63−69. (7) Benedetti, S.; Iametti, S.; Bonomi, F.; Mannino, S. Head space sensor array for the detection of aflatoxin M1 in raw ewe’s milk. J. Food Prot. 2005, 68, 1089−1092. (8) van Herwaarden, A. E.; Schinkel, A. H. The function of breast cancer resistance protein in epithelial barriers, stem cells and milk secretion of drugs and xenotoxins. Trends Pharmacol. Sci. 2006, 27, 10−16. (9) Magliulo, M.; Mirasoli, M.; Simoni, P.; Lelli, R.; Portabti, O.; Roda, A. Development and validation of an ultrasensitive chemiluminescent enzyme immunoassay for aflatoxin M1 in milk. J. Agric. Food Chem. 2005, 53, 3300−3305. (10) Cortés, G.; Carvajal, M.; Méndez-Ramírez, I.; Avila-González, E.; Chilpa-Galván, N.; Castillo-Urueta, P.; Flores, C. M. Identification and quantification of aflatoxins and aflatoxicol from poultry feed and their recovery in poultry litter. Poult. Sci. 2010, 89, 993−1001. (11) Cui, S. J.; Chen, C. M.; Tong, G. Z. A simple and rapid immunochromatographic strip test for monitoring antibodies to H5 subtype avian influenza virus. J. Virol. Methods 2008, 152, 102−105. (12) Zhang, D. H.; Li, P. W.; Zhang, Q.; Zhang, W. Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts. Biosens. Bioelectron. 2011, 26, 2877−2882. (13) Wang, Z. H.; Li, H.; Li, C. L.; Yu, Q.; Shen, J. Z.; De Saeger, S. Development and application of a quantitative fluorescence-based immunochromatographic assay for fumonisin B1 in maize. J. Agric. Food Chem. 2014, 62, 6294−6298. (14) Shephard, G. S.; Berthiller, F.; Burdaspal, P.; Crews, C.; Jonker, M. A.; Krska, R.; MacDonald, S.; Malone, B.; Maragos, C.; Sabino, M.; Solfrizzo, M.; Egmond, H. P.; Whitaker, T. B. Developments in mycotoxin analysis: an update for 2008−2009. World Mycotoxin J. 2010, 3, 3−23. (15) Dzantiev, B. B.; Byzova, N. A.; Urusov, A. E.; Zherdev, A. V. Immunochromatographic methods in food analysis. TrAC, Trends Anal. Chem. 2014, 55, 81−93. (16) Anklam, E.; Stroka, J.; Boenke, A. Acceptance of analytical methods for implementation of EU legislation with a focus on mycotoxins. Food Control 2002, 13, 173−183. (17) Hemmilá, I.; Mukkala, V. M. Time-resolution in fluorometry: Technologies, labels, and applications in bioanalytical assays. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441−519. (18) Huang, B.; Tao, W.; Shi, J.; Tang, L.; Jin, J. Determination of ochratoxin A by polyclonal antibodies based sensitive time-resolved fluoroimmunoassay. Arch. Toxicol. 2006, 80, 481−485. (19) Talha, S. M.; Salminen, T.; Swaminathan, S.; Soukka, T.; Pettersson, K.; Khanna, N. A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2. J. Virol. Methods 2011, 173, 24−30. (20) Bunzli, J. C. G.; Piguet, C. Taking advantage of luminescent lanthanide ions. Chem. Soc. Rev. 2005, 34, 1048−1077. (21) Shen, J. Z.; Zhang, Z.; Yao, Y.; Shi, W. M. A monoclonal antibody-based time-resolved fluoroimmunoassay for chloramphenicol in shrimp and chicken muscle. Anal. Chim. Acta 2006, 575, 262−266.
(22) Huang, B.; Xiao, H. L.; Zhang, X. R.; Zhu, L.; Liu, H. Y.; Jin, J. Ultrasensitive detection of pepsinogen I and pepsinogen II by a timeresolved fluoroimmunoassay and its preliminary. Anal. Chim. Acta 2006, 571, 74−78. (23) Hakala, H.; Mukkala, V. M.; Sutela, T.; Hovinen, J. Synthesis and properties of nanosphere copolymerized with luminescent europium(III) chelates. Org. Biomol. Chem. 2006, 4, 1383−1386. (24) Hai, X. D.; Tan, M. Q.; Wang, G. L.; Yuan, J. L. Preparation and a time-resolved fluoroimmunoassay application of new europium fluorescent nanoparticles. Anal. Sci. 2004, 20, 245−246. (25) Liu, J.; Du, B.; Zhang, P.; Haleyurgirisetty, M.; Zhao, J.; Ragupathy, V.; Lee, S. Development of a microchip europium nanoparticle immunoassay for sensitive point-of-care HIV detection. Biosens. Bioelectron. 2014, 61, 177−183. (26) Zhang, Z. W.; Wang, D.; Li, J.; Zhang, Q.; Li, P. W. Monoclonal antibody europium conjugate-based lateral flow time-resolved fluoroimmunoassay for quantitative determination of T-2 toxin in cereals and feed. Anal. Methods 2015, 7, 2822−2829. (27) Sun, Y. N.; Hu, X. F.; Zhang, Y.; Yang, J. F.; Wang, F. Y.; Wang, Y. Development of an immunochromatographic strip test for the rapid detection of zearalenone in corn. J. Agric. Food Chem. 2014, 62, 11116−11121. (28) Yu, L.; Li, P. W.; Zhang, Q.; Zhang, W.; Ding, X. X. Graphene oxide: an adsorbent for the extraction and quantification of aflatoxins in peanuts by high-performance liquid chromatography. J. Chromatogr., A 2013, 1318, 27−34. (29) Harma, H.; Soukka, T.; Lovgren, T. Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostatespecific antigen. Clin. Chem. 2001, 47, 561−568. (30) Tang, X. Q.; Zhang, Z. W.; Li, P. W.; Zhang, Q.; Jiang, J. Sample-pretreatment-free based high sensitive determination of aflatoxin M1 in raw milk using a time-resolved fluorescent competitive immunochromatographic assay. RSC Adv. 2015, 5, 558−564. (31) Rossi, C. N.; Takabayashi, C. R.; Ono, M. A.; Saito, G. H. Immunoassay based on monoclonal antibody for aflatoxin detection in poultry feed. Food Chem. 2012, 132, 2211−2216. (32) Ren, Y. P.; Zhang, Y.; Shao, S. L.; Cai, Z. X.; Feng, L. Simultaneous determination of multi-component mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography tandem mass spectrometry. J. Chromatogr., A 2007, 1143, 48−64. (33) Klich, M. A. Environmental and developmental factors influencing aflatoxin production by Aspergillus f lavus and Aspergillus parasiticus. Mycoscience 2007, 48, 71−80. (34) Cotty, P. J.; Jaime-Garcia, R. Influences of climate on aflatoxin producing fungi and aflatoxin contamination. Int. J. Food Microbiol. 2007, 119, 109−115. (35) Juodeikiene, G.; Basinskiene, L.; Bartkiene, E.; Matusevicius, P. Chapter 8: Mycotoxin decontamination aspects in food, feed and renewables using fermentation processes. In Agricultural and Biological Sciences: Structure and Function of Food Engineering; INTECH: Rijeka, Croatia, 2012; pp 171−20410.5772/46184. (36) Paterson, R. R. M.; Lima, N. How will climate change affect mycotoxins in food? Food Res. Int. 2010, 43, 1902−1914.
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DOI: 10.1021/acs.jafc.5b03746 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Europium Nanospheres-Based Time-Resolved Fluorescence for Rapid and Ultrasensitive Determination of Total Aflatoxin in Feed Du Wang,†,§,# Zhaowei Zhang,*,†,# Peiwu Li,*,†,‡,§ Qi Zhang,†,§,# Xiaoxia Ding,†,§ and Wen Zhang†,⊥ †
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China Key Laboratory of Biology and Genetic Improvement of Oil Crops, §Laboratory of Quality & Safety Risk Assessment for Oilseed Products (Wuhan), #Key Laboratory of Detection for Mycotoxins, and ⊥Quality Inspection & Test Center for Oilseed Products, Ministry of Agriculture, Wuhan 430062, China
‡
ABSTRACT: Immunochromatographic (IC) assays are considered suitable diagnostic tools for the determination of mycotoxins. A europium nanospheres-based time-resolved fluorescence immunoassay (Eu-Nano-TRFIA), based on a monoclonal antibody and a portable TRFIA reader, was developed to determine total aflatoxin (including aflatoxins B1, B2, G1, and G2) levels in feed samples. Under optimized conditions, the Eu-Nano-TRFIA method detected total aflatoxin within 12 min. It showed good linearity (R2 > 0.985), LOD of 0.16 μg/kg, a wide dynamic range of 0.48−30.0 μg/kg, recovery rates of 83.9−113.9%, and coefficients of variation (CVs) of 3.5−8.8%. In the 397 samples from company and livestock farms throughout China, the detection rate was 78.3%, concentrations were 0.50−145.30 μg/kg, the highest total aflatoxin content was found in cottonseed meal, and corn was found to be the most commonly contaminated feed. This method could be a powerful alternative for the rapid and ultrasensitive determination of total aflatoxin in quality control and meet the required Chinese maximum residue limits. KEYWORDS: Eu-Nano-TRFIA, europium label, nanospheres, total aflatoxin, feed
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INTRODUCTION Aflatoxins (AF), including AFB1, AFB2, AFG1, and AFG2, are mainly produced by Aspergillus flavus and Aspergillus parasiticus (Figure 1).1 They are commonly present in the food supply
avoid human exposure to aflatoxins, rigorous regulations have been set up by various international government agencies to monitor aflatoxin levels in feed.4,10 A number of methods have been employed for the determination of aflatoxins. IC assays are new, rapid detection technologies with several advantages, including simple and rapid operation, immediate results, and low cost.11−13 Labeled immunoassays, including fluorescence, bioluminescence, chemiluminescence, organic dyes, colloidal gold, magnetic beads, etc.,14 are widely used to detect binding events in many biomedical and diagnostic analyses. Although several efforts have been made toward the determination of aflatoxins using a test strip rapid methods are commercially available,15 they are not ultrasensitive and cannot quantitatively determine aflatoxins. Therefore, a rapid, quantitative determination method for aflatoxins sensing is needed. The time-resolved fluoroimmunoassay (TRFIA) was introduced in the early 1980s. This method uses lanthanide and fluorescent marker complexes, which have rapidly developed to become a new milestone in the development of labeled immunoassays for clinical medicine.16−19 The lanthanide and fluorescent complexes (e.g., Eu3+, Tb3+) provide longer relaxation times and reduced background signals with nanoto microsecond-long fluorescence,20,21 but their shortcoming is that the luminescence is in low quantum yields in comparison with those of organic fluorescence dyes. Due to the complex nature of feed matrices,22 enhancing sensitivity by improving
Figure 1. Chemical structures of the studied aflatoxins B1, B2, G1, and G2.
chain, such as in production, storage, transport, and consumables.2,3 Aflatoxins are regarded as one of the most harmful contaminants in feed, due to their carcinogenic, hepatotoxic, teratogenic, and mutagenic adverse effects toward animals,4,5 and have been shown to be related to the hepatic and extrahepatic carcinogenesis of humans and livestock.6,7 The order of toxicity, AFB1 > AFG1 > AFB2 > AFG2, indicates that the terminal furan moiety of AFB1 is the critical point for determining the degree of biological activity of these mycotoxins.5 Lactating animals can also be infected by aflatoxins by consuming contaminated feed, whereby metabolites of aflatoxins M1 and M2 are then excreted in milk.8,9 To © XXXX American Chemical Society
Received: July 30, 2015 Revised: November 11, 2015 Accepted: November 12, 2015
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DOI: 10.1021/acs.jafc.5b03746 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Diagrammatic description for principle of competitive assay in test strip format. lamp served as the excitation source at 365 ± 10 nm, and signal acquisition was obtained at 613 ± 10 nm using a photomultiplier tube (PMT). The light source and PMT tube were set at a 120° angle. When the exciting light was irradiated on the strip test area, the fluorescence signal was recorded by the PMT after a 400 μs delay. Meanwhile, the fluorescence with short relaxation time decayed rapidly, reducing the background noise and achieving a high signal-tonoise ratio. The fluorescence signals on test line (T line) and control line (C line) peak were processed using data processing software for quantitative analysis. Preparation of Eu-Nanospheres. The Eu-nanospheres were developed via a modified method.26,27 The carboxylated polystyrene nanospheres were diluted with a 10.0 mL mixture of DI water/acetone (1:1, v/v) to a final density of 1 × 1014 nanospheres in 10.0 mL. Then, a solution containing EuCl3 (100 μL, 0.1 mol/L), stearoyl benzoyl methane (400 μL, 0.1 M), phenanthroline (100 μL), and trioctylphosphine oxide (300 μL) was added and stirred for 10 h at 60 °C and subsequently for 2 h at room temperature. The resulting solution underwent vacuum distillation to remove the organic solvents and was dialyzed for 5 days. The dialysate was collected and stored at 4 °C with 0.05% NaN3. Coupling Antibody−Aflatoxins to Eu-Nanospheres. Two hundred microliters of Eu-nanospheres was added to 800 μL of boric acid buffer solutions (pH 8.2) and ultrasonicated (30 kHz frequency, 20% amplitude, 0.6 intermittent frequencies) at 20 °C for 10 min. Then, 40 μL of EDC (15 mg/mL) was added and stirred for 15 min. The suspension was separated by centrifugation at 17000g for 10 min. The precipitate was resuspended in 1.0 mL of boric acid buffer (pH 8.2) by ultrasonication for 2 min. After 1.0 ng/μL monoclonal anti-aflatoxins antibody (15, 25, 35, 45, and 55 μL, respectively) was added, the mixed solution was shaken for 12 h before pelleting at 17000g for 10 min. The residue was resuspended in 1.0 mL of boric acid buffer (0.5% BSA). The reaction was continued for another 2 h under agitation at 20 °C, and the solution was centrifuged at 17000g, resuspended in 1 mL of boric acid buffer (pH 8.2), and stored at 4 °C for further experiment. Fabrication of IC Strip. The IC strip is a single-step test based on a competitive immunoassay format (Figure 2). An IC strip contained a sample pad, an NC membrane, and an absorbent pad. The AFB1−BSA and rabbit anti-mouse IgG antibody were coated on the NC membrane as a T line and a C line by using Bio Dot XYZ Platform at an appropriate jetting rate. The distance between the T line and the C line was 10 mm. The coated membrane was dried at 37 °C for 2 h. The concentrations of AFB1−BSA and rabbit anti-mouse IgG antibody were optimized by a checkerboard method. The sample pad, NC membrane, and absorbent pad were then assembled on a plastic scale board. The whole assembled scale board was divided into 4.5 mm × 70 mm strips with a guillotine cutter (CM 4000) and stored at 4 °C. Optimization of the Eu-Nano-TRFIA Method. An indirect competitive immunoassay was performed on the strip. The antiaflatoxins−mAb Eu-nanospheres were diluted by 50, 100, 200, 300, and 500 times with protective reagents (6.0% sucrose, 4.0% bovine serum albumin, and 1.0% mannitol in 0.05 mol/L PBS buffer at pH 7.4). A 150 μL dilution was introduced in glass penicillin bottles before lyophilization. The concentrations of AFB1−BSA and rabbit anti-
either fluorescence or detection power for the purpose of rapid on-site sensing is required. One major signal amplification method is to pack the luminescence into a nanospheres capsule, thus enhancing its fluorescence up to thousands of times. After doping, the Eu of nanospheric particles and COOH-activated Eu-nanospheres surfaces were conjugated with monoclonal antibody against aflatoxins, to form an immunoreagent. This immunoreagent was further employed in the determination of aflatoxins by an IC strip via a homemade portable TRFIA reader. Herein, a Eu-Nano-TRFIA method is described, combining the advantages of the long fluorescent lifetime properties of lanthanide elements (Eu), signal amplifications of nanospheres, and IC assays.23,24 This protocol could dramatically enhance the sensitivity and linear range of the immunoreagent and could be employed in complex feed matrices.
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MATERIALS AND METHODS
Reagents and Apparatus. Standard solutions of aflatoxins (AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), zearalenone (ZEN), T2 toxin (T2), deoxynivalenol (DON), AFB1-conjugated BSA (AFB1− BSA), and rabbit anti-mouse IgG were purchased from Sigma-Aldrich (Urbana, IL, USA). Mannitol, sucrose, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), bovine serum albumin (BSA), and Tween 20 were purchased from Roche Applied Science (Roche, Indianapolis, IN, USA). The MycoSep 226 solid phase extraction (SPE) matrix was purchased from Romer Laboratories (Romer, Tulln, Austria). Deionized water (DI) was prepared using a Milli-Q quality water system (Bedford, MA, USA). All other inorganic chemicals and organic solvents were of analytical reagent grade or better. Anti-aflatoxin mAb4F12 was produced in our laboratory and further purified with a protein G immunoaffinity column.6 The cross-reactivity with four major aflatoxins was 100% of AFB1, 90.1% of AFB2, 84.6% of AFG1, and 20.7% of AFG2. Carboxylated polystyrene nanospheres were obtained from Shanghai Uni Biotech Ltd. (Shanghai, China), and polystyrene nanospheres (190 ± 10 nm particle size, 170−200 μequiv/ g surface charge, 25−35.7 sq.A/grp surface area) were obtained. Nitrocellulose (NC) membranes were obtained from Millipore Corp. (Millipore, Bedford, MA, USA). A 3050 dispensing platform and a CM 4000 Cutter (Bio Dot, Irvine, CA, USA) were used to prepare test strips. The HPLC apparatus consisted of an Agilent 1100 HPLC system (Palo Alto, CA, USA) including an isocratic pump (Agilent G1310A) connected to an automatic sampler (Agilent G1313A) and a fluorescence detector (Agilent G1321A). Postcolumn derivatization was carried out with electrochemically generated bromine in Cobra cells (Coring System Diagnostics GmbH, Gernsheim, Germany) using a reaction tube of 340 × 0.5 mm i.d. PTFE to enhance the fluorescence intensity. The analytical column Symmetry Alltima C18 (150 × 4.6 mm i.d., 5 μm; Agilent), kept at 25 °C, was used for aflatoxin determination. This system was piloted by Chemstation 3D software. TRFIA Apparatus. The portable TRFIA reader was homemade, and used for the quantitative determination of aflatoxins. A mercury B
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Journal of Agricultural and Food Chemistry mouse IgG were used for the test and control lines, respectively, and prepared with serial dilutions of 0.1−1.0 mg/mL. In line dispensing, the dispensed rate was evaluated from 4 to 10 μL/s, whereas the moving rate of the horizontal platform was optimized as 100 mm/s. Concentrations of immunoreagent were similarly screened as a checkerboard titration in an ELISA. An aflatoxin standard solution of 0.25 μg/kg (AFB1, AFG1, AFB2, and AFG2, 0.0625 μg/kg) was prepared using a blank feed sample (aflatoxin-free as determined by HPLC). Inhibitory rate values were recorded to determine the optimum concentrations. Aflatoxin-free feed sample extracts spiked with aflatoxins (0, 0.5, 1.5, 4.5, 9.0, 13.5, 22.5, and 30.0 μg/kg) were presented to the test strip to estimate the sensitivity and linear range of the method. A homemade TRFIA reader was used to measure the T line and C line peak areas. A calibration curve was fitted and expressed as T/C versus the natural logarithm of the aflatoxins concentration:
average light density (OD value) of a 10000 times diluted Eunanospheres sample was 0.356 at 340 nm. According to a multifunctional microplate reader, the average lifetime was 740 μs. To estimate the loading capacity of Eu on the nanospheres, the fluorescent intensities from Eu alone and Eu-nanospheres were measured.29 According to the linear relationship between Eu and the Eu-nanospheres, the Eu-nanospheres contained 150,000−160,000 Eu particles. Optimization of Preparation of Eu-Nanospheres Antibody−Aflatoxins. Carboxylated Eu-nanospheres served as carriers for the conjugation of mAbs against aflatoxins. Typical EDC conjugation methods were used because mild conditions maintained the antibody activity. After optimization, 40 μL (15 mg/mL) of EDC was used to activate a 1.0 mL Eunanospheres solution (containing ∼1012 particles). The mAb was then conjugated onto the Eu-nanospheres surfaces. After optimization, a 40 ng mAb against aflatoxins was conjugated with 1.0 mL of Eu-nanospheres, the solution was added to the strip, and the strip had the strongest fluorescence intensity at the same dilution. It was observed that with a lower density of mAb on the Eu-nanospheres, the coefficients of variation (CVs) of the T line increased; however, a higher mAb density could decrease the sensitivity. To obtain a minimum required dosage of Eu-nanospheres antibody and a clear T line, serial concentrations of 50, 100, 200, 300, and 500 times diluted 1.0 mL were used. The 200 times dilution was selected for further experimentation. Optimization of the IC Strip. Before fabricating the IC strip, nonspecific protein adsorption was prevented by using a blocking buffer on the sample pad. An optimized blocking buffer was used and contained 0.01 mol/L, pH 7.4, PBS, 1.0% BSA, 2.5% sucrose, 2.5% Tween 20, and 0.02% NaN3. The BSA prevented nonspecific protein adsorption, and sucrose aided the adsorption and desorption of immunoreagent for a more uniform chromatographic process; Tween 20 increased the specificity by maintaining an effective interaction between the mAb and the antigen, and NaN3 provided a preservation function. Using a checkerboard method, immunoreagent consumption was optimized at 7.5 μL/s of AFB1−BSA (0.5 mg/mL) and 6.0 μL/s of rabbit anti-mouse IgG (0.25 mg/mL) on the IC strip, whereas the moving rate was optimized as 100 mm/s, respectively. Optimization of Eu-Nano-TRFIA Method. Lyophilized Eu-nanospheres antibody−aflatoxins were pipetted into each glass penicillin bottle, and the extracting solution was then added. After mixing, an IC strip was inserted into the glass penicillin bottle and incubated at 37 °C for 8 min. For negative controls, the Eu-nanospheres antibody−aflatoxins present in the solution flowed laterally along the strip toward the T line, where the Eu-nanospheres antibody−aflatoxins are captured by the immobilized AFB1−BSA on the T line. Excess Eunanospheres antibody−aflatoxins flowed toward the C line and were captured by the rabbit anti-mouse IgG on the C line. The T line and C line were then inspected under UV exposure (Figure 3). For the positive feed sample, aflatoxins-conjugated Eu-nanospheres antibody−aflatoxins did not interact with the immobilized AFB1−BSA on the T line, but reached the C line, as indicated by a faint red color (i.e., a positive sample with 10.0 μg/kg of the aflatoxins) or no color (i.e., a positive sample with 30.0 μg/kg of the aflatoxins) on the T line under UV exposure (Figure 3). The ratio of the fluorescent intensities on the T line and C line was inversely proportional to the aflatoxins
Y (value T /C) = aX (ln Caflatoxins) + b Twenty blank samples were analyzed using standard curves to calculate the LOD according to previous reports25 with the homemade TRFIA reader. The LOQ was calculated as the 3 times the LOD. The recovery was studied to evaluate the accuracy of the Eu-NanoTRFIA method. Random aflatoxin concentration in samples was further determined by both Eu-Nano-TRFIA and HPLC methods, to calculate their recovery. Intra-batch repeatability was studied using five strips from one batch. Inter-batch accuracy was measured over five batches by calculating the mean value of five batches. The recovery was calibrated according to previous reports.26 Specificity was evaluated by investigating the cross-reactivity of the Eu-Nano-TRFIA method with mycotoxins in 30.0 μg/kg in blank feed sample, respectively. Sample Preparation for Eu-Nano-TRFIA Method. Feed samples were finely ground with a laboratory mill and sieved through a 20-mesh screen. A 20 g sample in 100 mL of 70% methanol (v/v) was homogenized for 2 min. Then, a 5 mL suspension was centrifuged at 8000g for 2 min. The extracted solutions were purified by MycoSep 226 SPE column, in which 1 mL of supernatant was diluted with 7 mL of 0.4% Tween 20 solution before measurement by the Eu-NanoTRFIA method. Comparison between Eu-Nano-TRFIA and HPLC Method. To validate the Eu-Nano-TRFIA method, the results between Eu-NanoTRFIA and HPLC methods were compared. Samples with random aflatoxin concentrations were compared. The HPLC method was according to the national standard.12 Extracts were filtered through double-layered filter paper. A 10.0 mL sample of the extract was diluted with 20.0 mL of water. The resulting solution was filtered via a 0.45 μm filter membrane before loading onto a homemade immunoaffinity chromatography column packed with anti-aflatoxins mAb. Aflatoxins were eluted using 1.0 mL of methanol and collected in a tube, followed by injection into an HPLC equipped with a fluorescence detector (λex = 360 nm, λem = 440 nm). Aflatoxin analysis was performed with an isocratic elution mode using a mixture of water/methanol (45:55, v/v) at a flow rate of 1.0 mL/min (run time of 18 min). The injection volume of suspended sample was 10.0 μL.28 Application in Real Feed Sample. A total of 397 feed samples were purchased from feed companies and livestock farms throughout China. The optimized method was used to analyze various feed samples, such as corn, wheat bran, soybean meal, peanut meal, cottonseed meal, distillers dried grains with soluble (DDGS), alfalfa forage, silage, and swine and poultry feed.
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RESULTS AND DISCUSSION Characterization of Eu-Nanospheres. The solid content of Eu-nanospheres was 0.99%, as determined by weighing blank nanospheres and Eu-nanosphere complexes. The morphology of the Eu-nanospheres was obtained by transmission electron microscopy. The Eu-nanospheres were uniform and had a welldistributed size, with an average diameter of 204 nm. The C
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(Figure 4), which showed that the Eu-Nano-TRFIA method has better specificity. Sample Preparation. Because aflatoxins were in trace amounts in feed, and there is an external restriction factor of uneven distribution and complex matrix nature of the feed, sample pretreatment must ensure representativeness and remove interference. To ensure the sample representativeness, the point-quartering method to take samples for extraction was applied. The extracted solutions were purified by MycoSep 226 SPE, by pushing the column into a test tube containing the sample extract, forcing the extract to filter upward through the packing material of the column. Interferences such as pigments, salt, and other proteins adhere to the chemical packing in the column, and the purified extract, containing the analytes of interest, passes through the column and is detected by the EuNano-TRFIA method.32 To improve the reaction efficiency of extracted solutions and Eu-Nano-TRFIA, 0.4% Tween 20 was used to dilute the extract solution. Tween 20 can increase the identification ability of EuNano-TRFIA, as well as reduce interactions between proteins. The results of comparison of the Eu-Nano-TRFIA and HPLC methods are shown in Table 3. The average recovery was 96.3%, ranging from 82.1 to 115.4%, with a mean CV of 6.1% (3.8−7.9%). Determination and Evaluation of Real Feed Samples. A total of 397 samples from feed companies and livestock farms were collected. These feed samples were categorized as feedstuffs and feed products. Feedstuffs included cottonseed meal, DDGS, corn, peanut meal, soybean meal, and wheat bran. Feed products included swine feed, poultry feed, alfalfa forage, and silage. As shown in Table 4, a total of 311 samples (78.3% of 397 samples) were found to contain aflatoxins having a concentration of 0.50−145.30 μg/kg. Furthermore, 254 samples (64.0% of the 397 samples) have aflatoxin concentrations 50 μg/kg. Aflatoxins were found in 93.3% of 45 corn samples, 59.4% of 32 wheat bran samples, 83.0% of 47 peanut meal samples, 34.9% of 43 soybean meal samples, 89.7% of 29 cottonseed meal samples, and 90.0% of 30 DDGS samples, respectively. The highest aflatoxin concentrations were found in cottonseed meal samples.33 Corn samples held the highest detection rates among the feedstuffs. Aflatoxin contamination was found in 45.7% of 45 alfalfa forage samples, 69.0% of 29 silage samples, 100% of 52 swine feed samples, and 100% of 55 poultry feed samples. Lower concentrations of aflatoxins in the alfalfa forage were likely due to higher moisture content, which may prevent
Figure 3. Gradient concentration of the aflatoxin-spiked feed sample under UV light.
concentration. On the basis of this relationship, the levels of aflatoxins were determined in the feed sample. To establish a standard curve, the aflatoxins-spiked feed sample was employed. By plotting the ratio of T line value to C line value versus the natural logarithm of the concentration (ln c), dependent on the different feed matrices, as seen in Table 1, a linear correlation was found between the T line and C line value ratio and the aflatoxins concentration. Figure 3 shows the gradient concentration of the aflatoxins-spiked feed sample under UV light. For the LOD evaluation, the mean value of five repeated experiments was recorded using 20 blank feed samples. On the basis of the calibration curve, the aflatoxin concentration was further calculated and defined as the LOD. The LOQ was calculated at 3 times the LODs. For different feed matrices, LODs were recorded from 0.16 to 0.25 μg/kg, whereas LOQs were found to be 0.48−0.75 μg/kg. The LOQs of a 0.5 μg/kg strip are shown in Figure 3. Linear wide dynamic ranges of 0.48−30.0 μg/kg were obtained, and a positive sample with 30.0 μg/kg aflatoxins showed no color on the T line.30 This established the Eu-Nano-TRFIA method had a 3 times lower LOD when compared with the ELISA method for broiler feed (1.25 μg/kg).31 For recovery, random feed samples were first measured using HPLC. Average recoveries for inter-batch reproducibility of 101.3% ranged from 83.9 to 113.9%, with a mean CV of 5.3% (3.5−7.9%). For inter-batch reproducibility, the test strips of five different batches were used for replicate testing of samples, with average recoveries of 96.0%, ranging from 89.7 to 99.6%, and mean CVs of 6.0% (3.9−8.8%). Results are shown in Table 2. This demonstrated that the Eu-Nano-TRFIA method was able to meet the regulatory standards in feed sample. To evaluate the method’s specificity, AFB1, AFB2, AFG1, AFG2, ZEN, OTA, DON, and T2 were tested for crossreactivity at spiked concentrations of 30.0 μg/kg in blank feed sample, respectively. Each toxin was measured by UV exposure Table 1. Standard Curves for Feed Sample Matrices sample corn wheat bran soybean meal peanut meal cottonseed meal DDGS alfalfa forage silage swine feed
standard curve Y Y Y Y Y Y Y Y Y
= = = = = = = = =
−0.341X −0.364X −0.347X −0.338X −0.403X −0.345X −0.338X −0.354X −0.391X
+ + + + + + + + +
1.264 1.291 1.211 1.173 1.414 1.287 1.329 1.305 1.328
LOD (μg/kg)
LOQ (μg/kg)
linear range (μg/kg)
R2
0.16 0.20 0.21 0.18 0.25 0.19 0.17 0.24 0.22
0.48 0.60 0.63 0.54 0.75 0.57 0.51 0.72 0.66
0.48−30.0 0.60−30.0 0.63−30.0 0.54−30.0 0.75−30.0 0.57−30.0 0.51−30.0 0.72−30.0 0.66−30.0
0.985 0.992 0.987 0.991 0.985 0.990 0.995 0.986 0.987
D
DOI: 10.1021/acs.jafc.5b03746 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 2. Recovery and CVs for the Eu-Nano-TRFIA Methoda intra-batch (n = 5)
a
inter-batch (n = 5)
sample
HPLC found aflatoxins (μg/kg)
av found (μg/kg)
av CV (%)
recovery (%)
av found (μg/kg)
av CV (%)
recovery (%)
corn wheat bran peanut meal soybean meal cottonseed meal DDGS alfalfa forage silage swine feed poultry feed
18.80 6.50 14.25 3.22 22.65 20.63 1.16 1.87 10.24 8.92
18.22 6.21 13.87 2.70 22.60 21.08 1.31 2.13 10.97 9.24
4.2 5.1 4.6 6.8 3.5 4.1 7.2 7.9 4.9 5.1
96.9 95.5 97.3 83.9 99.8 102.2 112.9 113.9 107.1 103.6
18.64 6.42 14.19 2.97 21.98 20.10 1.04 1.78 9.65 8.60
4.8 5.8 5.2 7.5 3.9 4.5 8.2 8.8 5.5 5.7
99.1 98.8 99.6 92.2 97.0 97.4 89.7 95.2 94.2 96.4
CV, coefficient of variation.
aflatoxin contamination.34 Because corn is the raw material for the preparation of DDGS, the DDGS sample was heavily contaminated with aflatoxins. These observations were in agreement with results reported by Paterson and Grazina.35,36 Higher concentrations of contaminants were found in feedstuffs than in feed products. This Eu-Nano-TRFIA method can be widely applied as a rapid, ultrasensitive determination method for aflatoxins in feed samples. Figure 4. Cross-reactivities with AFB1, AFB2 AFG1, AFG2, ZEN, OTA, DON, and T2 at the concentration of 30.0 μg/kg in feed sample.
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Table 3. Comparison of the Eu-Nano-TRFIA and HPLC Methods for Aflatoxins in Feed Samplesa
*(ZW.Z.) Phone: +86 27 86812943. Fax: +86 27 86812862; Email: [email protected]. *(PW.L.) E-mail: [email protected].
Corresponding Authors
Funding
aflatoxins found (μg/kg) sample
HPLC
Eu-Nano-TRFIA
RSD (%)
recovery (%)
corn wheat bran peanut meal soybean meal cottonseed meal DDGS alfalfa forage silage swine feed poultry feed
15.31 8.76 11.64 3.24 2.78 0.64 nd nd 19.57 3.64
15.13 9.30 10.94 2.66 2.46 0.56 nd nd 19.11 4.20
4.1 6.3 6.0 7.5 6.9 7.9
98.8 106.2 94.0 82.1 88.5 87.5
3.8 6.1
97.6 115.4
6.1
96.3
av a
AUTHOR INFORMATION
This project was supported by the Special Fund for Agroscientific Research in the Public Interest (201203094-4), Special Fund for Grain-scientific Research in the Public Interest (201513006-02), and National Natural Science Foundation of China (31171702, 31401601). Notes
The authors declare no competing financial interest.
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nd, not detected.
Table 4. Sample Detection Data Distribution by the Eu-Nano-TRFIA Methoda
a
sample
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45 32 47 43 29 30 35 29 52 55
9.21 1.46 6.51 3.21 9.81 8.66 0.82 2.61 7.59 8.66
0.65−81.21 0.78−15.25 0.91−103.21 0.70−43.21 0.90−145.30 0.92−102.24 0.70−12.51 1.00−19.82 0.50−81.69 0.80−67.08
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6.7 0.0 10.6 7.0 13.8 23.3 0.0 0.0 15.4 16.4
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