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Mar 3, 2016 - method has potential for on-site detection of total AFs in rice when using a ... desirable to develop antibody-free analytical methods t...
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Antibody-Free Colorimetric Detection of Total Aflatoxins in Rice Based on a Simple Two-Step Chromogenic Reaction Bibai Du,† Xiaoou Su,‡ Kunhao Yang,† Long Pan,† Qingju Liu,† Lingling Gong,† Peilong Wang,*,‡ Jingkui Yang,*,† and Yujian He*,†,§ †

School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China Key Laboratory of Agro-Product Safety and Quality, Ministry of Agriculture, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing 100081, China § State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China Downloaded via UNIV OF SUSSEX on August 10, 2018 at 07:44:09 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: The prevalently used immunoassays for fast screening of aftatoxins (AFs) usually cannot meet the requirement for simultaneous determination of total AFs (aflatoxin B1 + aflatoxin B2 + aflatoxin G1 + aflatoxin G2) due to the deficiency of highly group-specific antibodies. This paper describes a two-step chromogenic reaction based method to quantitatively detect total AFs in rice using colorimetric measurement without antibody. In the method, colorless AFs transform into greencolored indophenol products through the reaction with sodium hydroxide and 2,6dibromoquinone-4-chloroimide (DBQC) successively, allowing selectively determining total AFs up to 3.9 μg/kg over other competitive mycotoxins under optimal conditions by a UV−vis spectrophotometer. In addition, the colorimetric measurement results of the rice samples agree well with that of a standard HPLC method, demonstrating the good reliability and applicability of the method. Uniquely, the method has potential for on-site detection of total AFs in rice when using a nylon membrane-based device. flatoxins (AFs), a group of naturally occurring mycotoxins in food, are secondary metabolites of Aspergillus fungi and have been evaluated as Group 1 carcinogens by the International Agency for Research on Cancer.1 Up until now, more than 20 types of AFs have been identified, of which aflatoxin B1, B2, G1, and G2 are four major AFs existing in cereals and usually occur simultaneously under favorable environmental conditions with proportions of 1.0:0.1:0.3:0.03.2 The four types of AFs also have related chemical structures containing furofuran rings, lactone rings, a pentanone ring, and an aromatic six-membered ring moiety (Figure 1). In consideration of the harm of AFs to human health, many countries have set maximum admissible levels to control AFs contamination in food and feed. For example, action levels for AFs at 20 μg/kg have been set by the United States.3 To fulfill the above limitations, many researchers have focused on the development of analytical methods to detect AFs. Liquid chromatography with fluorescence detector (HPLC−FLD)4 or mass spectrometry (LC−MS/MS)5 have been regarded as the “gold standard” analytical methods for AFs due to their good accuracy and the capability to simultaneously detect total AFs in a single test. Nevertheless, chromatographic-based methods need expensive instruments and a time-consuming process for sample preparation and instrumental analysis, which are not suitable for testing in rural areas or screening a large number of samples.

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© 2016 American Chemical Society

Alternatively, various antibody-based immunoassay systems have been developed for the detection of AFs in recent decades, such as enzyme-linked immunosorbent assay (ELISA),6−8 chemiluminescent immunoassay,9 lateral flow immunoassay (LFA),10 fluorescence immunoassay,11 immunochip,12 membrane,13 and immunosensor.14−16 These methods can realize fast screening of a great quantity of samples and some of the methods can be even used for an on-site test. However, one shortcoming of immunoassays is the requirement of highquality AFs-specific antibodies, such as monoclonal antibody,6,8,10−15 polyclonal antibody,9,16 or recombinant antibody.7 Compared with chromatographic-based methods, antibody generation is also an expensive (e.g., $50 000−100 000 per ELISA assay17) and time-consuming (about 1 year17) process, coexisting with high batch-to-batch variability.18 Furthermore, antibodies are susceptible to degradation and denaturation toward extreme environmental conditions and external treatment,18 making the methods unreliable and difficult to standardize. Generally, immunoassays are highly specific for an individual aflatoxin,6−16 so another shortcoming of immunoassays is the incapability to meet the requirement to detect total AFs simultaneously, for AFs of more than one kind Received: December 13, 2015 Accepted: March 3, 2016 Published: March 3, 2016 3775

DOI: 10.1021/acs.analchem.5b04720 Anal. Chem. 2016, 88, 3775−3780

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Analytical Chemistry

Figure 1. Schematic representation of colorimetric detection of total AFs based on two-step chromogenic reaction. Step 1, alkaline ring-opening of lactone and step 2, Gibbs reaction using DBQC as the Gibbs reagent.

usually occur together2 and identification of total AFs in a single test can reduce costs and time. Hence, it is highly desirable to develop antibody-free analytical methods that can detect total AFs simultaneously company with the advantages of simplicity, rapidity, and stability. In recent years, some spectroscopy-based analytical methods have been reported for the detection of AFs, such as Raman spectroscopy,19 near-infrared spectroscopy,20 and a hyperspectral system,21 which are promising and excellent tools for screening of AFs contaminated samples due to their inherent rapidity, selectivity, and possibility to be computerized.19 However, there are few antibody-free methods for the detection of AFs based on ultraviolet−visible (UV−vis) spectrophotometry, which is the most available analytical method for chemical analysts. The main reason ascribes to the unsatisfied sensitivity and selectivity to detect AFs by UV−vis spectrophotometry when just utilizing inherent ultraviolet absorption of AFs. In this paper, we design a simple two-step chromogenic reaction, though which colorless AFs change into green-colored indophenols. On the basis of the reaction, we develop an antibody-free colorimetric method that allows the detection of total AFs simultaneously by a UV−vis spectrophotometer or even the naked eye. Our results can provide a new strategy for rapid detection of AFs.

Ultrapure water (18.2 MΩ cm) was prepared using a Milli-Q purification system (Millipore, U.S.). The individual standard solutions of the above mycotoxins and total AFs standard solution containing AFB1, AFB2, AFG1, and AFG2 with proportions of 1.0:0.1:0.3:0.032 were prepared in methanol at a concentration of 50 μg/mL, respectively. All needed working solutions were prepared by appropriate dilution. UV−vis absorption spectra were acquired on a UV-2550 spectrophotometer (Shimadzu, Japan). The chromatographic analysis of AFs was performed by a 1200 HPLC system (Agilent) equipped with a FLD detector. 13C NMR spectra were run on an Avance III spectrometer (Bruker, Germany) at 600 MHz. MALDI-TOF MS spectra were recorded by an Autoflex III instrument (Bruker, Germany). The photographs were taken by a DSC-W150 camera (Sony, Japan). Extraction and Preconcentration of AFs in Rice Samples by HLB Cartridge. Rice samples obtained from Chinese Academy of Inspection and Quarantine were ground and weighed (5 g) into a 150 mL conical flask, then extracted with 60 mL of methanol by shaking for 10 min at 200 rpm on a orbital shaker. All methanol extract were then filtered into a 200 mL pear-shaped flask and dried by vacuum rotary evaporation, and the residue was redissolved with 10 mL of methanol−water (4 + 6, v/v) by vortexing for 30 s at 2800 rpm on a vortex mixer. After that, all of the solutions were loaded into a HLB cartridge,5 which was previously conditioned with 5 mL of methanol and 5 mL of water in sequence. After washing with 5 mL of methanol−water (0.5 + 9.5, v/v), the AFs were eluted with 3 mL of methanol, and the eluate was collected in a test tube and evaporated to complete dryness under a nitrogen stream. Colorimetric Detection of AFs through Chromogenic Reaction. A volume of 170 μL of sodium hydroxide solution (0.01 M, dissolved in water) was pipetted into the above the completely dry test tube. After vortexing for 3 min, 30 μL of boric acid solution (0.1 M, dissolved in water) was added to adjust the pH of the solution to 9.24. Then, 5 μL of 2,6dibromoquinone-4-chloroimide solution (0.8 mg/mL, dissolved in methanol) was added to the mixed solution and the

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EXPERIMENTAL SECTION Materials and Instrumentation. Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), patulin (PAT), deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), fumonisin B1 (FB1), and T-2 toxin were purchased from Sigma (U.S.). 2,6-Dibromoquinone-4-chloroimide (DBQC, CAS No. 537-45-1) was bought from Tokyo Chemical Industry (Japan). The Oasis HLB cartridge (12 mL/200 mg) was purchased from Waters Corporation (U.S.). Sodium hydroxide was obtained from Xilong Chemical Co., Ltd. (Shantou, China). Boric acid and methanol (chromatographic grade) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 3776

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Analytical Chemistry solution turned green-colored after 5 min. At that time, the color of the solution was recorded by photograph and measured with UV−vis spectrophotometry.

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RESULTS AND DISCUSSION Mechanism of the Colorimetric Method. Figure 1 illustrates the proposed mechanism for colorimetric detection of total AFs based on a two-step chromogenic reaction. According to the previous reports,22,23 under the hydrolysis of alkali, the lactone ring of AFs will open to form substituted coumaric acids. In our study, AFs were previously extracted from rice samples and preconcentrated by a HLB cartridge, and then we designed the first step reaction where AFs (AFB1 + AFB2 + AFG1 + AFG2) react with sodium hydroxide to form corresponding substituted coumaric acids with the hydrolytic opening of the lactone rings. The structure-changing of AFs in the first step reaction was evidenced by UV−vis spectral conversion and 13C NMR analysis. As shown in Figure S1, the UV−vis spectra of AFB1 and its substituted coumaric acids can interconvert with alkalinization and acidification, which accords with the reaction rule of ring-opening of lactone.24 Similar results were obtained using AFG1 (Figure S2). The C2 and C12 signal of AFB1 in 13C NMR spectra were both shifted to the low field after reaction with sodium hydroxide with Δδ = 12.24 ppm and Δδ = 20.60 ppm, respectively (Figure S3), which further confirms the lactone ring is opened.25 In addition to having the similar changes like AFB1, the C4 signal of AFG1 in 13C NMR spectra is shifted to the high field, and C1 and C3 have the same chemical shifts (Figure S4). This shows that both the two lactone rings of AFG1 are opened. As the substituted coumaric acid is also a kind of substituted phenolate anion, which is able to react with Gibbs reagent to form indophenol.26 Consequently, we designed the generated substituted coumaric acids to react with Gibbs reagent (DBQC) as the second step reaction and green-colored indophenol products had been formed. MALDI-TOF MS spectrometry was used to identify the indophenol products, as shown in Figures S5 and S6, the peaks at m/z 614.15 and m/z 647.17 correspond to the indophenol products of AFB1 and AFG2, respectively. This indicates that the Gibbs reaction has occurred as designed. As can be seen from Figure 2, AFs solution is colorless and has no absorption band in the visible region (curve a). If AFs react only with sodium hydroxide or only mix with DBQC, no absorption band in the visible region appears as well (curves b and c). However, when AFs react with sodium hydroxide and DBQC successively, the mixed reaction solution demonstrates a green color with the absorption band at 673 nm (curve d), yet no absorption band at 673 nm can be found when sodium hydroxide only mix with DBQC without AFs (curve e). The above results indicate colorless AFs can transform into greencolored indophenol products through the reaction with sodium hydroxide and DBQC step by step. On the basis of this fact, we devised this colorimetric method based on two-step chromogenic reaction for detection of total AFs in rice. Optimization of Experimental Conditions. To obtain a highly sensitive response, it is necessary to optimize the conditions of the reactions. In the first step of the reaction, the concentration of sodium hydroxide and reaction time that are key factors for AFs to completely convert into substituted coumaric acids were optimized together. As shown in Figure 3a, the absorbance at 673 nm of the final generated indophenol products increased with the increasing concentration of sodium

Figure 2. UV−vis spectra of AFs (standard solution, 5 μg/mL) (a), mixed solution of AFs after reaction with only NaOH (b), only DBQC (c), and both NaOH and DBQC (d), mixed solution of NaOH and DBQC without AFs (e). Inset photographic images were the corresponding colorimetric response.

hydroxide and reaction time, but it remained unchanged with a further increase of the concentration and time to 0.02 mol/L and 3 min, respectively. The maximum response can be obtained when the concentration of sodium hydroxide was higher than 0.01 mol/L and the reaction time was more than 3 min. Considering saving time and getting the maximum response, 0.01 mol/L sodium hydroxide and reaction time of 3 min were selected as the optimal conditions in the first step reaction. The reaction conditions of the second step were further optimized. As pH is a key factor in the Gibbs reaction,26 experiments by adding different concentration of boric acid solution to adjust pH were performed to investigate the influence of pH on the reaction of substituted coumaric acids with DBQC. As illustrated in Figure 3b, the rate of the conversion of substituted coumaric acids to indophenols increased with the increase of pH. This is because it is easy for the hydrolysis of DBQC to yield monoamine in high pH, which results in undergoing the Gibbs reaction quickly.27 However, the absorbance at 673 nm decreased with time after reaching the maximum, indicating the resulting indophenol products were not stable. This is probably because the conformation of substituted coumaric acids needs to greatly change in order to be suitable for indophenols formation, and some energy will consume for the conformation conversion, resulting in the instability of the generated indophenol products.28 Moreover, the stability of the products varied significantly in different pH. The higher the pH was, the worse the stability of the indphenol products became, indicating that the pH should be adjusted as low as possible. Considering the reaction rate and the stability of the indophenol products, pH 9.24 was chosen as the optimal pH, where the indophenol products can be generated in 5 min and were stable for at least 5 min. The Gibbs reagent (DBQC) acts as one of the reactant in the second step reaction, its concentration will influence the generation of indophenol products. The effect of the concentration of DBQC on the second step reaction was further studied by measuring the absorbance of the resulting indophenol products at wavelength of 673 nm. As shown in Figure 3c, in the concentration range of DBQC at 0.2−0.8 mg/ 3777

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Figure 3. Optimization of experimental conditions. (a) The effect of concentration of sodium hydroxide and reaction time on the first-step reaction (other conditions, AFs standard solution 5 μg/mL, pH 9.24, 0.8 mg/mL DBQC, and second step reaction time 5 min); (b) effect of pH and reaction time on the second step reaction (other conditions, AFs standard solution 5 μg/mL, 0.01 M NaOH, first step reaction time 3 min 0.8 mg/mL DBQC); (c) effect of concentration of DBQC on the second reaction (other conditions, AFs standard solution 5 μg/mL, 0.01 M NaOH, first step reaction time 3 min, pH 9.24, second step reaction time 5 min).

regularly over the AFs concentration and a linear correlation with R2 = 0.9995 was obtained (Figure 4b). The detection limit for AFs standard solutions was calculated to be 0.092 μg/mL (3σ). The above results acquired from AFs standard solutions indicate that the colorimetric method has potential applications in the detection of total AFs in real samples. Rice is a vital foodstuff in Asia, but aflatoxin contamination in rice has been reported in many countries.29−31 To investigate the attainable sensitivity of the established colorimetric method in rice samples, different concentration of AFs standard solutions were added into the aflatoxin-free rice samples (previously analyzed by HPLC−FLD32) to make the level of AFs in the samples range from 0 to 200 μg/kg. The spiked rice samples were then pretreated by the steps listed in Extraction and Preconcentration of AFs in Rice Samples by HLB Cartridge and underwent the same chromogenic reaction as AFs standard solutions. Figure 4c showed the A673 value and solution color changes of different concentration of AFs, which were similar to the above standard solutions. A linear detection range of 0−200 μg/kg with a detection limit of 3.9 μg/kg can be achieved (3σ) (Figure 4d).The detection limit is lower than the action levels of AFs in rice defined by U.S. (20 ppb) and China (10 ppb). As shown in Table 1, the sensitivity of this method may be not so good as the chromatographic-based methods (HPLC−FLD and LC−MS/MS) and some antibodybased immunoassays. However, the approach avoids the use of expensive apparatus required in chromatographic-based methods. More importantly, compared with immunoassays that rely on antibodies and are specific for an individual aflatoxin, the method has the capability to simultaneously detect total AFs in a single test without using expensive and instable antibody. These results imply that the colorimetric method is a promising method for the detection of AFs in rice. Selectivity of the Colorimetric Method. To evaluate the selectivity of the colorimetric method for AFs, other mycotoxins were tested with the colorimetric method like other previous reports.33,34 Rice samples spiked with AFs (AFB1, AFB2, AFG1, and AFG2) at the level of 3.9 μg/kg and other mycotoxins (ZEN, PAT, T-2 toxin, DON, NIV, FB1, and OTA) at the level of 50 μg/kg were carried out to undergo the chromogenic reaction after sample pretreatment, respectively. As shown in Figure 5, in the presence of four individual aflatoxin down to the detection limit (3.9 μg/kg), the solutions after reaction showed a light green color and an appreciable absorption at 673 nm can be observed, implying AFs transform

mL, the absorbance at 673 nm of indophenol products increased with the increase of DBQC. When the concentration was more than 0.8 mg/mL, the absorbance of indophenol products scarcely increased, but the absorbance of the blank sample where no AFs was added increased, indicating interference of reagent blank had occurred. Thus, the concentration of 0.8 mg/mL DBQC was used as a condition in the second step reaction. Sensitivity of the Colorimetric Method. To examine the feasibility of the colorimetric method for determining total AFs, a series of different concentration of AFs standard solutions which contained AFB1, AFB2, AFG1, and AFG2 with the natural occurred proportions of 1.0:0.1:0.3:0.032 were added to undergo the two-step chromogenic reaction. As shown in Figure 4a, the absorbance band at 673 nm increased systematically with the concentration of AFs increasing from 0 to 5 μg/mL. In addition, a gradual color change from colorless to green can also be observed (the inset portion of Figure 4a). The absorbance at 673 nm (A673) increased

Figure 4. (a) UV−vis spectra of indophenol products generated by different concentration of AFs standard solutions. Insert portion was the corresponding photographic images. (b) Linear dependence of A673 versus the concentration of AFs standard solutions. (c) UV−vis spectra of indophenol products generated by aflatoxin-free rice samples spiked with different concentration of AFs. Insert portion was the corresponding photographic images. (d) Linear dependence of A673 versus the concentration of AFs in rice samples. 3778

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Analytical Chemistry Table 1. Comparison of Different Analytical Methods for the Determination of AFs methods

linear range (ppb)

detection limit (ppb)

selectivity

use of antibody

ref

HPLC−FLD LC−MS/MS direct competitive ELISA indirect competitive ELISA indirect competitive ELISA chemiluminescent immunoassay lateral flow immunoassay fluorescence immunoassay immunochip membrane electrochemical immunosensor piezoelectric immunosensor optical immunosensor colorimetric method

0.15−6 2−100 0.1−10 0.117−5.676 not given 0.001−1 not given 0.02−1 0.04−1.69 not given 10−500 0.1−100 3.0−98.0 0−200

0.046 0.1 0.05 0.754 0.006 0.00025 5 0.017 0.01 20 2 0.01

AFB1, B2, G1, G2 AFB1, B2, G1, G2 AFB1 AFB1 AFM1 AFM1 AFB1 AFB1 AFB1 AFB1 AFB1 AFB1 AFB1 AFB1, B2, G1, G2

monoclonal antibody antibody-free monoclonal antibody recombinant antibody monoclonal antibody polyclonal antibody monoclonal antibody monoclonal antibody monoclonal antibody monoclonal antibody monoclonal antibody monoclonal antibody polyclonal antibody antibody-free

4 5 6 7 8 9 10 11 12 13 14 15 16 this study

3.9

Table 2. Results Obtained by Colorimetric Method and HPLC−FLD for Determining Total AFs in Rice Samples rice samples

colorimetric method (μg/kg ± SD, n = 3)

HPLC−FLD (μg/kg ± SD, n = 3)

1 2 3 4 5 6

34.5 ± 2.0 11.1 ± 0.7 24.0 ± 1.3 15.1 ± 0.4 not detectable 14.2 ± 0.8

32.1 ± 1.1 9.2 ± 0.5 23.2 ± 0.5 15.3 ± 0.7 1.2 ± 0.2 12.6 ± 0.3

measure), demonstrating the good applicability of the colorimetric method for monitoring AFs in rice samples. However, for more complicated matrixes (e.g., peanuts, formulated foods), better sample cleanup procedures may be needed to eliminate interference. Extending the approach to detect AFs in other agro-products will be a direction for future work. To further access the possibility to utilize the newly developed method for on-site application, we constructed and examined a membrane and paper-based platform to semiquantify total AFs in rice. Nylon membrane, nitrocellulose membrane, and filter paper were chosen as the support, 15 μL of reaction solution by aflatoxin-free rice sample and spiked rice sample undergoing the two-step chromogenic reactions were dripped onto the support. As shown in the Figure 6, for a negative rice sample (aflatoxin-free), no color spot can be observed on the membrane or filter paper. For a positive

Figure 5. Absorbance at 673 nm of the mixed solutions after aflatoxinfree rice samples spiked with AFs (each at 3.9 μg/kg) and other mycotoxins (each at 50 μg/kg) undergoing the two-step chromogenic reactions. Insert portion was the corresponding photographic images.

into indophenol products through the two-step chromogenic reaction. However, the other seven mycotoxins with the spiked level 10 times higher than AFs still elicited similar response to the blank sample, the solutions were almost colorless and A673 values were below 0.0032, revealing that they cannot undergo the chromogenic reaction to generate green-colored indophenol products. This is significant because other mycotoxins contain no lactone rings in their chemical structures, thus other mycotoxins cannot produce colored indophenols and will not interfere with the detection. Moreover, the response of the blank rice sample also indicates that the possible interfering substances in rice that may produce colored indophenols do not exist either. These above results demonstrate the colorimetric method has excellent selectivity for the detection of AFs in rice. Rice Samples Analysis and Potential Application for on-Site Test. To evaluate the applicability of the colorimetric method, a total of 20 rice samples obtained from Chinese Academy of Inspection and Quarantine were analyzed, and the results were compared with HPLC−FLD (the procedures were shown in the Supporting Information).32 The results showed that six rice samples were tested as positive. As listed in Table 2, the results of the established method were in agreement with that of confirmatory HPLC−FLD method (R2 = 0.9858, removing the sample which the colorimetric method cannot

Figure 6. Photographic images of membrane or paper-based device (20 mm × 45 mm) showing its performance for detecting total AFs (50 μg/kg) in rice using the two-step chromogenic reaction. 3779

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Analytical Chemistry sample (50 μg/kg), a obvious visible green-colored spot can be observed in the center of the nylon membrane, but a obscure light green-colored spot can be observed on the nitrocellulose membrane and filter paper, indicating nylon membrane-based device may have potential application for the on-site detection. Future research will investigate the specific procedures including extraction, separation, and the reaction on the membrane to make the nylon membrane-based device perform as a spot screening tool for the determination of total AFs in rice.

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CONCLUSION In conclusion, we have designed a two-step chromogenic reaction specific to AFs. Colorless AFs can change into greencolored indophenol products through the reaction, allowing the detection of AFs by a UV−vis spectrophotometer or even the naked eye. The experimental results showed that AFs can be determined at a concentration as low as 3.9 μg/kg in rice with good selectivity against other mycotoxins. Compared with conventional antibody-based immunoassays for AFs, the established method can simultaneously detect total AFs and avoided the use of antibody. In addition, the established method can be successfully applied to the determination of total AFs and can also be exploited in the fabrication of a nylon membrane-based device that has potential application for onsite detection of total AFs in rice.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04720. Supplementary method description, experimental spectra, and reference (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *Phone: +86 10 88256827. Fax: +86 10 88256092. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was sponsored by the National Natural Science Foundation of China (Grants 21272263 and 31201832), the State Key Laboratory of Natural and Biomimetic Drugs (Grant K20140204), the University of Chinese Academy of Sciences (Grant O8JT011J01), and the National S&T Pillar Project (Grant 2011BAD26B0405).

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REFERENCES

(1) International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; IARC: Lyon, France, 2002; Vol. 82. (2) Kensler, T. W.; Roebuck, B. D.; Wogan, G. N.; Groopman, J. D. Toxicol. Sci. 2011, 120, S28−S48. (3) U. S. Food and Drug Administration. Compliance Policy Guide Sec. 683.100, Action Levels for Aflatoxins in Animal Feeds, 1994. (4) Kabak, B. Food Control 2012, 28, 1−6. 3780

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