Biosensors and Bioelectronics 56 (2014) 340–344
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Short communication
Development of an ultrasensitive aptasensor for the detection of aflatoxin B1$ Xiaodong Guo a,b,c,1, Fang Wen a,c,1, Nan Zheng a,c,d,n, Qiujiang Luo b, Haiwei Wang a,c, Hui Wang a,c, Songli Li a,c, Jiaqi Wang a,c,d a Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China b College of Animal Science and Technology, Xinjiang Agricultural University, Urumchi 830000, PR China c Ministry of Agriculture—Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, PR China d State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 5 November 2013 Received in revised form 10 January 2014 Accepted 24 January 2014 Available online 2 February 2014
Contamination of feed and food by aflatoxin B1 (AFB1), one of the most toxic of the mycotoxins, is a global concern. To prevent food safety scares, and avoid subsequent economic losses due to the recall of contaminated items, methods for the rapid, sensitive and specific detection of AFB1 at trace levels are much in demand. In this work, a simple, ultrasensitive, and reliable aptasensor is described for the detection of AFB1. An AFB1 aptamer was used as a molecular recognition probe, while its complementary DNA played a role as a signal generator for amplification by real-time quantitative polymerase chain reaction (PCR). Under optimal conditions, a wide linear detection range (5.0 10 5 to 5.0 ng mL 1) was achieved, with a high sensitivity (limit of detection (LOD)¼ 25 fg mL 1). In addition, the proposed aptasensor exhibited excellent specificity for AFB1 compared with eight other mycotoxins, with no obvious Ct value change. This aptasensor can also be used in quantifying AFB1 levels in Chinese wildrye hay samples and infant rice cereal samples, demonstrating satisfactory recoveries in the range of 88– 127% and 94–119%, respectively. This detection technique has a significant potential for high-throughput, quantitative determination of mycotoxin levels in a large range of feeds and foods. & 2014 Elsevier B.V. All rights reserved.
Keywords: Aflatoxin B1 Aptasensor RT-qPCR Feed and food safety
1. Introduction Mycotoxin feed and food contamination is a cause of global concern because of the toxic effects of mycotoxins for animals and human (Jolly et al., 2007; Pattono et al., 2011; Williams et al., 2004). Aflatoxins, one of the most important mycotoxins, are toxic metabolites produced mainly by molds, including Aspergillus flavus and Aspergillus parasiticus. Of the several types of aflatoxins (B1, B2, G1, G2, M1, and M2), aflatoxin B1 (AFB1) is the most toxic, and has been designated as a primary carcinogenic compound by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) (Bakirci, 2001). AFB1 can appear in human foodstuffs through direct contamination of items such as cereals or nuts, under hot and humid conditions, or through indirect contamination from ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. n Corresponding author at: State Key Laboratory of Animal Nutrition, Ruminant Nutrition Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuan Ming Yuan West Road, Haidian District, Beijing 100193, PR China. Tel.: þ 86 106 281 5859; fax: þ 86 106 289 7587. E-mail address: [email protected] (N. Zheng). 1 The authors contributed equally.
0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2014.01.045
animals feeds leading to the presence of metabolites such as aflatoxin M1 in milk (Diaz and Espitia, 2006; Decastelli et al., 2007). In order to prevent food safety scares, and subsequent economic losses due to the recall of contaminated feeds and foods, many countries have set maximum safety levels for aflatoxins. The limits for AFB1 in different foodstuffs have been typically set between 0.05 and 20 ng mL 1 (Babu and Muriana, 2011). The low permissible limits, combined with the frequent occurrence and high toxicity of AFB1, require rapid, sensitive and specific analytical methods to quantify even trace levels. Methods have been developed for the confirmatory and quantitative determination of AFB1 based on thin layer chromatography (TLC) (Var et al., 2007), high-performance liquid chromatography (HPLC), and LC combined with mass spectrometry (MS) (Corcuera et al., 2011; Njumbe Ediage et al., 2011; Souheib Oueslatia et al., 2012). Meanwhile, rapid screening methods, such as enzyme-linked immune sorbent assay (ELISA), immunosensors, and surface plasmon resonance (SPR) techniques (Liu et al., 2006, 2013a; Jae Hong Parka et al., 2013; Piermarini et al., 2007) have also been developed for aflatoxin detection. While antibody-based rapid screening methods are widely used, problems with antibody stability during transport and storage mean that this technique is limited to highly controlled environments. Aptamers, with a similar function to antibodies, have the advantages of low cost, high stability, ease of modification, easy synthesis, without the requirement for live animals or cells, repeated use, and can be
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preserved for a long time. In addition, aptamers can be exponentially amplified as a template of real-time quantitative polymerase chain reaction (RT-qPCR) to greatly improve detection sensitivity (Kubista et al., 2006; Mestdagh et al., 2008; Schmittgen and Livak, 2008). Previously, the research into aptamer-based sensors for mycotoxins mainly focused on ochratoxin A (OTA) and fumonisin B1 (FB1) because of the limited usable aptamers available for mycotoxins (Hayat et al., 2013; Ma et al., 2012; McKeague et al., 2010; Sheng et al., 2011; Vidal et al., 2013; Zhang et al., 2012). Neoventures Biotechnology Inc. (Canada) has patented specific aptamers to AFB1 and zearalenone (Patent:PCT/CA2010/001292). To the best of our knowledge, aptamerbased sensors for AFB1 detection have not been previously reported. Here, a new biosensor for the detection of AFB1 is described, based on an aptamer specific to AFB1. The aptamer is employed as a molecular recognition probe, and this is coupled with real-time quantitative PCR amplification of complementary single-stranded DNA (ssDNA), which acts as a signal generator. The chemicallysynthesized aptamer is immobilized onto a PCR tube through biotin-streptavidin coupling and hybridized with the complementary ssDNA, forming double-stranded DNA (dsDNA) on the surface. In the presence of AFB1, the process of binding between AFB1 and the aptamer induces the release of the complementary ssDNA, resulting in the reduction of the amount of the PCR template. As a consequence, the change in the PCR amplification signal is related to the concentration of AFB1. The whole sensing procedure is accomplished in a single PCR tube. This aptasensor is rapid, low cost, easy to use, and has excellent sensitivity and stability for the detection of AFB1. To the best of our knowledge, this is the first time an aptamer-based sensor has been developed for the detection of AFB1 with 25 fg mL 1 measurable levels.
2. Materials and methods 2.1. Materials and reagents Aflatoxin B1 (AFB1) was purchased from the National Standard Reference Center (Beijing, China). Ochratoxin A (OTA), zearalenone (ZEN), α-zearalenol, and aflatoxin M1 (AFM1) were purchased from Sigma-Aldrich (USA). Aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and fumonisin (FB1) were purchased from Pribolab Co. Ltd (Singapore). Streptavidin was obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). Other chemicals such as sodium chloride (NaCl), potassium chloride (KCl), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), anhydrous calcium chloride (CaCl2), sodium carbonate (Na2CO3), ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate (NaHCO3), and sodium citrate (C6H5Na3O7) were purchased from the Shanghai Chemical Reagent Company (Shanghai, China). Water was purified using a Milli-Q purification system. The SYBRs Premix Ex Taq™ II [includes 2X SYBRs Premix Ex Taqs(2 ) (SYBRs Premix Ex Taq™ II (Perfect Real Time)) and ROX Reference Dye II(50 )] were obtained from Takara Bio Co. Ltd. (Dalian, China). The specific aptamer, with 3'-terminal biotin groups and a complementary DNA fragment, were chemically synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) and purified by HPLC. Their sequences are as follows: AFB1 Aptamer (Patent:PCT/CA2010/001292): 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-biotin-3' Complementary DNA (AFB1 DNA): 5'-ACACGTGCCCAACAATCTGGTTTAGCTACGCCTTCCCCGTGGCGATGTTTCTTAGCGCCTTAC-3' Upstream primer: 5'-AATCTGGTTTAGCTACGCCTTC-3' Downstream primer: 5'-GTAAGGCGCTAAGAAACATCG-3'
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2.2. Immobilization of aptamer The immobilization of the aptamer was carried out according to Ma et al. (2012), with some modifications. In order to improve adsorbability, PCR tubes were treated with 50 μL 0.8% glutaraldehyde solution at 37 1C for 5 h before use. After washing three times with ultrapure water, 50 μL of streptavidin dissolved in 0.01 M carbonate buffer solution was added and incubated at 37 1C for 2 h. The tubes were then washed twice with phosphate buffer solution with tween20 (PBST) (10 mM PBS, pH 7.2, 0.05% Tween-20). The aptamer and its complementary DNA fragment were mixed intensively in a hybridization buffer (750 mM NaCl, 75 mM C6H5Na3O7, pH 8.0) in a 1:1 (v/v) ratio, and 50 μL of the mixture then added to each tube and incubated at 37 1C for about 1 h. In order to remove the uncombined DNA fragments, the tubes were subsequently washed three times with hybridization buffer, leaving the aptamer and modified DNA on the surface of the PCR tubes. 2.3. RT-qPCR assay for AFB1 detection The whole process of detection was finished in the modified PCR tubes, with 50 μL of AFB1 standard solution added and incubated with Tris buffer (10 mM Tris, 120 mM NaCl, 5 mM KCl, 20 mM CaCl2, pH 7.0) for 1 h at 45 1C. All tubes were washed three times with the Tris buffer to remove the uncombined AFB1 and the released complementary DNA. Finally, RT-qPCR was applied using the ABI 7500 Real-Time PCR System (USA). The 50 μL PCR mixture consisted of 2 μL of 10 μM upstream and downstream primer, respectively, 25 μL SYBRs Premix Ex Taqs(2 ), 1 μL of ROX Reference Dye II (50 ), and 20 μL water. The real-time PCR cycle parameters were as follows: initial denaturation for 30 s at 95 1C, followed by 40 cycles of denaturation for 5 s at 95 1C, and annealing for 34 s at 60 1C. Fluorescence measurements were taken after each annealing step. A melting curve analysis was performed from 60 1C to 95 1C to detect potential nonspecific products. The conditions were as follows: initial denaturation for 15 s at 95 1C, followed by 40 cycles of denaturation for 1 min at 60 1C, and annealing for 15 s at 95 1C. The reaction efficiency was calculated using the formula E¼ 10( 1/slope) 1 and estimated to reach the quantification requirements of RT-qPCR (Babu and Muriana, 2011). 2.4. Specificity analysis To evaluate the ability of this assay for highly selective detection of AFB1, eight critical mycotoxins (OTA, ZEN, α-ZOL, FB1, AFM1, AFB2, AFG1, and AFG2) were selected for testing. All of the mycotoxins were used at the same concentration (5 ng mL 1). All other detection conditions were identical to those used in the AFB1 procedure, allowing a comparison of the cycle numbers for these toxins. 2.5. Method validation The method developed was validated for the detection of AFB1 in Chinese wildrye hay samples and infant rice cereal samples. Chinese wildrye hay samples were contaminated with AFB1 at 5 10 5, 1 10 4, 0.01, and 0.1 ng mL 1 concentrations. Each sample was accurately weighed (0.5 g) after drying into 10 mL centrifuge tubes and 2.5 mL of 70% methanol in water then added to extract AFB1 from the sample. The entire mixture was vortexed for 5 min using a Vortex-Genie 2 (Scientific Industries, USA) and then centrifuged at 10,000g for 10 min. The supernatant was collected and concentrated to 0.5 mL under a nitrogen stream. Finally, each of the residues was re-dissolved in 2 mL of aqueous methanol solution (5%) and subjected to RT-qPCR. The
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pretreatment of the infant rice cereal samples, which were contaminated with AFB1 at concentrations of 5 10 4, 1 10 3, 0.005, and 0.01 ng mL 1, was similar to that used for the Chinese wildrye hay samples.
3. Results and discussion The schematic for the method of detection of AFB1 is shown in Fig. 1. The principle behind the sensing strategy is based on conformational change as the result of the formation of an AFB1/ aptamer complex and signal amplification by RT-qPCR. The biotinylated aptamer was initially immobilized on the surface of the streptavidin-coated PCR tubes as a result of the significantly strong specific binding of biotin-streptavidin, with a dissociation constant (KD) of 10 15 M (Waner and Mascotti, 2008). The complementary ssDNA was partly hybridized with the single-strand aptamer to form dsDNA on the surface of the PCR tubes. In the presence of AFB1, a structural switch of the aptamer was induced through the binding of the aptamer and AFB1. The complementary ssDNA was then released as a result of the formation of the AFB1/aptamer complex (Yang et al., 2012), leading to a reduction in the amount of the complementary ssDNA template for RT-qPCR amplification. Thus, the concentration of AFB1 was related to the change in the PCR amplification signal, which can be used for the quantification of the level of AFB1. As the complementary ssDNA forms the PCR template, the concentration of the complementary ssDNA, and specificity of the primer, are important in this assay and should be optimized. As shown in Fig. S1(A), the amplification curve indicated that the cycle number increased with decreasing concentration of the complementary ssDNA. Corresponding to the amplification curve (Fig. S1), the standard curve between the cycle threshold (Ct) and
Fig. 1. Schematic illustration of the aptasensor for detection of Aflatoxin B1.
the concentrations of the complementary ssDNA shows that the RT-qPCR assay performed sensitive and quantitative detection of the complementary ssDNA with a high amplification efficiency (98.2%). A good linear relationship was obtained, in the range of 1 10 4 to 10 nM, with a high correlation coefficient (R2 ¼ 0.996). The linear regression equation was described by Ct ¼ –3.3661 lg Cþ 38.127, where Ct is cycle threshold number and C is the concentration of AFB1 DNA. The optimal concentration of the complementary ssDNA was selected as 10 nM since the Ct values reached their lowest level at this concentration. The PCR melting curve, as shown in Fig. S2, indicated a clear single peak at 80 1C, demonstrating the amplification to be specific, without the appearance of primer dimers or other nonspecific DNA fragments. The concentrations of streptavidin and the biotinylated aptamer, as well as the complementary ssDNA, greatly affect the performance of this aptasensor. Under a fixed 10 nM concentration of complementary ssDNA, the concentrations of streptavidin and the biotinylated aptamer were optimized by analyzing the change in the PCR amplification signal. First, the adsorptive ability of the streptavidincoated tubes for the biotinylated aptamer was assessed by varying the concentrations of streptavidin. As Fig. S3 shows, an obvious difference in Ct values was obtained between the streptavidin-coated and unmodified tubes, demonstrating the strong adsorptive power of streptavidin-coated tubes for the biotinylated aptamer. Comparing Ct values at different concentrations of streptavidin, it was lowest when the concentration of streptavidin was 2.5 ng mL 1. When the concentrations of aptamer were below 5.0 nM, the Ct values decreased with the increase in the amount of aptamer. However, at aptamer levels above 5.0 nM, the Ct values increased with an increase in aptamer, which might be due to steric hindrance. Therefore, concentrations of 2.5 ng mL 1 of streptavidin and 5.0 nM of aptamer were selected as the optimal conditions for RT-qPCR assay. Under optimal conditions, the typical amplification curves of this aptasensor against different concentrations of AFB1 were determined using RT-qPCR (Fig. 2). The cycle number increased with increasing concentration of AFB1, as indicated in Fig. 2(A). The more AFB1 present, the more complementary ssDNA released, resulting in a decrease in the template levels, and an increase in Ct values. The calibration curve of Ct values versus AFB1 concentrations was linear over the range of 5 10 5 to 5 ng mL 1 (R2 ¼0.9932). The linear regression equation was described by Ct¼3.816 lg Cþ24.622, where Ct is cycle threshold number and C is the concentration of AFB1. The limit of detection of AFB1 (S/N¼ 3) was 25 fg mL 1, approximately 400 times lower than the methods previously reported. Compared with the currently available instrumental and rapid screening methods, the results in this work clearly indicate an excellent sensitivity for the detection of AFB1 (Table 1). In order to determine the specificity of this method, eight mycotoxins (OTA, ZEN, α-ZOL, FB1, AFM1, AFB2, AFG1, and AFG2) were selected as interferences. As indicated in Fig. 3, no obvious Ct value changes were detected at the concentration of 5 ng mL 1 for these eight mycotoxins separately, which was similar to the control result, without any mycotoxin present. Similar response was obtained toward the mixture of those eight mycotoxins altogether in the absence of AFB1. The corresponding Ct value of AFB1 in the presence of those eight mycotoxins was slightly lower than the Ct value of the only separated AFB1. The results indicated that the aptamer would not interact with other mycotoxins besides AFB1. However, the presence of the other mycotoxins might interfere the binding reaction between AFB1 and the aptamer, resulting in the detected concentration of AFB1 was lower. Our results suggest that this aptasensor has perfect specificity for the detection of AFB1 as the biotin-labeled aptamer did not recognize other interferences. It is important that the results obtained are repeatable and this was evaluated by measuring the Ct values of the same sample (5.0 10 4 ng mL 1 AFB1) seven times. A relative standard deviation
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30 25
Ct
20 15 10 5 0 control AFB1 OTA ZEA a -ZOL AFM1 AFB2 AFG1 AFG FB1 Mix1 Mix2 2
Fig. 3. The Ct values in the absence and presence of 5 ng mL 1 mycotoxins including AFB1, OTA, ZEA, α-ZOL, AFM1, AFB2, AFG1, AFG2, FB1, Mix1 (AFB1, OTA, ZEA, α-ZOL, AFM1, AFB2, AFG1, AFG2, and FB1) and Mix2 (OTA, ZEA, α-ZOL, AFM1, AFB2, AFG1, AFG2, and FB1). The experiment conditions are as following: complementary ssDNA 10 nM, aptamer 5 nM, and streptavidin 2.5 ng mL 1.
Table 2 Determination of AFB1 spiked into infant rice cereal and Chinese wildrye hay samples.
Fig. 2. (A) The amplification curves at different concentrations of AFB1 in the range of 5 10 5 to 5 ng mL 1, including the negative control without AFB1. (B) The standard curves between the AFB1 concentration and the Ct value in the range of 5 10 5 to 5 ng mL 1.
Table 1 Comparison of the sensitivity of currently available methods for the detection of AFB1. No. Method
LOD
Reference
2.5 ng mL 1 0.3 ng mL 1 5 ng mL 1
4
Immunochromatography assay Indirect competitive immunoassay Clean-up tandem immunoassay column Electrochemical immunosensor
5 6 7
Enzyme immunoassay Piezoelectric immunosensor Real time quantitative PCR
2 ng mL 1 0.01 ng mL 1 0.1 ng mL 1
8 9
UHPLC-FLD Surface plasmon resonance biosensors LC-ESI-MS/MS RT-qPCR based aptasensor
2 ng mL 1 0.94 ng mL 1
Xiulan et al. (2006) Sapsford et al. (2006) Goryacheva et al. (2007) Piermarini et al. (2007) Saha et al. (2007) Jin et al. (2009) Babu and Muriana (2011) Corcuera et al. (2011) Puiu et al. (2012)
1 2 3
10 11
0.03 ng mL 1
0.02 ng mL 1 Liu et al. (2013b) 25 fg mL 1 This work
(RSD) of 2.0% was obtained, as shown in Fig. S4, indicating a good repeatability of the measurements. To evaluate the practical applicability and accuracy of this method, it was validated for detection of AFB1 in Chinese wildrye hay samples and infant rice cereal samples. Chinese wildrye hay samples were spiked with AFB1 at 5 10 5, 1 10 4, 0.01, and 0.1 ng mL 1 concentrations, while infant rice cereal samples were spiked with AFB1 at 5 10 4, 1 10 3, 0.005 and 0.01 ng mL 1
Sample
Spiked concentration (pg mL 1)
Detected Recovery concentrations (%) a b 1 Mean 7 SD (pg mL )
Infant rice cereal
10.0 5.0 1.0 0.50 100.0 10.0 0.10 0.05
9.4 71.1 5.7 70.5 1.197 0.21 0.577 0.07 105.0 7 0.9 12.7 7 1.2 0.107 70.034 0.0447 0.009
Chinese wildrye hay
a b
94 114 119 114 105 127 107 88
The mean of three experiments. SD ¼ standard deviation.
levels. The recoveries were in the range of 88–127% and 94–119%, respectively (Table 2). The experimental results demonstrate that the ultrasensitive aptasensor can be used for rapid AFB1 detection in feeds and foods.
4. Conclusions A rapid and ultrasensitive sensing platform has been developed for the detection of AFB1. An aptamer of AFB1 was used as a molecular recognition probe, with its complementary DNA acting as a template for real-time quantitative PCR amplification to generate signals. The formation of the AFB1/aptamer complex leads to conformational change of the aptamer, resulting in release of the complementary DNA and an increase in Ct values. When AFB1 is added, under optimal conditions, the Ct values increase linearly over the range of 5 10 5 to 5 ng mL 1, with high sensitivity (LOD ¼25 fg mL 1). The aptasensor developed in this work shows excellent selectivity when tested against eight other mycotoxins. Satisfactory recoveries were obtained in the Chinese wildrye hay and infant rice cereal samples that were spiked with different concentrations of AFB1. Compared to the previously reported methods for AFB1 detection found in the literature, the present RT-qPCR-based aptasensor has the advantages of ultrahigh sensitivity, specificity, and relatively low cost, and is a highly
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promising approach for the high-throughput detection of trace levels of AFB1 in feeds and foods. However, the proposed method cannot be considered for in-field analysis since it requires pretreatment and the instrumentation is not portable but useful for standard laboratories tests. In the future, we would focus on developing rapid, portable and sensitive portable aptasensor for determination of mycotoxins. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 21305158), Special Fund for Agroscientific Research in the Public Interest (201403071), Project of risk assessment on raw milk, Modern Agro-Industry Technology Research System of the PR China (nycytx-04-01), and International Collaboration and Communication in Agriculture. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.01.045. References Babu, D., Muriana, P.M., 2011. J. Microbiol. Methods 86, 188. Bakirci, I., 2001. Food Control 12, 47. Corcuera, L.A., Ibanez-Vea, M., Vettorazzi, A., Gonzalez-Penas, E., Cerain, A.L., 2011. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879, 2733. Decastelli, L., Lai, J., Gramaglia, M., Monaco, A., Nachtmann, C., Oldano, F., Ruffier, M., Sezian, A., Bandirola, C., 2007. Food Control 18, 1263. Diaz, G.J., Espitia, E., 2006. Food Addit. Contam. 23, 811. Goryacheva, I.Y., De Saeger, S., Delmulle, B., Lobeau, M., Eremin, S.A., Barna-Vetró, I., Van Peteghem, C., 2007. Anal. Chim. Acta 590, 118. Hayat, A., Sassolas, A., Marty, J.L., Radi, A.E., 2013. Talanta 103, 14.
Jae Hong Parka, Y.-P.K., Kimc, In-Ho, Kod, Sungho, 2013. Food Control 36, 183. Jin, X., Jin, X., Chen, L., Jiang, J., Shen, G., Yu, R., 2009. Biosens. Bioelectron. 24, 2580. Jolly, P.E., Jiang, Y., Ellis, W.O., Awuah, R.T., Appawu, J., Nnedu, O., Stiles, J.K., Wang, J.S., Adjei, O., Jolly, C.M., 2007. J. Nutr. Environ. Med. 16, 242. Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, A., Jonák, J., Lind, K., Sindelka, R., Sjöback, R., Sjögreen, B., Strömbom, L., Ståhlberg, A., Zoric, N., 2006. Mol. Aspects Med. 27, 95. Liu, B.-H., Hsu, Y.-T., Lu, C.-C., Yu, F.-Y., 2013a. Food Control 30, 184. Liu, S., Qiu, F., Kong, W., Wei, J., Xiao, X., Yang, M., 2013b. Food Control 29, 156. Liu, Y., Qin, Z., Wu, X., Jiang, H., 2006. Biochem. Eng. J. 32, 211. Ma, W., Yin, H., Xu, L., Xu, Z., Kuang, H., Wang, L., Xu, C., 2012. Biosens. Bioelectron. 42, 545. McKeague, M., Bradley, C.R., De Girolamo, A., Visconti, A., Miller, J.D., Derosa, M.C., 2010. Int. J. Mol. Sci. 11, 4864. Mestdagh, P., Feys, T., Bernard, N., Guenther, S., Chen, C., Speleman, F., Vandesompele, J., 2008. Nucleic Acids Res. 36, e143. Njumbe Ediage, E., Diana Di Mavungu, J., Monbaliu, S., Van Peteghem, C., De Saeger, S., 2011. J. Agric. Food. Chem. 59, 5173. Patent:PCT/CA2010/001292,DNA Ligands for Aflatoxin and Zearalenone, NeoVentures Biotechnology Inch. Pattono, D., Gallo, P., Civera, T., 2011. Food Chem. 127, 374. Piermarini, S., Micheli, L., Ammida, N.H.S., Palleschi, G., Moscone, D., 2007. Biosens. Bioelectron. 22, 1434. Puiu, M., Istrate, O., Rotariu, L., Bala, C., 2012. Anal. Biochem. 421, 587. Saha, D., Acharya, D., Roy, D., Shrestha, D., Dhar, T.K., 2007. Anal. Chim. Acta 584, 343. Sapsford, K.E., Taitt, C.R., Fertig, S., Moore, M.H., Lassman, M.E., Maragos, C.M., Shriver-Lake, L.C., 2006. Biosens. Bioelectron. 21, 2298. Schmittgen, T.D., Livak, K.J., 2008. Nat. Protoc. 3, 1101. Sheng, L., Ren, J., Miao, Y., Wang, J., Wang, E., 2011. Biosens. Bioelectron. 26, 3494. Souheib Oueslatia, R.R.-G., Salma Lasrama, Antonia Garrido, Frenichb, José Luis, Martínez, Vidalb, 2012. Food Chem. Toxicol. 50, 2376. Var, I., Kabak, B., Gök, F., 2007. Food Control 18, 59. Vidal, J.C., Bonel, L., Ezquerra, A., Hernández, S., Bertolín, J.R., Cubel, C., Castillo, J.R., 2013. Biosens. Bioelectron. 49, 146. Waner, M.J., Mascotti, D.P., 2008. J. Biochem. Biophys. Methods 70, 873. Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., Aggarwal, D., 2004. Am. J. Clin. Nutr. 80, 1106. Xiulan, S., Xiaolian, Z., Jian, T., Xiaohong, G., Jun, Z., Chu, F.S., 2006. Food Control 17, 256. Yang, C., Lates, V., Prieto-Simon, B., Marty, J.L., Yang, X., 2012. Biosens. Bioelectron. 32, 208. Zhang, J., Zhang, X., Yang, G., Chen, J., Wang, S., 2012. Biosens. Bioelectron. 41, 704.
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Short communication
Development of an ultrasensitive aptasensor for the detection of aflatoxin B1$ Xiaodong Guo a,b,c,1, Fang Wen a,c,1, Nan Zheng a,c,d,n, Qiujiang Luo b, Haiwei Wang a,c, Hui Wang a,c, Songli Li a,c, Jiaqi Wang a,c,d a Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China b College of Animal Science and Technology, Xinjiang Agricultural University, Urumchi 830000, PR China c Ministry of Agriculture—Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, PR China d State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 5 November 2013 Received in revised form 10 January 2014 Accepted 24 January 2014 Available online 2 February 2014
Contamination of feed and food by aflatoxin B1 (AFB1), one of the most toxic of the mycotoxins, is a global concern. To prevent food safety scares, and avoid subsequent economic losses due to the recall of contaminated items, methods for the rapid, sensitive and specific detection of AFB1 at trace levels are much in demand. In this work, a simple, ultrasensitive, and reliable aptasensor is described for the detection of AFB1. An AFB1 aptamer was used as a molecular recognition probe, while its complementary DNA played a role as a signal generator for amplification by real-time quantitative polymerase chain reaction (PCR). Under optimal conditions, a wide linear detection range (5.0 10 5 to 5.0 ng mL 1) was achieved, with a high sensitivity (limit of detection (LOD)¼ 25 fg mL 1). In addition, the proposed aptasensor exhibited excellent specificity for AFB1 compared with eight other mycotoxins, with no obvious Ct value change. This aptasensor can also be used in quantifying AFB1 levels in Chinese wildrye hay samples and infant rice cereal samples, demonstrating satisfactory recoveries in the range of 88– 127% and 94–119%, respectively. This detection technique has a significant potential for high-throughput, quantitative determination of mycotoxin levels in a large range of feeds and foods. & 2014 Elsevier B.V. All rights reserved.
Keywords: Aflatoxin B1 Aptasensor RT-qPCR Feed and food safety
1. Introduction Mycotoxin feed and food contamination is a cause of global concern because of the toxic effects of mycotoxins for animals and human (Jolly et al., 2007; Pattono et al., 2011; Williams et al., 2004). Aflatoxins, one of the most important mycotoxins, are toxic metabolites produced mainly by molds, including Aspergillus flavus and Aspergillus parasiticus. Of the several types of aflatoxins (B1, B2, G1, G2, M1, and M2), aflatoxin B1 (AFB1) is the most toxic, and has been designated as a primary carcinogenic compound by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) (Bakirci, 2001). AFB1 can appear in human foodstuffs through direct contamination of items such as cereals or nuts, under hot and humid conditions, or through indirect contamination from ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. n Corresponding author at: State Key Laboratory of Animal Nutrition, Ruminant Nutrition Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuan Ming Yuan West Road, Haidian District, Beijing 100193, PR China. Tel.: þ 86 106 281 5859; fax: þ 86 106 289 7587. E-mail address: [email protected] (N. Zheng). 1 The authors contributed equally.
0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2014.01.045
animals feeds leading to the presence of metabolites such as aflatoxin M1 in milk (Diaz and Espitia, 2006; Decastelli et al., 2007). In order to prevent food safety scares, and subsequent economic losses due to the recall of contaminated feeds and foods, many countries have set maximum safety levels for aflatoxins. The limits for AFB1 in different foodstuffs have been typically set between 0.05 and 20 ng mL 1 (Babu and Muriana, 2011). The low permissible limits, combined with the frequent occurrence and high toxicity of AFB1, require rapid, sensitive and specific analytical methods to quantify even trace levels. Methods have been developed for the confirmatory and quantitative determination of AFB1 based on thin layer chromatography (TLC) (Var et al., 2007), high-performance liquid chromatography (HPLC), and LC combined with mass spectrometry (MS) (Corcuera et al., 2011; Njumbe Ediage et al., 2011; Souheib Oueslatia et al., 2012). Meanwhile, rapid screening methods, such as enzyme-linked immune sorbent assay (ELISA), immunosensors, and surface plasmon resonance (SPR) techniques (Liu et al., 2006, 2013a; Jae Hong Parka et al., 2013; Piermarini et al., 2007) have also been developed for aflatoxin detection. While antibody-based rapid screening methods are widely used, problems with antibody stability during transport and storage mean that this technique is limited to highly controlled environments. Aptamers, with a similar function to antibodies, have the advantages of low cost, high stability, ease of modification, easy synthesis, without the requirement for live animals or cells, repeated use, and can be
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preserved for a long time. In addition, aptamers can be exponentially amplified as a template of real-time quantitative polymerase chain reaction (RT-qPCR) to greatly improve detection sensitivity (Kubista et al., 2006; Mestdagh et al., 2008; Schmittgen and Livak, 2008). Previously, the research into aptamer-based sensors for mycotoxins mainly focused on ochratoxin A (OTA) and fumonisin B1 (FB1) because of the limited usable aptamers available for mycotoxins (Hayat et al., 2013; Ma et al., 2012; McKeague et al., 2010; Sheng et al., 2011; Vidal et al., 2013; Zhang et al., 2012). Neoventures Biotechnology Inc. (Canada) has patented specific aptamers to AFB1 and zearalenone (Patent:PCT/CA2010/001292). To the best of our knowledge, aptamerbased sensors for AFB1 detection have not been previously reported. Here, a new biosensor for the detection of AFB1 is described, based on an aptamer specific to AFB1. The aptamer is employed as a molecular recognition probe, and this is coupled with real-time quantitative PCR amplification of complementary single-stranded DNA (ssDNA), which acts as a signal generator. The chemicallysynthesized aptamer is immobilized onto a PCR tube through biotin-streptavidin coupling and hybridized with the complementary ssDNA, forming double-stranded DNA (dsDNA) on the surface. In the presence of AFB1, the process of binding between AFB1 and the aptamer induces the release of the complementary ssDNA, resulting in the reduction of the amount of the PCR template. As a consequence, the change in the PCR amplification signal is related to the concentration of AFB1. The whole sensing procedure is accomplished in a single PCR tube. This aptasensor is rapid, low cost, easy to use, and has excellent sensitivity and stability for the detection of AFB1. To the best of our knowledge, this is the first time an aptamer-based sensor has been developed for the detection of AFB1 with 25 fg mL 1 measurable levels.
2. Materials and methods 2.1. Materials and reagents Aflatoxin B1 (AFB1) was purchased from the National Standard Reference Center (Beijing, China). Ochratoxin A (OTA), zearalenone (ZEN), α-zearalenol, and aflatoxin M1 (AFM1) were purchased from Sigma-Aldrich (USA). Aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and fumonisin (FB1) were purchased from Pribolab Co. Ltd (Singapore). Streptavidin was obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). Other chemicals such as sodium chloride (NaCl), potassium chloride (KCl), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), anhydrous calcium chloride (CaCl2), sodium carbonate (Na2CO3), ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate (NaHCO3), and sodium citrate (C6H5Na3O7) were purchased from the Shanghai Chemical Reagent Company (Shanghai, China). Water was purified using a Milli-Q purification system. The SYBRs Premix Ex Taq™ II [includes 2X SYBRs Premix Ex Taqs(2 ) (SYBRs Premix Ex Taq™ II (Perfect Real Time)) and ROX Reference Dye II(50 )] were obtained from Takara Bio Co. Ltd. (Dalian, China). The specific aptamer, with 3'-terminal biotin groups and a complementary DNA fragment, were chemically synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) and purified by HPLC. Their sequences are as follows: AFB1 Aptamer (Patent:PCT/CA2010/001292): 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-biotin-3' Complementary DNA (AFB1 DNA): 5'-ACACGTGCCCAACAATCTGGTTTAGCTACGCCTTCCCCGTGGCGATGTTTCTTAGCGCCTTAC-3' Upstream primer: 5'-AATCTGGTTTAGCTACGCCTTC-3' Downstream primer: 5'-GTAAGGCGCTAAGAAACATCG-3'
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2.2. Immobilization of aptamer The immobilization of the aptamer was carried out according to Ma et al. (2012), with some modifications. In order to improve adsorbability, PCR tubes were treated with 50 μL 0.8% glutaraldehyde solution at 37 1C for 5 h before use. After washing three times with ultrapure water, 50 μL of streptavidin dissolved in 0.01 M carbonate buffer solution was added and incubated at 37 1C for 2 h. The tubes were then washed twice with phosphate buffer solution with tween20 (PBST) (10 mM PBS, pH 7.2, 0.05% Tween-20). The aptamer and its complementary DNA fragment were mixed intensively in a hybridization buffer (750 mM NaCl, 75 mM C6H5Na3O7, pH 8.0) in a 1:1 (v/v) ratio, and 50 μL of the mixture then added to each tube and incubated at 37 1C for about 1 h. In order to remove the uncombined DNA fragments, the tubes were subsequently washed three times with hybridization buffer, leaving the aptamer and modified DNA on the surface of the PCR tubes. 2.3. RT-qPCR assay for AFB1 detection The whole process of detection was finished in the modified PCR tubes, with 50 μL of AFB1 standard solution added and incubated with Tris buffer (10 mM Tris, 120 mM NaCl, 5 mM KCl, 20 mM CaCl2, pH 7.0) for 1 h at 45 1C. All tubes were washed three times with the Tris buffer to remove the uncombined AFB1 and the released complementary DNA. Finally, RT-qPCR was applied using the ABI 7500 Real-Time PCR System (USA). The 50 μL PCR mixture consisted of 2 μL of 10 μM upstream and downstream primer, respectively, 25 μL SYBRs Premix Ex Taqs(2 ), 1 μL of ROX Reference Dye II (50 ), and 20 μL water. The real-time PCR cycle parameters were as follows: initial denaturation for 30 s at 95 1C, followed by 40 cycles of denaturation for 5 s at 95 1C, and annealing for 34 s at 60 1C. Fluorescence measurements were taken after each annealing step. A melting curve analysis was performed from 60 1C to 95 1C to detect potential nonspecific products. The conditions were as follows: initial denaturation for 15 s at 95 1C, followed by 40 cycles of denaturation for 1 min at 60 1C, and annealing for 15 s at 95 1C. The reaction efficiency was calculated using the formula E¼ 10( 1/slope) 1 and estimated to reach the quantification requirements of RT-qPCR (Babu and Muriana, 2011). 2.4. Specificity analysis To evaluate the ability of this assay for highly selective detection of AFB1, eight critical mycotoxins (OTA, ZEN, α-ZOL, FB1, AFM1, AFB2, AFG1, and AFG2) were selected for testing. All of the mycotoxins were used at the same concentration (5 ng mL 1). All other detection conditions were identical to those used in the AFB1 procedure, allowing a comparison of the cycle numbers for these toxins. 2.5. Method validation The method developed was validated for the detection of AFB1 in Chinese wildrye hay samples and infant rice cereal samples. Chinese wildrye hay samples were contaminated with AFB1 at 5 10 5, 1 10 4, 0.01, and 0.1 ng mL 1 concentrations. Each sample was accurately weighed (0.5 g) after drying into 10 mL centrifuge tubes and 2.5 mL of 70% methanol in water then added to extract AFB1 from the sample. The entire mixture was vortexed for 5 min using a Vortex-Genie 2 (Scientific Industries, USA) and then centrifuged at 10,000g for 10 min. The supernatant was collected and concentrated to 0.5 mL under a nitrogen stream. Finally, each of the residues was re-dissolved in 2 mL of aqueous methanol solution (5%) and subjected to RT-qPCR. The
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pretreatment of the infant rice cereal samples, which were contaminated with AFB1 at concentrations of 5 10 4, 1 10 3, 0.005, and 0.01 ng mL 1, was similar to that used for the Chinese wildrye hay samples.
3. Results and discussion The schematic for the method of detection of AFB1 is shown in Fig. 1. The principle behind the sensing strategy is based on conformational change as the result of the formation of an AFB1/ aptamer complex and signal amplification by RT-qPCR. The biotinylated aptamer was initially immobilized on the surface of the streptavidin-coated PCR tubes as a result of the significantly strong specific binding of biotin-streptavidin, with a dissociation constant (KD) of 10 15 M (Waner and Mascotti, 2008). The complementary ssDNA was partly hybridized with the single-strand aptamer to form dsDNA on the surface of the PCR tubes. In the presence of AFB1, a structural switch of the aptamer was induced through the binding of the aptamer and AFB1. The complementary ssDNA was then released as a result of the formation of the AFB1/aptamer complex (Yang et al., 2012), leading to a reduction in the amount of the complementary ssDNA template for RT-qPCR amplification. Thus, the concentration of AFB1 was related to the change in the PCR amplification signal, which can be used for the quantification of the level of AFB1. As the complementary ssDNA forms the PCR template, the concentration of the complementary ssDNA, and specificity of the primer, are important in this assay and should be optimized. As shown in Fig. S1(A), the amplification curve indicated that the cycle number increased with decreasing concentration of the complementary ssDNA. Corresponding to the amplification curve (Fig. S1), the standard curve between the cycle threshold (Ct) and
Fig. 1. Schematic illustration of the aptasensor for detection of Aflatoxin B1.
the concentrations of the complementary ssDNA shows that the RT-qPCR assay performed sensitive and quantitative detection of the complementary ssDNA with a high amplification efficiency (98.2%). A good linear relationship was obtained, in the range of 1 10 4 to 10 nM, with a high correlation coefficient (R2 ¼ 0.996). The linear regression equation was described by Ct ¼ –3.3661 lg Cþ 38.127, where Ct is cycle threshold number and C is the concentration of AFB1 DNA. The optimal concentration of the complementary ssDNA was selected as 10 nM since the Ct values reached their lowest level at this concentration. The PCR melting curve, as shown in Fig. S2, indicated a clear single peak at 80 1C, demonstrating the amplification to be specific, without the appearance of primer dimers or other nonspecific DNA fragments. The concentrations of streptavidin and the biotinylated aptamer, as well as the complementary ssDNA, greatly affect the performance of this aptasensor. Under a fixed 10 nM concentration of complementary ssDNA, the concentrations of streptavidin and the biotinylated aptamer were optimized by analyzing the change in the PCR amplification signal. First, the adsorptive ability of the streptavidincoated tubes for the biotinylated aptamer was assessed by varying the concentrations of streptavidin. As Fig. S3 shows, an obvious difference in Ct values was obtained between the streptavidin-coated and unmodified tubes, demonstrating the strong adsorptive power of streptavidin-coated tubes for the biotinylated aptamer. Comparing Ct values at different concentrations of streptavidin, it was lowest when the concentration of streptavidin was 2.5 ng mL 1. When the concentrations of aptamer were below 5.0 nM, the Ct values decreased with the increase in the amount of aptamer. However, at aptamer levels above 5.0 nM, the Ct values increased with an increase in aptamer, which might be due to steric hindrance. Therefore, concentrations of 2.5 ng mL 1 of streptavidin and 5.0 nM of aptamer were selected as the optimal conditions for RT-qPCR assay. Under optimal conditions, the typical amplification curves of this aptasensor against different concentrations of AFB1 were determined using RT-qPCR (Fig. 2). The cycle number increased with increasing concentration of AFB1, as indicated in Fig. 2(A). The more AFB1 present, the more complementary ssDNA released, resulting in a decrease in the template levels, and an increase in Ct values. The calibration curve of Ct values versus AFB1 concentrations was linear over the range of 5 10 5 to 5 ng mL 1 (R2 ¼0.9932). The linear regression equation was described by Ct¼3.816 lg Cþ24.622, where Ct is cycle threshold number and C is the concentration of AFB1. The limit of detection of AFB1 (S/N¼ 3) was 25 fg mL 1, approximately 400 times lower than the methods previously reported. Compared with the currently available instrumental and rapid screening methods, the results in this work clearly indicate an excellent sensitivity for the detection of AFB1 (Table 1). In order to determine the specificity of this method, eight mycotoxins (OTA, ZEN, α-ZOL, FB1, AFM1, AFB2, AFG1, and AFG2) were selected as interferences. As indicated in Fig. 3, no obvious Ct value changes were detected at the concentration of 5 ng mL 1 for these eight mycotoxins separately, which was similar to the control result, without any mycotoxin present. Similar response was obtained toward the mixture of those eight mycotoxins altogether in the absence of AFB1. The corresponding Ct value of AFB1 in the presence of those eight mycotoxins was slightly lower than the Ct value of the only separated AFB1. The results indicated that the aptamer would not interact with other mycotoxins besides AFB1. However, the presence of the other mycotoxins might interfere the binding reaction between AFB1 and the aptamer, resulting in the detected concentration of AFB1 was lower. Our results suggest that this aptasensor has perfect specificity for the detection of AFB1 as the biotin-labeled aptamer did not recognize other interferences. It is important that the results obtained are repeatable and this was evaluated by measuring the Ct values of the same sample (5.0 10 4 ng mL 1 AFB1) seven times. A relative standard deviation
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30 25
Ct
20 15 10 5 0 control AFB1 OTA ZEA a -ZOL AFM1 AFB2 AFG1 AFG FB1 Mix1 Mix2 2
Fig. 3. The Ct values in the absence and presence of 5 ng mL 1 mycotoxins including AFB1, OTA, ZEA, α-ZOL, AFM1, AFB2, AFG1, AFG2, FB1, Mix1 (AFB1, OTA, ZEA, α-ZOL, AFM1, AFB2, AFG1, AFG2, and FB1) and Mix2 (OTA, ZEA, α-ZOL, AFM1, AFB2, AFG1, AFG2, and FB1). The experiment conditions are as following: complementary ssDNA 10 nM, aptamer 5 nM, and streptavidin 2.5 ng mL 1.
Table 2 Determination of AFB1 spiked into infant rice cereal and Chinese wildrye hay samples.
Fig. 2. (A) The amplification curves at different concentrations of AFB1 in the range of 5 10 5 to 5 ng mL 1, including the negative control without AFB1. (B) The standard curves between the AFB1 concentration and the Ct value in the range of 5 10 5 to 5 ng mL 1.
Table 1 Comparison of the sensitivity of currently available methods for the detection of AFB1. No. Method
LOD
Reference
2.5 ng mL 1 0.3 ng mL 1 5 ng mL 1
4
Immunochromatography assay Indirect competitive immunoassay Clean-up tandem immunoassay column Electrochemical immunosensor
5 6 7
Enzyme immunoassay Piezoelectric immunosensor Real time quantitative PCR
2 ng mL 1 0.01 ng mL 1 0.1 ng mL 1
8 9
UHPLC-FLD Surface plasmon resonance biosensors LC-ESI-MS/MS RT-qPCR based aptasensor
2 ng mL 1 0.94 ng mL 1
Xiulan et al. (2006) Sapsford et al. (2006) Goryacheva et al. (2007) Piermarini et al. (2007) Saha et al. (2007) Jin et al. (2009) Babu and Muriana (2011) Corcuera et al. (2011) Puiu et al. (2012)
1 2 3
10 11
0.03 ng mL 1
0.02 ng mL 1 Liu et al. (2013b) 25 fg mL 1 This work
(RSD) of 2.0% was obtained, as shown in Fig. S4, indicating a good repeatability of the measurements. To evaluate the practical applicability and accuracy of this method, it was validated for detection of AFB1 in Chinese wildrye hay samples and infant rice cereal samples. Chinese wildrye hay samples were spiked with AFB1 at 5 10 5, 1 10 4, 0.01, and 0.1 ng mL 1 concentrations, while infant rice cereal samples were spiked with AFB1 at 5 10 4, 1 10 3, 0.005 and 0.01 ng mL 1
Sample
Spiked concentration (pg mL 1)
Detected Recovery concentrations (%) a b 1 Mean 7 SD (pg mL )
Infant rice cereal
10.0 5.0 1.0 0.50 100.0 10.0 0.10 0.05
9.4 71.1 5.7 70.5 1.197 0.21 0.577 0.07 105.0 7 0.9 12.7 7 1.2 0.107 70.034 0.0447 0.009
Chinese wildrye hay
a b
94 114 119 114 105 127 107 88
The mean of three experiments. SD ¼ standard deviation.
levels. The recoveries were in the range of 88–127% and 94–119%, respectively (Table 2). The experimental results demonstrate that the ultrasensitive aptasensor can be used for rapid AFB1 detection in feeds and foods.
4. Conclusions A rapid and ultrasensitive sensing platform has been developed for the detection of AFB1. An aptamer of AFB1 was used as a molecular recognition probe, with its complementary DNA acting as a template for real-time quantitative PCR amplification to generate signals. The formation of the AFB1/aptamer complex leads to conformational change of the aptamer, resulting in release of the complementary DNA and an increase in Ct values. When AFB1 is added, under optimal conditions, the Ct values increase linearly over the range of 5 10 5 to 5 ng mL 1, with high sensitivity (LOD ¼25 fg mL 1). The aptasensor developed in this work shows excellent selectivity when tested against eight other mycotoxins. Satisfactory recoveries were obtained in the Chinese wildrye hay and infant rice cereal samples that were spiked with different concentrations of AFB1. Compared to the previously reported methods for AFB1 detection found in the literature, the present RT-qPCR-based aptasensor has the advantages of ultrahigh sensitivity, specificity, and relatively low cost, and is a highly
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promising approach for the high-throughput detection of trace levels of AFB1 in feeds and foods. However, the proposed method cannot be considered for in-field analysis since it requires pretreatment and the instrumentation is not portable but useful for standard laboratories tests. In the future, we would focus on developing rapid, portable and sensitive portable aptasensor for determination of mycotoxins. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 21305158), Special Fund for Agroscientific Research in the Public Interest (201403071), Project of risk assessment on raw milk, Modern Agro-Industry Technology Research System of the PR China (nycytx-04-01), and International Collaboration and Communication in Agriculture. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.01.045. References Babu, D., Muriana, P.M., 2011. J. Microbiol. Methods 86, 188. Bakirci, I., 2001. Food Control 12, 47. Corcuera, L.A., Ibanez-Vea, M., Vettorazzi, A., Gonzalez-Penas, E., Cerain, A.L., 2011. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879, 2733. Decastelli, L., Lai, J., Gramaglia, M., Monaco, A., Nachtmann, C., Oldano, F., Ruffier, M., Sezian, A., Bandirola, C., 2007. Food Control 18, 1263. Diaz, G.J., Espitia, E., 2006. Food Addit. Contam. 23, 811. Goryacheva, I.Y., De Saeger, S., Delmulle, B., Lobeau, M., Eremin, S.A., Barna-Vetró, I., Van Peteghem, C., 2007. Anal. Chim. Acta 590, 118. Hayat, A., Sassolas, A., Marty, J.L., Radi, A.E., 2013. Talanta 103, 14.
Jae Hong Parka, Y.-P.K., Kimc, In-Ho, Kod, Sungho, 2013. Food Control 36, 183. Jin, X., Jin, X., Chen, L., Jiang, J., Shen, G., Yu, R., 2009. Biosens. Bioelectron. 24, 2580. Jolly, P.E., Jiang, Y., Ellis, W.O., Awuah, R.T., Appawu, J., Nnedu, O., Stiles, J.K., Wang, J.S., Adjei, O., Jolly, C.M., 2007. J. Nutr. Environ. Med. 16, 242. Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, A., Jonák, J., Lind, K., Sindelka, R., Sjöback, R., Sjögreen, B., Strömbom, L., Ståhlberg, A., Zoric, N., 2006. Mol. Aspects Med. 27, 95. Liu, B.-H., Hsu, Y.-T., Lu, C.-C., Yu, F.-Y., 2013a. Food Control 30, 184. Liu, S., Qiu, F., Kong, W., Wei, J., Xiao, X., Yang, M., 2013b. Food Control 29, 156. Liu, Y., Qin, Z., Wu, X., Jiang, H., 2006. Biochem. Eng. J. 32, 211. Ma, W., Yin, H., Xu, L., Xu, Z., Kuang, H., Wang, L., Xu, C., 2012. Biosens. Bioelectron. 42, 545. McKeague, M., Bradley, C.R., De Girolamo, A., Visconti, A., Miller, J.D., Derosa, M.C., 2010. Int. J. Mol. Sci. 11, 4864. Mestdagh, P., Feys, T., Bernard, N., Guenther, S., Chen, C., Speleman, F., Vandesompele, J., 2008. Nucleic Acids Res. 36, e143. Njumbe Ediage, E., Diana Di Mavungu, J., Monbaliu, S., Van Peteghem, C., De Saeger, S., 2011. J. Agric. Food. Chem. 59, 5173. Patent:PCT/CA2010/001292,DNA Ligands for Aflatoxin and Zearalenone, NeoVentures Biotechnology Inch. Pattono, D., Gallo, P., Civera, T., 2011. Food Chem. 127, 374. Piermarini, S., Micheli, L., Ammida, N.H.S., Palleschi, G., Moscone, D., 2007. Biosens. Bioelectron. 22, 1434. Puiu, M., Istrate, O., Rotariu, L., Bala, C., 2012. Anal. Biochem. 421, 587. Saha, D., Acharya, D., Roy, D., Shrestha, D., Dhar, T.K., 2007. Anal. Chim. Acta 584, 343. Sapsford, K.E., Taitt, C.R., Fertig, S., Moore, M.H., Lassman, M.E., Maragos, C.M., Shriver-Lake, L.C., 2006. Biosens. Bioelectron. 21, 2298. Schmittgen, T.D., Livak, K.J., 2008. Nat. Protoc. 3, 1101. Sheng, L., Ren, J., Miao, Y., Wang, J., Wang, E., 2011. Biosens. Bioelectron. 26, 3494. Souheib Oueslatia, R.R.-G., Salma Lasrama, Antonia Garrido, Frenichb, José Luis, Martínez, Vidalb, 2012. Food Chem. Toxicol. 50, 2376. Var, I., Kabak, B., Gök, F., 2007. Food Control 18, 59. Vidal, J.C., Bonel, L., Ezquerra, A., Hernández, S., Bertolín, J.R., Cubel, C., Castillo, J.R., 2013. Biosens. Bioelectron. 49, 146. Waner, M.J., Mascotti, D.P., 2008. J. Biochem. Biophys. Methods 70, 873. Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., Aggarwal, D., 2004. Am. J. Clin. Nutr. 80, 1106. Xiulan, S., Xiaolian, Z., Jian, T., Xiaohong, G., Jun, Z., Chu, F.S., 2006. Food Control 17, 256. Yang, C., Lates, V., Prieto-Simon, B., Marty, J.L., Yang, X., 2012. Biosens. Bioelectron. 32, 208. Zhang, J., Zhang, X., Yang, G., Chen, J., Wang, S., 2012. Biosens. Bioelectron. 41, 704.
