Biosensors and Bioelectronics 62 (2014) 288–294 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1 Won-Bo Shim a,1, Min Jin Kim a,1, Hyoyoung Mun a, Min-Gon Kim a,b,n a b Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea Advanced Photonics Research Institute, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea art ic l e i nf o a b s t r a c t Article history: Received 25 March 2014 Received in revised form 10 June 2014 Accepted 30 June 2014 Available online 4 July 2014 A rapid and simple dipstick assay based on an aptamer has been developed for the determination of aflatoxin B1 (AFB1). The dipstick assay format was based on a competitive reaction of the biotin-modified aptamer specific to AFB1 between target and cy5-modified DNA probes. Streptavidin and anti-cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. After optimization, the limit of detection for the dipstick assay was 0.1 ng/ml AFB1 in buffer. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min. Aqueous methanol (20%) provided a good extraction efficiency, and the matrix influence from corn extracts was successfully reduced through 2-fold dilution. The results of AFB1 analysis for corn samples spiked with known concentration of AFB1 by the dipstick assay and ELISA showed good agreement. The cut-off value of the dipstick assay for corn samples was 0.3 ng/g AFB1. Therefore, the dipstick assay is first reported and considered as a rapid, simple, on-site and inexpensive screening tool for AFB1 determination in grains as well as a corn. & 2014 Elsevier B.V. All rights reserved. Keywords: Aflatoxin B1 Dipstick assay Aptamer On-site detection Corn 1. Introduction Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and A. parasiticus and the most predominant and toxic of mycotoxins. Since aflatoxins have been frequently detected in food and agricultural commodities, they cause significant health and economic problems in many countries (Bhatnagar et al., 2002; Blesa et al., 2003). Among the aflatoxins, aflatoxin B1 (AFB1) possesses the highest toxicity and is listed as group I carcinogens by the International Agency for Research on Cancer (IARC, 2002). AFB1 is generally detected in agricultural commodities, such as grains, peanuts, corn, and feedstuffs. In order to protect human health from exposure to AFB1, many countries have set regulation limits. For examples, permissible levels set by the European Union are 2 mg/kg for AFB1 and 4 mg/kg for total aflatoxins in groundnuts, nuts, dried fruits, and cereals (Commission Regulation, 2001), whereas Korean maximum levels is 10 mg/kg for AFB1 and 15 mg/kg for total aflatoxins in food, respectively (MFDS, 2014). n Corresponding author at: Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea. Tel.: þ 82 62 715 2874; fax: þ82 62 715 3419. E-mail address: mkim@gist.ac.kr (M.-G. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bios.2014.06.059 0956-5663/& 2014 Elsevier B.V. All rights reserved. Therefore, analytical methods for AFB1 are essential to prevent exposure to the mycotoxin. Various methods based on chromatographic method and immunoassay have been developed and practically used for the determination of AFB1 in real samples. Chromatographic methods including a high performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS) have been officially accepted for the quantification of AFB1 but are time-consuming, unsuitable for large numbers of samples, laborious, and not amenable to on-site detection (Shim et al., 2007). Immunoassays are simpler and easier-to-use methods compared with chromatographic methods and are being used increasingly for screening of AFB1 in food and agricultural commodities. Especially, enzyme-linked immunosorbent assay (ELISA) has significantly grown in the development of methods for AFB1. However, ELISA often requires long reaction times and involves multiple incubation and washing steps and its utilization has been confined to laboratories equipped with tools and special devices for analysis (Paek et al., 2000). Thus, these disadvantages make it difficult for on-site detection. On-site detection technology has received great attention in the development of analytical methods for mycotoxins determination as well as diagnosing human diseases and hazards in environmental and food samples (Wang et al., 2011a; Cella et al., 2010). Therefore, there are many studies that have been conducted for the development of rapid, simple, and easy-to-use detection methods to detect mycotoxins. Immunochromatographic and W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294 dipstick assays are representative on-site detection technologies and are based on a membrane containing detector and capture reagents. Both methods combine several benefits such as a userfriendly format, long-term stability, and cost-effectiveness. These properties make both methods attractive for on-site screening by untrained personnel (Lattanzio et al., 2012; Wang et al., 2011b). Immunochromatographic and dipstick assays for various mycotoxins such as AFB1, ochratoxin A, zearalenone, fumonisins, T-2 and HT-2 toxins, and deoxynivalenol have been well developed. However, the assays reported involve the use of antibodies specific to targets (Wang et al., 2011b) and the utilization of antibodies has many disadvantages in terms of the high cost of production (economically, labor-wise, and time involved) and their stability as they are easily denatured during the storage in a buffer solution (Huang et al., 2012). Aptamers have been considered as a good alternative to antibodies. Aptamers are single-stranded DNA or RNA that can specifically bind to a target molecule and offer significant advantages over antibodies as their production is less expensive and labor-intensive. Aptamers are easier to label with fluorescent dyes, enzymes, biotin, and DNA ligands (Jayasena, 1999), and to regenerate by heating (McKeague et al., 2010). An added advantage of using aptamers is the option of hybridization with complementary DNA and the deconstruction of hybridization once aptamers meet a target molecule (Chen et al., 2012). These advantages make aptamers attractive in the development of low-cost, reusable, and robust analytical methods. Recently, several aptamers which are specific to mycotoxins (McKeague et al., 2010; Cruz-Aguado and Penner, 2008) have been developed. Especially, ochratoxin A aptamer was firstly reported in 2008 and has been well investigated to develop various aptamer sensors. Membrane based chromatographic strip assay (lateral flow strip) with ochratoxin A aptamer was reported for on-site detection of ochratoxin A (Wang et al., 2011a, 2011b). In the method, two probes (complementary single strand DNAs) were immobilized on a membrane to hybridize with residue aptamers on gold nanoparticles. However, it is often difficult to hybridize an aptamer unbound with a target and a complementary single strand DNA used as a probe since a sample solution quickly passed the membrane within 10 min and this period was not enough to get hybridization form on the membrane. Wei et al. (2005) reported that 500 s as a minimum time to hybridize DNA on a microarray using a microfluidic device was required in liquid solution. Although lateral flow strip is simple and user-friendly format intended to detect the target analyte without the need for specialized and costly equipment, we considered that a lateral flow strip based on an aptamer may be limited with the hybridization of the aptamer and a complementary single strand DNA. On the other hand, a dipstick assay usually includes two steps, prereaction with a target molcule and a receptor (antibody and aptamer) and dipping a dipstick composed with an absorbent pad and a membrane treated with capture reagents that can bind the receptor. At the pre-reaction step, sufficient hybridization of the aptamer and complementary single strand DNA was formed for negative samples whereas interaction of the aptamer to the target analyte for positive samples are performed in liquid solution. When a dipstick is dipped into the mixtures, the complexes (target–aptamer and hybridized aptamer/complementary DNA) were trapped by the capture reagents immobilized on the membrane. However, to the best of our knowledge, no dipstick assay based on an aptamer is reported for mycotoxin and any analyte analysis. In this study, we first reported novel aptamer-based dipstick assay for the rapid, easy-to-perform, and on-site detection of AFB1. For this study, we used a biotin-modified aptamer specific to AFB1 and cyanine 5 (Cy5)-modified a single strand-DNA probe, which can hybridize with the aptamer, used as the fluorescent 289 reporter. Streptavidin and anti-cy5 antibody were treated as test and control zones, respectively. The aptamer-based dipstick assay was successfully optimized to detect AFB1 and applied to corn samples artificially contaminated with AFB1. 2. Materials and methods 2.1. Chemicals and reagents AFB1 and related mycotoxins (AFB2, AFG1, AFG2, ochratoxin A, zearalenone, citrinin, T-2 toxin, deoxynivalenol, and patulin), bovine serum albumin (BSA), and sodium chloride were purchased from Sigma (St. Louis, MO, USA). 96-Microwell plates (flat bottom) were purchased from Nunc (Roskilde, Denmark). An anti-cy5 antibody was obtained from abcam (Cambridge, UK). A nitrocellulose membrane and an absorbent pad were purchased from Millipore Co. (Bedford, MA, USA). Semirigid polyethylene sheets were obtained from a local market. All chemicals and organic solvents were reagent grade. Water used in all experiments was purified with a Purelab Option Water Purification System (ELGA, Marlow, UK). Phosphate buffer saline (0.1 M, pH 7.4), carbonate buffer (25 mM, pH 9.6), borate buffer (1 mM, pH 7.4), Tris–HCl (0.1 M, pH 7.4 and 8.8), MES buffer (25 mM, pH 5.0), acetate buffer (0.1 M, pH 5.0), and deionized water were tested as working buffers for the dilution of aptamers and DNA probes. Standards of mycotoxins were prepared by diluting stock solutions of each mycotoxin (1 mg/ml) in absolute methanol. The ChemiDocTM MP System (BioRad, Hercules, CA, USA) was used for the measurement of fluorescent intensities on a dipstick assay. 2.2. Aptamer and DNA probes Biotin-modified aptamer to AFB1 and cy5-modified DNA probes with different length (14 mer and 23 mer) were purchased from GenoTech Corp. (Daejeon, Korea). The sequences of the aptamer and DNA probes were as follows: Biotin-modified aptamer: 5′-biotin-AAA AAA AAA AGT TGG GCA CGT GTT GTC TCT CTG TGT CTC GTG CCC TTC GCT AGG CCC ACA -3′ Cy5-modified DNA probe 1 (14 mer): 5′-cy5-AAA TGT GGG CCT AGC GA-3′ Cy5-modified DNA probe 2 (23 mer): 5′-cy5-AAA TGT GGG CCT AGC GAA GGG CAC GA-3′ The sequences in italic type mean the real sequences of aptamer specific to AFB1 which was presented by NeoVenture Bitechnologyj Inc. (Canada) in 2012 (Patent:PCT/CA2010/001292). Ten adenines at 5′ end were used as a linker to modify biotin. Additionally, appropriate lengths of complementary single strand DNA to an aptamer are needed to form a swithching structures from aptamer/complementary DNA (a duplex DNA) to aptamer/ target complex (Nutiu and Li, 2003, 2005; Chen et al., 2012) Therefore, two DNA probes, DNA probes 1 and 2, were designed for the development of the dipstick assay. The sequences underlined on the DNA probes were portions which can hybridize with the aptamer. 2.3. Sample preparation Corn samples were purchased from local markets and tested by LC/MS to gain AFB1-free corn samples for the preparation of AFB1positive corn samples. For the AFB1 analysis by the dipstick assay, 1 g of corn samples was extracted with 3 ml of methanol/water (20:80, v/v) for 15 min at room temperature and then centrifuged at 3075g for 5 min at 4 °C. The supernatants were filtered through 290 W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294 a disposable syringe filter (0.45 μm and diluted 2-fold with 0.1 M Tris–HCl (pH 7.4) to minimize matrix and methanol influence. The diluted extracts were analyzed by the dipstick assay. 2.4. Development of the dipstick assay for AFB1 The format of the dipstick assay was based on an indirect competitive assay. AFB1 competes with a cy5-modified DNA probes to bind a biotin-modified aptamer specific to AFB1. Fig. 1 shows a construction scheme and the principle of the dipstick assay for AFB1. The dipstick assay was consisted of an absorbance pad and a nitrocellulose (NC) membrane. A nitrocellulose membrane was treated with streptavidin and anti-cy5 antibody for test and control lines, respectively and soaked into 1% BSA in order to prevent non-specific binding of the cy5-modified DNA probe on a residue surface of the NC membrane. The treated NC membrane was dried at 37 °C for 20 min. The pad and NC membrane were placed on a semirigid polyethylene sheets. The aptamer (0.1 mM), AFB1 standard solutions, and DNA probes (0.5 mM) were sequentially added into wells of microplate. The mixtures were incubated for 20 min at 37 °C, and a dipstick was placed into the wells containing the mixture. In case of the presence of AFB1, the biotin-modified aptamer firstly reacts to AFB1, and cy5-modified DNA probes cannot hybridize with the aptamer. The complex of biotin-modified aptamer and AFB1 was migrated to the NC membrane and trapped by streptavidin at the test line, and free cy5-modified DNA probes migrated up to anticy5 antibody on the control line. Therefore, one fluorescent dot was observed on the NC membrane by the ChemiDocTM MP System. Whereas, the biotin-modified aptamer and DNA-cy5 probes could be completely hybridized under the absence of AFB1 that caused the formation of double stranded DNA (hybridized biotin-aptamer/cy5-DNA probe). The formed double stranded DNA migrated up to the NC membrane and was captured by streptavidin at the test line, and residue free cy5-modified DNA probes was trapped by anti-cy5 antibody immobilized at control line. Consequently, two fluorescent dots on the membrane were observed. 2.5. Sensitivity and specificity of the dipstick assay The sensitivity and specificity of the dipstick assay were conducted by analyzing AFB1 standards (0, 0.1, 0.3, 1, 3, and 10 ng/ml) and other mycotoxin standards such as AFB2, AFG1, AFG2, ochratoxin A, zearalenone, citrinin, T-2 toxin, deoxynivalenol, and patulin at the concentration of 50 ng/ml. The performance of the dipstick assay was as described above. 2.6. Analysis of AFB1 in corn samples AFB1-positive corn samples to validate the aptamer-based dipstick assay were prepared by spiking known amounts of AFB1 at 1, 0.1, 0.5, 1, 5, and 10 ng/g to AFB1-free corn samples. The spiked samples were kept at room temperature for 4 h under dark condition to evaporate methanol used to prepare AFB1 standards. The corn samples (1 g) were extracted 3 ml of 20% methanol/water Fig. 1. Schematic illustration of the dipstick assay for the simple and rapid detection of AFB1. A construction of dipstick assay is shown on Upperpart. The procedures and results of the dipstick assay for negative (bottom left) and positive tests are presented on bottom of the schematic illustration. W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294 291 (v/v) for 15 min at room temperature and then centrifuged at 2906g for 5 min at 4 °C. Further steps (filtration and dilution) for the sample preparation were as previously described. The sample extracts were directly applied to the dipstick assay. The results were compared with those obtained by the enzyme-linked immuosorbent assay (ELISA). The ELISA analysis for corn samples spiked with AFB1 was performed according to that described previously (Kolosova et al., 2007). 3. Results and disscussion 3.1. Optimization of the dipstick assay for AFB1 The dipstick assay developed in this study was based on the competition reaction of the biotin-modified aptamer between AFB1 and cy5-modified DNA probes. In the dipstick assay, the biotin-modified aptamer and cy5-modified DNA probe were used as a detector and competitor to mycotoxin replacing the roles of antibodies and mycotoxin–protein conjugates respectively in traditional dipstick assays based on a competitive reaction for mycotoxin. In the general competitive immunoassay, appropriate amount of immunoreagents such as antibody and antigen or target is required to develop the sensitive detection method. In addition, reaction mode and incubation period are also key parameter for the development of sensitive immunoassays. For the optimization of the dipstick assay to AFB1, several experimental parameters such as, the length of DNA probe, amount of the biotin-modified aptamer and cy5-modified DNA probe, working buffer, and incubation step, time and temperature were investigated. One of aptamer properties is that an aptamer can bind target or can be hybridized with complementary DNA, and the length of complementary DNA may affect the interaction of aptamer and target (Nutiu and Li, 2003, 2005; Chen et al., 2012). For example, if the length of complementary DNA is the same with that of an aptamer, the aptamer tends to strongly hybridize with complementary DNA than a target, while too short length of the complementary DNA may be difficult to hybridize. In the present study, we synthesized two types of complementary DNA modified with cy5, 14 mer (DNA probe 1) and 23 mer (DNA probe 2) as DNA probes. Both DNA probes were tested to select optimal length of DNA probe producing expected performance on the dipstick assay explained in Fig. 1. As shown in Fig. 2, both DNA probes worked well on the dipstick system, but a decrease of fluorescence intensity performed with biotin-modified aptamer (1 mM), cy5-modified DNA probe 1 (1 mM) and AFB1 10 ng/ml was much higher compared to that with biotin-modified aptamer (1 mM), cy5-modified DNA Fig. 2. Comparison test with two cy5-modified DNA probes. The lengths of DNA probe 1 and probe 2 are 14 mer and 23 mer. Both DNA probes were linked with cy5-AAA at the 5′ end. The sequences of DNA probes 1 and 2 and biotin-modified aptamer are described in Section Materials and methods. Fig. 3. Selection of appropriate concentrations of biotin-modified aptamer (A) and cy5-modified DNA probe 1 (B) for the development of the aptamer-based dipstick assay for AFB1 determination. probe 2 (1 mM), and AFB1 10 ng/ml. Thus, cy5-modified DNA probe 1 was chosen for further experiments. In typical competitive immunoassay, increasing concentration of immunoreagents decreases the sensitivity of immunoassays for small molecules as well as mycotoxins. Therefore, the utilization of appropriate amount of immunoreagent is a key factor to develop a sensitive assay. In this study, various concentrations of biotinmodified aptamer (0.1. 0.5, 1, and 2 mM) and cy5-modified DNA probe l (0.1, 0.5, and 1 mM) were tested. For the determination of optimal amount of the biotin-modified aptamer, the different concentrations of aptamers were mixed with 0, 10, and 100 ng/ ml AFB1 and cy5-modified DNA probe 1 (1 mM) and incubated for 30 min at room temperature. The bottom of the dipstick assay treated with 0.5 mg streptavidin and 0.5 mg anti-cy5 antibody was placed into wells containing the mixture and kept for 15 min. Fig. 3A shows the results of dipstick assay performed with different concentrations of biotin-modified aptamer. Fluorescent intensities between AFB1 negative (0 ng/ml) and positives (10 and 100 ng/ml) did not differ on the dipstick assay performed with the aptamer at 2 mM, while the fluorescent on the dipstick assay with 0.1, 0.5, and 1 mM aptamer was on the decrease with the increase of AFB1 concentration. Among three concentrations (0.1, 0.5 and 1 mM) of the aptamer, the dipsticks with 0.1 mM aptamer resulted in the highest different of fluorescent between AFB1 negative and positive, so we selected 0.1 mM aptamer for further studies. Increasing the concentration of the aptamer decreased fluorescent intensities on the dipstick assay. The reason is that free biotinmodified aptamer can more faster migrate to streptavidin on the membrane than hybridized biotin-modified aptamer and cy5modified DNA probe 1 and first bind to streptavidin resulting in lower fluorescent intensities at test zone on the membrane. Three concentrations (0.1, 0.5, and 1 mM) of cy5-modified DNA probe 1 was also tested with the biotin-modified aptamer at 0.1 mM. As shown in Fig. 3B, increasing the concentration of the probe enhanced the intensities of fluorescent on dipstick assay as we expected. The dipstick assay performed with cy5-modified DNA probe of 0.5 mM showed the clear and high difference in fluorescent intensities between AFB1 negative (0 ng/ml) and positives (10 and 100 ng/ml). Consequentially, the concentrations of biotinmodified aptamer (0.1 mM) and cy5-modified DNA probe 1 (0.5 m M) were chosen as optimal amounts for further studies. In the development of analytical methods based on aptamer, buffers for the dilution of reagents are also an important parameter, since binding of target to aptamer depened on the presence 292 W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294 of cations, pH, and component of buffers (Wang et al., 2011a; CruzAguado and Penner, 2008). Therefore, several buffers such as phosphate buffer saline (PBS; 0.1 M, pH 7.4), carbonate buffer (CB; 25 mM, pH 9.6), borate buffer (BB; 1 mM, pH 7.4), Tris–HCl (0.1 M, pH 7.4 and 8.8), MES buffer (25 mM, pH 5.0), acetate buffer (AB; 0.1 M, pH 5.0), and deionized water were tested in this study as working buffers. CB, MES, and Tris–HCl (pH 8.8) buffer did not produce a fluorescent dot on the dipstick, three buffers (BB, AB, and deionized water) induced the absorption of the cy5-modified DNA probe 1 on the membrane of dipstick assay. PBS exhibited adequate fluorescent dots, but no difference in CL values between positive and negative samples. Only Tris–HCl buffer (pH 7.4) showed the best performance on the dipstick assay resulting in different fluorescent intensities for AFB1 negative and positive tests (Fig. S1). According to the above result, we recognized that pH of buffer is also an important factor to develop aptamer assay, since results from Tris–HCl buffers (pH 7.4 and 8.8) were completely different. Finally, competition modes (direct and indirect competitions) and period for the competitive reaction of biotin-modified aptamer to AFB1 or cy5-modified probe 1. The competition modes were investigated with the three reagents (the aptamer, DNA probe, and AFB1) mixed at a time (direct competition) and time lag (indirect competition). For the direct competition mode, three reagents were mixed together, incubated for 30 min, and subjected to the dipstick assay. For the indirect competition mode, the biotin-modified aptamer and AFB1 were mixed and incubated for 30 min. The cy5-modified DNA probe 1 was then added to the mixture of the aptamer and AFB1 and additionally incubated for 30 min at room temperature. The mixtures from direct and indirect competition modes were tested by the dipstick assay. As shown in Fig. S2A, the fluorescence at the test zone on the dipstick assay was gradually decreased with the increase of AFB1 concentration from the direct competition mode, while the indirect competition mode showed entire diminishment of fluorescence on at the test zone on the dipstick assays. Therefore, we chose the direct competition mode for the interaction of biotin-modified aptamer, AFB1, and cy5-modified probe 1 to develop the rapid and simple dipstick assay.The incubation period of the mixture of three reagents were tested. After mixing, the mixture was incubated from 10 to 30 min and tested by the dipstick assay. The mixture incubated for 10 min produced unstable fluorescence on the dipstick assay with the increase of AFB1 concentration, while the mixture incubated longer than 20 min exhibited the gradual decrease of fluorescent intensities at the test zone on dipstick assay with increasing the concentration of AFB1. Thus, we chose 20 min as an optimal incubation time for the mixture of three reagents (Fig. S2B). Final results of the dipstick assay was obtained at 10 min after the mixture was subjected to the dipstick assay. Consequentially, total time to get a result by the dipstick is 30 min. 3.2. Sensitivity and specificity of the dipstick assay The dipstick assay optimized in this study was evaluated by testing the sensitivity using AFB1 standard solutions at concentrations of 0, 0.1, 0.3 1, 3, and 10 ng/ml in methanol/Tris–HCl (10:90, v/v), and the specificity of the assay was studied using other mycotoxins at a concentration of 50 ng/ml. When mixed three reagents (biotin-modified aptamer, AFB1 or no AFB1, and cy5modified DNA probe 1), AFB1 was first bound to the biotinmodified aptamer resulting in free cy5-modified DNA probe 1, whereas no AFB1 resulted in sufficient hybridization of biotinmodified aptamer and cy5-modified DNA probe 1, which can be trapped by the streptavidin immobilized at test line on the dipstick assay and produce a fluorescent dot at the test zone on the dipstick assay. The sensitivity of the dipstick assay was shown Fig. 4. Sensitivity of the aptamer-based dipstick assay for AFB1 determination. (A) Results of dipstick assay performed with AFB1 standards and (B) calibration curve corresponding to AFB1 standard. in Fig. 4A. Increasing the concentration of AFB1 decreased the fluorescent intensities at the test line, produced by hybridized biotin-modified aptamer and cy5-modified DNA probe 1, on the dipstick assay. The fluorescent intensities from the test line were converted to numeric values to produce a typical standard curve (Fig. 4B). A brighter fluorescent intensity was obtained from the negative test, and fluorescent intensities gradually decreased with increasing the concentration of AFB1. The difference of fluorescent intensities between AFB1 negative (0 ng/ml) and AFB1 positive at 0.1 ng/ml was clear, and the fluorescent intensity from 0 ng/ml AFB1 was much brighter compared to that of 0.1 ng/ml AFB1. The fluorescent intensity almost disappeared at 10 ng/ml AFB1. The sensitivity of our aptamer based dipstick assay was similar to or better than antibody based dipstick assay (LOD: 0.1 ng/ml) and antibody based lateral flow strip assay (LOD: 0.5 ng/ml) reported previously (Shim et al., 2007; Liao and Li, 2010 respectively). In previous reports, different cross-reactivities to ocharatoxin B by the several methods used the same aptamer (or antibody) were often reported (Chen et al., 2012; Cruz-Aguado and Penner, 2008). On the other hand, in a previous study about comparative study of three immunoassays, fluorescence polorization immunoassay (FPIA), indirect and direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA) based on the same monoclonal antibody specific to parathion-methyl, different cross-reactivities to parathion-ethyl (36% from FPIA, 61% from IC-ELISA, 125% from DC-ELISA) were observed (Kolovsova et al., 2004). These indicated that cross-reactivity of analytical methods based on bioreceptors such as aptamers and antibodies could be different even if the methods were developed with the same bioreceptor. The aptamer used in this study was developed by a Canadian Company (NeoVentures Biotechnology Inc.) in 2012. The company presents that the aptamer is specific to not only AFB1 but also zearalenone. Even if the company reported a cross-reaction to zearalenone, the cross-reactivity of the dipstick optimized was investigated to other mycotoxins. Fig. 5 exhibits a cross-reactivity of the dipstick assay. The dipstick assay was confirmed to be highly W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294 specific to AFB1, and no cross-reaction to other mycotoxin was observed. In our previous report with a chemiluminescence assay based on the same aptamer to AFB1 modified with DNAzyme and performed on a wells of microtitre plate (Shim et al., 2014), the chemiluminescence assay based on the same aptamer used in this study showed cross-reaction to AFG1 and zearalenone. Although both methods used the same aptamer specific to AFB1, the different results of both methods with cross-reactivity test were reasonable, since the performances and formats of the both assays were different. Therefore, the result of the cross-reaction test demonstrated that the dipstick assay can be used as a rapid and simple screening tools for AFB1 in grain and other agricultural commodities. Table 1 shows the comparison of the sensitivity, rapidity and simplicity in the dipstick assay and others based an aptamer or antibody. There are two aptasensors for AFB1 analysis reported. One of both methods, an aptasensor based on real time PCR can detect 25 fg/ml of AFB1 but requires time-consuming (4 2 h) and complicated steps (4 4; pre-incubation, washing, and real time PCR, etc.). The sensitivity of the dipstick assay suggested in this study is also comparable to those of other immunosensors except ELISA requiring complicated experimental steps (6 steps). To the best of our knowledge, no dipstick assay based on an aptamer has not yet been reported and the present study is the first report for the development of an aptamer based-dipstick assay. 3.3. Analysis of corn samples spiked with AFB1 The analysis of corn samples artificially spiked with AFB1 at 0, 0.1, 0.5, 1, 5, and 10 ng/g was conducted in order to validate the dipstick assay. Before the analysis, the tolerance of the dipstick assay to methanol was explored because AFB1 possesses low solubility in aqueous solution, and methanol is generally used to extract AFB1 from real samples such as food and agricultural commodities. Therefore, the dipstick assay was subjected with different concentration (10%, 20%, and 30% in water) of methanol containing AFB1 at 0, 10, and 100 ng/ml. As shown in Fig. S3, a fluorescent intensity on the dipstick assay tested with AFB1 negative gradually diminished by increasing the concentration of 293 methanol. The results of the dipstick assay with AFB1 standard solution in 10% methanol showed the gradual decrease of fluorescent intensities by increasing the concentration of AFB1 as we expected. However, 20% and 30% containing different concentration of AFB1 showed inappropriate performance on the dipstick. This results meant that the sample extracts obtained from general extraction methods for AFB1 using 60% methanol cannot be directly used for the dipstick assay and is required to be diluted with an appropriate buffer resulting in the diminishment of initial concentration of AFB1 in samples. Therefore, a modified extraction method using lower methanol is necessary for the dipstick assay. In our previous report (Shim et al., 2014), a modified extraction method using methanol/water (20:80, v/v) as an extraction solvent was well established for the extraction of AFB1 in corn samples, and matrix effects by co-extracted various substances in corn samples was effectively removed by the dilution. For analysis of corn samples spiked with AFB1 by the dipstick assay, we utilized the modified extraction method in this study. An AFB1 free corn sample (1 g) was extracted with the extract buffer of 3 ml (20% methanol/water) for 20 min at room temperature and diluted with 0.1 M Tris–HCl (pH 7.4). The results of the dipstick assay tested with 2-fold diluted corn extracts containing different concentration of AFB1 were similar to those of the dipstick assay performed with AFB1 standards in 10% methanol/0.1 M Tris–HCl (Fig. S4). The results indicated that the matrix effect successfully disappeared after a 2-fold dilution. The spiked corn samples were prepared as described above, and the diluted extracts were tested by the dipstick assay. Table 2 shows the results for the analysis of spiked corn samples by the dipstick assay. According to the results, fluorescent intensities at test line on dipstick assays gradually decreased with increasing the concentration of AFB1 spiked in the corn samples, and the dipstick assay can detect the corn samples spiked with AFB1 at Z0.3 ng/g. Although recovery ratio of spiked corn samples by the dipstick assay was 74–112%, these values are acceptable for the range required by Directive 98/53/EC (European Commission, 2002). The results of AFB1 determination in artificially contaminated corn samples by the dipstick assay showed very good agreement with those of ELISA analysis. These results demonstrated that the dipstick assay developed in this study can be useful as a quantitative and qualitative methods for AFB1 detection in real samples. 4. Conclusions Fig. 5. Result of cross-reactivity test with AFB1-related compounds and other mycotoxins by the aptamer-based dipstick assay. (1) Negative test (10% MeOH); (2) AFB1; (3) aflatoxin G1; (4) aflatoxin G2; (5) ochratoxin A; (6) zearalenone; (7) T2 toxin; (8) patulin; (9) dexoynivalenol; and (10) citrinin. The concentration of mycotoxins is all 50 ng/ml. Although lateral flow strip based on an aptamer has been reported and is simple and user-friendly format intended to detect the target analyte, a dipstick assay is also representative on-site detection technologies and has several benefits such as a userfriendly format, long-term stability, and cost-effectiveness. Therefore, the dipstick assay is still attractive for on-site screening by untrained personnel. Dipstick assays based on antibodies have been frequently reported for rapid detection of mycotoxins. However, a dipstick assay based on an aptamer has not been developed Table 1 Comparison of the method developed in this study with other methods for aflatoxin B1. Detection technique Recognition probe Step required Limit of detection Time Reference Immunochromatography Optical biosensor based on competitive dispersion of gold nanorod Conductometric biosensor based on acetylcholoineserase ELISA Aptasensor based on ELAA Aptasensor based on real time PCR Dipstick assay Antibody Antibody Enzyme Antibody Aptamer Aptamer Aptamer 1 3 3 6 4 44 2 0.5 ng/ml 0.16 ng/ml 50 ng/ml 4 pg/ml 0.11 ng/ml 25 fg/ml 0.1 ng/ml 15 min 4 45 min 5 min 110 min 4 20 min 42 h o 30 min Shim et al. (2007) Xu et al. (2013) Soldatkin et al. (2013) Shim et al. (2008) Shim et al. (2014) Guo et al. (2014) This work 294 W.-B. Shim et al. / Biosensors and Bioelectronics 62 (2014) 288–294 Appendix A. Supplementary material Table 2 Recovery of AFB1 from spiked samples by the dipstick assay. AFB1 spiked Dipstick assay (ng/g) AFB1 detected (ng/g) 0 0.1 0.3 1 3 10 a b NDa ND 0.222 7 0.18 0.8127 0.09 3.361 70.36 8.943 7 1.42 Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.06.059. ELISA Recovery (%) AFB1 detected (ng/g) Recovery (%) –b – 747 6 817 9 1127 12 897 14 – 68 7 15 108 78 917 8 847 13 817 7 ND 0.068 7 0.02 0.32470.02 0.9127 0.08 4.1857 0.39 8.1297 0.68 AFB1 was not detected by the method. Recovery was not calculated. yet. In this study, we first reported a dipstick assay based on an aptamer specific to AFB1 and successfully used the assay for AFB1 detection in corn samples. The detection limits of the dipstick assay were 0.1 ng/ml of AFB1 in buffer and 0.3 ng/g of AFB1 in a corn sample. Although the dipstick assay requires an instrument for fluorescence measurement, the method is easy to perform, and the results could be obtained within 30 min without the need of washing and/or separation step. Recently, since aptamers have been considered as a good candidate to replace antibodies which are used in other immunoassays, the aptamer-based dipstick assay developed in this study is superior to other immunoassays with respect to its setting speed and stability. The membrane treated with streptavidin and anti-cy5 antibody in the dipstick assay can easily capture the hybridized biotin-modified aptamer/cy5-modified DNA probe, biotin-modified aptamer/target complex, and free cy5-modified DNA probe and can be universally used to develop dipstick assay with any biotin-modified aptamer to other mycotoxins and cy5-modified DNA probe specific to respective aptamers. The results in this study indicated that the dipstick assay was sufficiently sensitive and reliable to be useful for rapid screening of AFB1 in real samples. Acknowledgment This study was supported by grants from Advanced Production Technology Development Program, Ministry of Agriculture, Food and Rural Affairs and from the NLRL Program (NRF-2011-0028915) and the GRL Program (NRF-2013K1A1A2A02050616) funded by the Ministry of Science, ICT and Future Planning. References Bhatnagar, D., Yu, J., Ehrlich, K.C., 2002. Chem. Immunol. 81, 167–206. Blesa, J., Soriano, J.M., Molto, J.C., Marin, R., Manes, J., 2003. J. Chromatogr. A 1011, 49–54. Cella, L.N., Sanchez, P., Zhong, W., Myung, N.V., Chen, W., Mulchandani, A., 2010. Anal. Chem. 82, 2042–2047. Chen, J., Fang, Z., Liu, J., Zeng, L., 2012. Food Control 25, 555–560. Commission Regulation (EC)Off. J. Eur. Union. L77, 1–6. Cruz-Aguado, J.A., Penner, G., 2008. J. Agric. Food Chem. 56, 10456–10461. European CommissionOff. J. Eur. Commun. 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Food Control 36 (2014) 30e35 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Chemiluminescence competitive aptamer assay for the detection of aflatoxin B1 in corn samples Won-Bo Shim a, c, Hyoyoung Mun a, Hyo-Arm Joung a, Jack Appiah Ofori b, Duck-Hwa Chung c, Min-Gon Kim a, * a b c School of Physics and Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32310, USA Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongnam 660-701, Republic of Korea a r t i c l e i n f o a b s t r a c t Article history: Received 29 May 2013 Received in revised form 19 July 2013 Accepted 27 July 2013 In this study, we developed a chemiluminescence competitive aptamer assay for aflatoxin B1 (AFB1) using a hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) linked with an aptamer specific to AFB1. Single, double, and triple HRP-DNAzymes coupled to the AFB1 aptamer were tested, and the AFB1 aptamer linked with double HRP-DNAzymes that produced sufficient chemiluminescence (CL) values when binding to AFB1-ovalbumin (OVA) used as a coating antigen, was selected. Under conditions optimized by testing key parameters, the aptamer assay exhibited a wide dynamic range from 0.1 to 10 ng/mL and showed a limit of detection of 0.11 ng/mL. Cross-reaction to aflatoxin G1, aflatoxin M1, and zearalenone was observed but no cross-reaction to other mycotoxins or the herbicide (atrazine) was observed. Aqueous methanol (20%) gave a good extraction efficiency and the matrix influence from corn extracts was successfully reduced through 4-fold dilution with water. Recovery from spiked corn samples averaged from 60.4 to 105.5%. Thus, the aptamer linked with HRPDNAzymes can be useful as a reagent in the development of a biosensor for the rapid and simple detection of AFB1. Results from this study provide the basis for research into the development of various aptasensors for AFB1 analysis in foods. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. Keywords: Aflatoxin B1 Aptamer Chemiluminescence HRP-DNAzyme Corn 1. Introduction Mycotoxins, the secondary metabolites of Aspergillus, Penicillium, and Fusarium spp. are highly toxic substances in food and agricultural products. Aflatoxin B1 (AFB1), the predominant and most toxic mycotoxin, is produced by Aspergillus flavus and Aspergillus parasiticus and is a carcinogenic and mutagenic compound. This mycotoxin is classified as a group 1 carcinogen (carcinogenic to humans and animals) by the International Agency for Research in Cancer (IARC, 2002). AFB1 represents a high risk as it contaminates a variety of agricultural commodities, such as tree nuts and derived products, peanuts, corn, cereals, grains, cottonseed, milk, animal feeds, and herbal medicines (Boyacioglu & Gonul, 1990; Nilufer & Boyacioglu, 2002; Shim, Kim, Ofori, Chung, & Chung, 2012; Steiner, Brunschweiler, Leimbacher, & Schneider, 1992; Trucksess, Stack, Nesheim, Albert, & Romer, 1994). The occurrence of AFB1 in food is therefore considered to be one of the * Corresponding author. Tel.: þ82 62 715 2874; fax: þ82 62 715 3419. E-mail address: mkim@gist.ac.kr (M.-G. Kim). most important global food safety issues (Shim et al., 2007). Therefore, more than 99 countries have set regulations for AFB1 or the sum of AFB1, aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2) in foods and agricultural stuffs. The most common limits for AFB1 and total aflatoxins are 5 and 20 mg/kg, respectively (Shim et al., 2012) but more rigorous regulations for AFB1 (2 mg/kg) and the sum of total aflatoxins (4 mg/kg) in groundnuts, nuts, dried fruits, and cereals were set by European Union regulations (Commission Regulation, 2006). Therefore, the ability to detect AFB1 is vitally important. Many approaches based on chromatographic and immunological principles have been developed and employed. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) as representative instrumental methods have been officially accepted for the quantification of AFB1 but are time-consuming, laborious, and unsuitable for on-site detection (Shim et al., 2007). Many analytical methods based on immunoassays such as enzymelinked immunosorbent assays (ELISA), fluorescence polarization immunoassays, immunochromatographic assays, and immunosensors have been introduced for rapid and on-site detection of AFB1 in food 0956-7135/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.07.042 W.-B. Shim et al. / Food Control 36 (2014) 30e35 and agricultural products. These assays involve the use of antibodies specific to AFB1 and are considered as rapid and simple methods for mycotoxin detection (Wang et al., 2011). However, the use of antibodies has many disadvantages in terms of the high cost of production and their stability, as they are easily denatured during storage (Huang, Zhao, Chen, Shi, & Liang, 2012). In recent years, an innovative method based on aptamers has been developed in the field of analytical methods. Aptamers are single-stranded oligonucleotides that can strongly and selectively bind to a target molecule and offer significant advantages over antibodies as their production is less expensive and laborintensive, and they are easier to label with fluorescent dyes, enzymes, biotin, and DNA ligands (Jayasena, 1999). In addition, aptamers immobilized on solid phases can easily be regenerated by heating and therefore can be recycled (McKeague et al., 2010). These advantages make aptamers attractive in the development of low-cost, reusable, and robust analytical methods. Several aptamers for small molecules such as mycotoxins (CruzAguado & Penner, 2008; McKeague et al., 2010), antibiotics (Jeong & Rhee Paeng, 2012), and pesticides (Wang et al., 2012) have been recently reported. Among them, an aptamer against ochratoxin A is well-known and has been studied for the development of biosensors (Barthelmebsa, Joncab, Hayata, Prieto-Simonc, & Martya, 2011; Chen, Fang, Liu, & Zeng, 2012; Wu et al., 2012). In 2010, McKeague et al. (2010) reported 6 oligonucleotides as candidates for a fumonisin B1 aptamer and presented the use of those aptamers in a fumonisin biosensor and affinity column. In addition, enzyme-linked aptamer assays (ELAAs) for the detection of mycotoxins (Barthelmebsa et al., 2011), antibiotics (Jeong & Rhee Paeng, 2012), and biomolecules (Park & Rhee Paeng, 2011) detection have been reported, and their experimental procedures are almost identical to those of ELISAs. Most ELAAs recently reported generally used the enzyme horseradish peroxidase (HRP) to determine the amount of aptamer and analyte bound. Although enzymes can be linked to aptamers, this is a time-consuming approach and furthermore, the enzyme becomes unstable when stored in an aqueous solution for long periods (Miranda-Castro, Lobo-Castañón, Miranda-Ordieres, & Tuñón-Blanco, 2010). Currently, there are several oligonucleotides that act like enzymes and are known as DNAzymes. DNAzymes can be easily synthesized and stored for long periods in solution compared to enzymes. Thus, there is growing interest in using DNAzymes in the development of biosensors and aptasensors as amplifying labels. Among the DNAzymes currently being used, the hemin/Gquadruplex horseradish peroxidase-like DNAzyme (HRP-DNAzyme) has been frequently used as a catalytic nucleic acid label to develop ultrasensitive biosensing platforms for the analysis of proteins and heavy metals and can be an alternative to HRP (Pelossof, Tel-Vered, Elbaz, & Willner, 2010). In 2012, a Canadian company (NeoVentures Biotechnology Inc.) obtained a patent for an AFB1 aptamer and has produced a commercial affinity column and detection kit using the AFB1 aptamer (NeoVentures Biotechnology, 2013). To the best of our knowledge, analytical methods based on aptamers for AFB1 detection have not yet been developed. In this study, we report the first instance of the use of a chemiluminescence competitive aptamer assay for the detection of AFB1 using HRP-DNAzyme linked to an AFB1 aptamer. In addition, this study represents the first approach in respect to using fully synthesized oligonucleotides instead of using enzymelinked antibodies or aptamers in a competitive assay. The AFB1 aptamer linked with HRP-DNAzyme used as a detector reagent in this study was synthesized according to the full-length sequences of the AFB1 aptamer presented by NeoVentures Biotechnology Inc. Optimization of the aptamer assay was carried out by testing key 31 parameters, and the optimized AFB1 aptamer assay was validated by analyzing corn samples spiked with known concentrations of AFB1. 2. Materials and Methods 2.1. Materials and reagents AFB1, related mycotoxins (AFB2, AFG1, AFG2, ochratoxin A (OTA), zearalenone, citrinin, T-2 toxin, deoxynivalenol, and patulin), atrazine (herbicide), bovine serum albumin (BSA), OVA, skim milk, luminol, hemin, r-comaric acid, Tween 20, and sodium chloride were purchased from Sigma (St Louis, MO, USA). White 96microwell plates (flat bottom) were purchased from Nunc (Roskilde, Denmark). All chemicals and organic solvents were reagent grade. Water used in all experiments was purified with a Purelab Option Water Purification System (ELGA, Marlow, UK). Phosphate buffer saline (PBS, 0.1 M, pH 7.4), borate buffer (0.1 M, pH 7.2), 0.1 M TriseHCl (pH 7.4 and 8.8), 0.1 M NaCl, and 0.1 M KCl were used in the present study as working buffers for the dilution of aptamers against AFB1. The ChemiDocTM MP System (Bio-Rad, Hercules, CA, USA) was used to determine the intensities of chemiluminescence (CL). A microplate well washer was purchased from Nunc. Standards of mycotoxins and herbicide were prepared by diluting stock solutions of these compounds (1 mg/mL) in absolute methanol. 2.2. AFB1-protein conjugates AFB1-protein conjugates (AFB1-BSA and AFB1-OVA), which were used as coating antigens in this study, were prepared by first derivating AFB1 into AFB1-oxime according to the method previously described (Chu, Hsia, & Sun, 1977). Carrier proteins (BSA and OVA) were then attached to AFB1 using the activated ester method (Kolosova, Shim, Yang, Eremin, & Chung, 2006). 2.3. AFB1 aptamers linked with HRP-DNAzyme In this study, three lengths of oligonucleotides as aptamers linked with HRP-DNAzyme were designed and used for the development of the chemiluminescence competitive aptamer assay for AFB1. The oligonucleotides were obtained from GenoTech Corp. (Daejeon, Korea) and their sequences are shown in Table 1. CL was obtained when the aptamer linked with HRP-DNAzyme bound to coating antigens on the wells reacted to the luminol solution (2 mM luminol, 2 mM H2O2, and 0.5 mM r-comaric acid in 0.1 M TriseHCl, pH 8.8). 2.4. Chemiluminescence competitive aptamer assay The aptamer assay developed in this study was based on similar procedures as pertains to competitive ELISA. A schematic diagram of the aptamer assay is shown in Fig. 1. For optimization of the aptamer assay, selection of the coating antigen (AFB1-BSA and AFB1-OVA), kind of blocking reagent (1% BSA and 1% skim milk), selection of the optimal aptamer linked with different numbers of HRP-DNAzymes and working buffers for the aptamer, and concentration of the aptamer were tested as key parameters. The assay was conducted on White 96-microtitre wells to determine the CL produced from the interaction between the AFB1 aptamer linked with HRP-DNAzymes and luminol. Briefly, 25 ng AFB1-OVA conjugate in PBS (100 mL) was coated on the wells and incubated for 1 h at 37  C. After washing 3 times with PBS containing 0.05% Tween 20 (PBST), the wells were blocked with 1% BSA (200 mL), kept overnight 32 W.-B. Shim et al. / Food Control 36 (2014) 30e35 Table 1 Sequences of oligonucleotides used in this study. Aptamers Number of HRP-DNAzyme on oligonucleotides Sequence (50 / 30 ) Bare AF-1 0 1 AF-2 2 AF-3 3 GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA C AAA GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CAA AAA GGT AGG GCG GGT TGG GTA AAT AAA GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CAA AAA GGT AGG GCG GGT TGG GTA AAT AAA AAG GGT AGG GCG GGT TGG GTA AAT AAA GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CAA AAA GGT AGG GCG GGT TGG GTA AAT AAA AAG GGT AGG GCG GGT TGG GTA AAT AAA AAG GGT AGG GCG GGT TGG GTA AAT Size (bp) 56 85 107 134 Aptamer nucleotides are italicized, and the underlined nucleotide sequences are HRP-DNAzymes. at 4  C, and washed 4 times again. The competition step was performed by adding 50 mL of a standard or 50 mL of a sample and 50 mL of aptamer solution (30 fM) containing 0.1 mM hemin and incubated at 30  C for 15 min. The plate was then washed 10 times and 100 mL of the luminol solution prepared as described above was added to the wells and the CL values were measured using the ChemiDocTM MP System. 2.5. Validation of the aptamer assay The optimized aptamer assay was validated by testing its sensitivity with using various concentrations of AFB1 (0, 0.1, 0.3, 1, 3, and 10 ng/mL) prepared in methanol/water (10:90, v/v). Crossreactivity for AFB1-related compounds (AFB2, AFG1, AFG2, AFM1, OTA, zearalenone, citrinin, T-2 toxin, deoxynivalenol, and patulin) and atrazine, each prepared at concentrations of 10 ng/mL in methanol/water (10:90, v/v) were also tested. 2.6. Analysis of AFB1 spiked in a corn sample AFB1-free corn samples confirmed by LC/MS used in our previous report (Shim et al., 2012) were ground, and AFB1-positive corn samples were prepared by spiking AFB1-free corn with known amounts of AFB1 to achieve final concentrations of 0, 0.5, 1, 5 and 10 mg/kg. The spiked corn samples were dried overnight at room temperature in a dark place and 1 g of each spiked sample was extracted with 3 mL of methanol/water (20:80, v/v) for 20 min at room temperature and then centrifuged at 3075 g for 15 min at 4  C. The supernatants were filtered through a disposable syringe filter (0.45 mm), diluted 4-fold with deionized water to reduce the matrix and methanol influence, and directly applied to the aptamer assay. 3. Results and discussion 3.1. Selection of appropriate length of aptamer linked with HRPDNAzymes One of the advantages of aptamers is easy modification and synthesis with other oligonucleotides, proteins, fluorescent dyes or other tags. In this study, we designed and tested three different aptamers linked with single (AF-1), dual (AF-2), and triple (AF-3) HRP-DNAzymes. 2 nM of each aptamer was mixed with the luminol solution, and the CL values were measured. As shown in Fig. 2, AF-3 produced a higher CL value compared to the CL values for the AF-2 and AF-1 aptamers. However, the AF-2 aptamer was chosen for the development of the chemiluminescence competitive aptamer assay to detect AFB1 in corn because, although the AF-3 aptamer showed the higher signal, the length of the AF-3 aptamer (more than 130 bp) is relatively long, which makes its synthesis costly and also less efficient. Meanwhile, the AF-2 aptamer is easier and cheaper to synthesize, and also produces sufficient CL signals to distinguish between positive and negative tests. 3.2. Optimization of the aptamer assay Optimization of the method was carried out by testing some key parameters. For the selection of coating antigens in the aptamer assay, AFB1-BSA and AFB1-OVA conjugates (100 ng/100 mL/well) as Fig. 1. Schematic diagram of chemiluminescence competitive aptamer assay for the detection of AFB1. Fig. 2. Comparison test for three lengths of AFB1 aptamer linked with single (AF-1), double (AF-2), and triple (AF-3) HRP-DNAzymes used in this study. W.-B. Shim et al. / Food Control 36 (2014) 30e35 3.3. Properties of the aptamer assay After optimal conditions for the aptamer assay for AFB1 were determined, the detection limit was investigated using AFB1 standard Fig. 4. Specificity of the aptamer assay to AFB1 with other mycotoxins and chemicals. AFB2: aflatoxin B2, AFG1: aflatoxin G1, AFG2: aflatoxin G2, AFM1: aflatoxin M1, CIT: citrinin, OTA: ochratoxin A, ZEA: zearalenone, T-2: T-2 toxin, DON: deoxynivalenol, PAT: patulin, and AT: atrazine. solutions at concentrations of 0, 0.1, 0.3, 1, 3, and 10 ng/mL in methanol/water (10:90, v/v), and the specificity of the method was studied using other mycotoxins and chemicals at a concentration of 10 ng/mL. The standard curve of the aptamer assay for AFB1 prepared in methanol/water (10:90, v/v) is presented in Fig. 5 and is similar to the typical standard curve of a competitive immunoassay. The IC50 value was 1.34 ng/mL, and the detection limit (10% inhibition) was 0.11 ng/ mL. Although NeoVentures Biotechnology Inc. reported that AFB1 aptamers exhibit cross-reaction to zearalenone, the cross-reactivity of the developed aptamer assay against other mycotoxins and chemicals was investigated. As shown in Fig. 4, the aptamer assay based on the AFB1 aptamer was specific not only to AFB1 and zearalenone but also to AFG1 and AFM1. Cross-reactivity of the aptamer assay to AFM1 means that the method can be used to analyze AFM1 in milk and dairy products. In a previous paper by the current author regarding a direct competitive ELISA (DC-ELISA) for AFB1 analysis, the detection limit of the DC-ELISA was 0.1 ng/mL. The sensitivity of the aptamer assay developed in this study was similar to that of the DC-ELISA reported. The total assay time of the aptamer assay was faster compared with that of the DC-ELISA because the aptamer assay for the determination of AFB1 in samples after the coating and blocking steps, which can be 120 100 % Binnding [(CL/CL0)X100] coating antigens were tested with the AF-2 aptamer and OTA aptamer linked with dual HRP-DNAzymes. The OTA aptamer was used as a control for the AF-2 aptamer. As shown in Fig. 3(a), even if the aptamer assay with the OTA aptamer and AFB1-OVA conjugate exhibited a weak signal, the AF-2 aptamer specifically bound to the AFB1-OVA conjugate. On the other hand, the aptamer assay performed with the AFB1-BSA conjugate and OTA aptamer showed strong non-specific binding. Thus, for the following studies, the AFB1-OVA conjugate was chosen as the coating antigen for further experiments, and the optimal concentration of AFB1-OVA per well was 25 ng. In addition, pure OVA protein, which was used as a carrier protein in the synthesis of the AFB1-OVA conjugate, was tested to see if the protein causes non-specific binding with the AF2 aptamer. A well was coated with 25 ng of pure OVA protein, and the aptamer assay was carried out with the AF-2 aptamer as previously described. As shown in Fig. 3(b), the AF-2 aptamer practically only bound to the AFB1 on the coating antigen (AFB1-OVA conjugate) and did not react to the pure OVA protein. In order to select the blocking reagent, 1% BSA and skim milk, commonly used as blocking reagents in typical immunoassays, were tested to avoid non-specific absorbance. The aptamer assay with 1% BSA produced sufficient CL signals. However, 1% skim milk showed a significantly lower signal which means that more aptamer and coating antigen are required (data not shown). Thus, for economic reasons, BSA was chosen as the blocking reagent in the development of the aptamer assay. In immunochemical methods based on the inhibition assay for mycotoxin, increasing the concentration of immunoreagents such as antibodies and coating antigens causes lower sensitivity of the analytical methods. Therefore, determination of the optimal immunoreagent concentration should be considered in the development of assays based on inhibition of the interaction between an antibody and a coating antigen due to the presence of the target. In this study, we determined the optimal concentration of the AF-2 aptamer (from 0 to 500 fM) for the AFB1OVA conjugate (25 ng per well) immobilized on the wells by analyzing AFB1 positive and negative samples. Increasing the concentration of the aptamer (50 fM) produced adequate CL values but showed no difference in CL values between positive and negative samples, while decreasing the concentration of the aptamer (15 fM) did not produce sufficient CL values. The most suitable AF-2 aptamer (30 fM) exhibited the highest difference in CL values between AFB1 negative (0 ng/mL) and positive (10 ng/mL) samples. Therefore, 25 ng of AFB1-OVA per well and a 30 fM AFB1 aptamer were selected as optimum conditions for the aptamer assay. Additionally, the various buffers mentioned in the Materials and Methods section were subjected to analysis to select the optimal working buffer for the aptamer, and accordingly, 0.1 M NaCl was chosen for the development of the aptamer assay (data not shown). 33 Aflatoxin B1 in 10% MeOH/PBS containing 0.1 M NaCl Aflatoxin B1 in corn extract 80 60 40 20 0 0.0001 0.0010.1 Fig. 3. Feasibility of the aptamer assay performed with AFB1 (AF-2) and ochratoxin A (control) aptamers. Two aptamers were coupled with double HRP-DNAzymes (a). Carrier protein, ovalbumin (OVA) was used to determine non-specific binding of AFB1 aptamer (b). 1 10 Aflatoxin B1 (ng/mL) Fig. 5. Standard curves of the aptamer assay performed with various concentrations of AFB1 prepared in working buffer and diluted corn extracts. 34 W.-B. Shim et al. / Food Control 36 (2014) 30e35 prepared before samples are obtained and pre-treated for the sample preparation, can be finished in just 20 min (competition step: 15 min, washing step: 2 min, substrate step: 2 min), while the DC-ELISA can be completed in 110 min. Several researches based on the enzyme-linked aptamer assay (ELAA) for the detection of mycotoxins (Barthelmebsa et al., 2011), antibiotics (Jeong & Rhee Paeng, 2012), and biomolecules (Park & Rhee Paeng, 2011) detection have been reported. However, these studies used the enzyme, horseradish peroxidase (HRP), as a marker in order to recognize the binding or immobilization of the aptamer to the immunoreagents or solid phase, respectively. Currently, DNAzymes, that which act like enzymes and can not only easily and multiply be linked to aptamers but are also stable in solution compared with enzymes, are a growing area of interest (Shim et al., 2007). In this study, we first developed the chemiluminescence aptamer assay for AFB1 using the aflatoxin B1 aptamer linked with dual HRP-DNAzymes (AF-2 aptamer). The sequences of HRP-DNAzyme used were 50 -GGT AGG GCG GGT TGG GTA AAT-30 . The main advantage for using HRP-DNAzymes instead of enzymes is that multiple DNAzymes can be linked to the aptamers required, and as a result signal enhancement is easily achieved from the multiple DNAzymes. The AF-2 aptamer produced sufficient CL values to allow signals between AFB1 positive and negative samples to be easily distinguished. 3.4. Effect of corn matrix on the aptamer assay A methanol concentration of greater than 60% has been generally used to extract AFB1 in real samples, but a high concentration of methanol may co-extract various substances existing in complex matrices that interrupt the antigeneantibody interaction in immunoassays and immunosensors (Kolosova et al., 2006). To minimize matrix interferences, two common approaches, sample cleanup and dilution, have been used. One of the major advantages of biosensors based on aptamers is simplicity. Therefore, the dilution method is more suitable to the aptamer assay optimized in this study, because the sample clean-up method is laborious and timeconsuming and requires complicated steps. In preliminary tests, the extraction method with methanol/water (60:40, v/v) was investigated, and the extracts were examined for the influence of interactions between the AF-2 aptamer and free AFB1 (or AFB1-OVA). The methanol concentration (60%) in the extracts may cause the denaturation of the OVA protein on the AFB1-OVA conjugate coated on the wells and hence dilution with an appropriate buffer to minimize methanol influence is necessary. Up to a 5-fold dilution of the extracts, the matrix effect was still observed. This indicated that the initial concentration of AFB1 in the corn samples should be diluted more than 30-fold after extraction by 60% methanol and dilution (sample:extraction solvent ¼ 1:5 and extract:diluent ¼ 1:4). Although the aptamer assay possesses high sensitivity, it is difficult to detect AFB1 in real samples at low concentrations. Therefore, an extraction method using a lower concentration of methanol was needed. In previous reports (Shim, Dzantiev, Eremin, & Chung, 2009; Wang, Liu, Xu, Zhang, & Wang, 2007), low methanol concentrations (5 and 30%) as extraction solvents for mycotoxin have been effectively used. Consequently, a new extraction method using methanol/water (20:80, v/v) as an extraction solvent was established in the present study. With 2 mL of the solvent for 1 g corn sample, the extraction was not only successfully conducted but also gave us approximately 1 mL of supernatant, which is sufficient volume for the aptamer assay for AFB1. Undiluted extracts also showed a matrix effect but it was not significant compared to extracts with 60% methanol. The matrix effect successfully disappeared after a 4-fold dilution with deionized water. Fig. 5 shows the standard curve of the aptamer assay Table 2 Recoveries of AFB1 spiked corn samples. AFB1 spiked (mg/kg) (n ¼ 3) AFB1 detected (mg/kg) Recoveries (%) 0 0.5 1 5 10 ND 0.30  0.009 1.06  0.18 5.26  0.32 10.19  1.49 e 60.42  8.5 105.52  18.0 105.16  6.45 101.86  14.9 ND: Not detected by the aptamer assay. performed with different concentrations of AFB1 in the diluted corn extracts. According to the standard curve, the limit of detection (10% inhibition) and IC50 values were 0.14 and 1.85 ng/mL, respectively. The results were compared with another standard curve prepared in a buffer at the same concentration. The aptamer assay with the diluted corn extracts resulted in slightly lower sensitivity than that performed with a buffer but still possessed adequate sensitivity to detect AFB1 at the low concentrations set as regulation limits for food and agricultural products by many countries (Shim et al., 2012). 3.5. Application of corn samples spiked with AFB1 to the aptamer assay In order to validate the feasibility of the aptamer assay to detect AFB1 in corn samples, AFB1-positive corn samples were prepared by spiking AFB1 into AFB1-free corn samples at concentrations of 0, 0.5, 1, 5, and 10 ng/g and these samples were tested using the method. Triple recovery tests were conducted on different days, and the results are shown in Table 2. Recovery of AFB1 spiked in corn samples was 60.4e105.5%, which is acceptable for the range required by Directive 98/53/EC (European Commission, 2002). These results indicate that the aptamer assay can be practically used as a quantitative method for AFB1 detection in corn samples. The results demonstrate that the method based on the aptamer-HRP DNAzyme and the new extraction method proposed in the present study have been successfully applied to corn samples and can be applied to other grains with the minimization of matrix influences. 4. Conclusions In this study, we successfully developed a chemiluminescence competitive aptamer assay for AFB1 using an aptamer-linked HRPDNAzyme and applied the method to real corn samples. To the best of our knowledge, no aptamer assay or biosenor-based aptamer for the analysis of AFB1 has yet been reported, and the aptamer assay developed is the first application of an aptamer assay for the detection of AFB1. Three different lengths (AF-1, AF-2, and AF-3) of aptamer-linked HRP-DNAzyme were investigated, and AFB1 aptamer-linked dual HRP-DNAzyme was chosen as the recognition element. After optimizing the aptamer assay by testing key parameters, the sensitivity of the aptamer assay was found to be sufficient to detect AFB1 at low concentrations (5 mg/kg), which have been set as the most common maximum permissible limits for food samples. Although the aptamer used in this study possessed cross-reaction to AFG1 and zearalenone, the aptamer assay is still an attractively useful tool to detect AFB1. The recovery values obtained in the present study met the requirements of the EU (European Commission, 2002) for newly developed analytical methods and demonstrated the feasibility of the aptamer assay for application on real samples. This study provides great opportunities to develop this aptamer assay for AFB1 detection into biosensors. In addition, since the method possessed cross-reactivity to AFM1, the aptamer assay could be a useful screening tool for AFM1 W.-B. Shim et al. / Food Control 36 (2014) 30e35 in milk and dairy products. Currently, studies are ongoing in our laboratory to develop the AFB1 (or AFM1) aptamer assay into a biosensor for rapid, on-site, inexpensive, easy-to-use, and ultrasensitive detection of AFB1 (or AFM1). Acknowledgments This study was supported by grants from Advanced Production Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, the NLRL Program (2011-0028915), and Public welfare & Safety Research Program (2011-0021115) through the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST). 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