Introduction

Zearalenone (ZEN; also known as F-2 toxin), chemically described as 6-[10-hydroxy-6-oxo -trans-1-undecenyl]-β-resorcyclic acid lactone, exhibits blue-green fluorescence when excited by UV light [1, 2]. ZEN is a nonsteroidal estrogenic mycotoxin biosynthesized through a polyketide pathway by many species of fungi of the genus Fusarium (e.g., Fusarium graminearum, Fusarium culmorum, Fusarium cerealis, Fusarium equiseti, Fusarium crookwellense, and Fusarium semitectum) [3]. As one of the most significant mycotoxins worldwide in maize, barley, oats, wheat, rice, sorghum, and their derived food and feed stuffs, ZEN is suspected to be associated with premature puberty syndrome, hyperplastic neoplastic endometrium, and human cervical cancer [4, 5]. ZEN is also deemed to be cytotoxic and exhibits a genotoxic potential in vitro and in vivo through the induction of micronuclei, chromosome aberrations, DNA fragmentation, and cell cycle arrest [6–10]. Therefore, the determination of ZEN in food samples is of great importance for food safety monitoring.

The current analytical methods available for ZEN measurement primarily rely on instrumental analysis, such as high-performance liquid chromatography with either fluorescence detector or mass spectrometric detection and immunoassays including enzyme-linked immunosorbent assay (ELISA) and gold immunochromatography assay [11–15]. Instrumental analytical methods provide good performance in terms of accuracy, precision, sensitivity, and reproducibility. However, they are unacceptable for large-scale and on-site fast analysis because they require tedious sample preparation, expensive equipment, and highly skilled operators. Immunoassays are used for rapid qualitative screening, but they often fail to provide accurate quantitative results and a definite confirmation of the mycotoxin ZEN because the antibodies in immunoassays are sensitive to temperature and require strict physiological conditions for use. In addition, antibodies require animals or cell cultures in a time-consuming and costly production. Therefore, it is imperative to develop some new recognition molecules for ZEN that are easier to produce and more stable.

Aptamers are single-stranded DNA or RNA ligands generated from a combinatorial random nucleic acid library of 1013–1016 molecules using systematic evolution of ligands by exponential enrichment (SELEX), an in vitro selection process involving iterative binding, separation, and amplification [16, 17]. Aptamers can target a variety of molecules, including small molecules such as acetamiprid, metal ions, peptides, proteins, bacteria, viruses, and cancer cells [18–26]. By folding into distinct secondary and tertiary structures, aptamers can recognize their targets with high affinities and specificities comparable to antibodies [27]. Moreover, aptamers have long shelf lives, show little to no immune response, are easily refolded if denatured, and are readily synthesized, allowing for precise chemical modification with little batch-to-batch variation [28].

Ochratoxin A aptamers selected by affinity column-based SELEX and fumonisin B1 (FB1) aptamers selected by magnetic bead-based SELEX have been reported [29, 30]. Various detection methods based on recognition between aptamers and mycotoxin targets have since been developed [31–33]. In the present report, we describe the selection of ZEN-specific single-stranded DNA (ssDNA) aptamers using magnetic bead-based SELEX, with some modifications, and the identification of specific binding between aptamer and ZEN using binding assays, indirect competition assays, and circular dichroism spectrum. A simple and rapid test based on magnetic separation/preconcentration was developed for the detection of ZEN. The selected aptamers could be powerful recognition ligands in the potential development of affinity columns, biosensors, or other analytical systems for field and laboratory determination of ZEN in food and agricultural products.

Experimental

Chemicals

ZEN, β-zearalenol (β-ZOL), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), fumonisin B1 (FB1), fumonisin B2 (FB2), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), acrylamide/bis-acrylamide (30 % solution), and streptavidin (SA) were all purchased from Sigma-Aldrich (Saint Louis, MO). Taq Plus DNA Polymerase (5 U/μL), dNTPmixs (each 25 mM), 10× polymerase chain reaction (PCR) buffer (containing Mg2+), and other electrophoresis components were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Lambda exonuclease enzyme (5,000 U/mL) and 10× lambda exonuclease reaction buffer were purchased from New England BioLabs (Ipswich, MA). Other unspecified chemicals and reagents were of analytical purity and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Solutions were prepared with ultrapure water processed with a Milli Direct-Q 3 ultrapure water system (Millipore, Bedford, MA). DNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). The ssDNA library contained a central random sequence of 40 nt flanked by two 20-nt primer hybridization sites (5′-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTG AA-3′). A forward primer (P1: 5′-AGCAGCACAGAGGTCAGATG-3′), a reverse primer (P2: 5′-TTCACGGTAGCACGCATAGG-3′), or a phosphorylated labeled reverse primer (phosphate-P2: 5′-P-TTCACGGTAGCACGCATAGG-3′) was used for the amplification of double-stranded DNA (dsDNA).

PCR amplification and ssDNA generation

All PCRs were performed in a C1000 Thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA). The amplification conditions were 5 min at 94 °C, 16–25 cycles of 30 s at 94 °C, 30 s at 57 °C, and 30 s at 72 °C, followed by 5 min at 72 °C after the last cycle. The PCR products throughout the selection were analyzed by 8 % native PAGE, stained with ethidium bromide, and analyzed using a Chemi DocTM XRS+ imaging system with Image Lab™ Software (Bio-Rad Laboratories, Inc.). After purification by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation, the completely dried dsDNA powders were dissolved in sterile water and the concentration was measured using an ND-1000 Spectrophotometer (Nanodrop Technologies, Inc., Wilmington, DE).

The purified phosphorylated dsDNA fragments were digested with lambda exonuclease according to the manufacturer’s instructions (first incubated at 37 °C for 60 min then heat-inactivated at 75 °C for 10 min to stop the enzymatic reaction). Ten-microliter samples of the digestion products were identified by 8 % denaturing PAGE with 7 M urea and the remainder was purified and precipitated. Finally, the dried ssDNA powders were dissolved in sterile binding buffer (BB) and the concentration was measured using a Nanodrop Spectrophotometer.

Immobilization of targets on magnetic beads

The amine magnetic beads used for the immobilization of targets were prepared in our own laboratory as described previously [34] according to the report of Wang et al. [35]. Because ZEN possesses no reactive group for coupling reactions, it was first converted to ZEN-6′-carboxymethyloxime (ZENO) using the method of Thouvenot and Morfin [36] and then immobilized on the amine magnetic beads with the catalysis of EDC. The blank beads used in the negative selection were treated the same as above, but without ZENO. ZEN coupling was verified by infrared (IR) spectrum using a Nicolet Nexus 470 FTIR spectrometer (Thermo Scientific, Inc., Waltham, MA) and by observing the fluorescence of ZEN-coated beads on an LSM 700 laser scan confocal microscope (Zeiss Co., Oberkochen, Germany).

In vitro selection

Before each SELEX round, the ssDNA library pool was dissolved in 500 μL of BB (pH 7.4, containing 100 mM NaCl, 20 mM Tris–HCl, 2 mM MgCl2, 5 mM KCl, 1 mM CaCl2, and 0.02 % Tween-20) and then denatured at 94 °C for 5 min. The pool was then immediately cooled on ice for 15 min and incubated at room temperature (RT) for 10 min. Meanwhile, 100 μL of blank beads for negative selection, or ZEN-coated beads for positive selection (approximately 1 × 108 beads), was washed six times in BB and separated from the supernatant by magnetic separation. Subsequently, the denatured ssDNA pool was transferred to the tube of magnetic beads; as a result, the ssDNAs that bound to the tube wall were eliminated.

For the first selection round, 2 nmol of random ssDNA pool was incubated with ZEN-coated beads at RT for 60 min with tilting and rotation. The unbound oligonucleotides were removed by six washings using 500 μL of BB each time. The bound oligonucleotides were eluted from the ZEN-coated beads by incubating the target-DNA complex in 100 μL of elution buffer (EB, pH 8.0, containing 20 mM Tris–HCl, 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.02 % Tween-20, and 0.1 % TritonX-100) at 90 °C for 10 min with shaking, followed by magnetic separation of beads and ssDNA supernatant. This process was repeated several times to retrieve all traces of bound ssDNA; the bound fractions were pooled as PCR templates.

To increase the specificity of aptamers, negative selections were performed from the second round. Notably, from the third round, free β-ZOL and/or AFB1, AFB2, FB1, and FB2 solutions were added into the incubation system during the positive selection for competitive binding to ssDNA, in which the total number of mycotoxin molecules was equal to the number of ssDNA molecules of the library pool. By using this modified counter selection, complex and time-consuming immobilization and the identification of other mycotoxin molecules on the magnetic beads were avoided. In the last two rounds, the bound ssDNA was eluted with EB containing 50 pmol of ZEN. Furthermore, the washing times before elution were increased from six to nine and the incubation time was shortened when the selection increased. The conditions used in each selection cycle are summarized in Electronic supplementary material (ESM) Table S1.

After round 14, the selected ssDNA pool was amplified with unmodified primers and the purified products were cloned into Escherichia coli using the TOPO TA Cloning Kit (Invitrogen). Thirty-nine colonies were randomly selected and sequenced by Sangon Biotechnology Co., Ltd. Multiple sequence alignment of the sequences was performed using DNAMAN software and the secondary structure of each sequence was predicted using RNA structure 3.0 software.

Binding assays and dissociation constant (K d) measurements

Twelve representative sequences selected from the 39 sequences were synthesized with 5′-biotin for further assays. The binding assays were conducted using a method similar to the SELEX conditions by incubating a constant number of ZEN-coated magnetic beads against various concentrations of ssDNA (0–1,600 nM) for 60 min. After rigorous washing, the bead-bound aptamers were eluted by heat treatment and the ssDNA amounts were measured using a Nanodrop Spectrophotometer. The same experiment was performed on blank beads to ensure that the observed binding of sequences was specific to the target. All of the above experiments were performed in triplicate for error analysis. GraphPad Prism 5.0 software was used to fit a nonlinear regression curve from which the K d values were calculated.

Determination of the specificity of aptamers

SA-coated magnetic beads were prepared according to the report of Lu et al. [37] (illustrated in the ESM). The specificity of the selected aptamers—5Z28, 8Z31, and 11Z1—was tested via indirect competition assays as follows: mixtures containing 100 μL of the 5′-biotin-labeled sequence (1 μM) and 100 μL of 1 × 108 SA-coated beads were reacted at RT for 60 min. Subsequently, the biotin aptamer/SA-coated beads were washed with PBS three times and 200 μL of 800 nM FAM-labeled short strands (5′-FAM-TTCACGGTAGCAC-3′) in BB was added to hybridize with the aptamer strands on the surface of the magnetic beads. After hybridization, the complexes were washed twice and resuspended in BB. Seven equal divisions from the resuspension were all separated from the supernatant by magnetic separation. Subsequently, 100 nmol of each mycotoxin or blank BB was added into the constructed aptamer-coated magnetic beads to incubate for 60 min at RT in the dark. The fluorescence of each supernatant obtained by magnetic separation was measured using an F-7000 fluorescence spectrophotometer (Hitachi Co., Tokyo, Japan) by exciting the samples at 485 nm.

Regarding the fact that the aptamers were selected against modified ZEN immobilized on magnetic beads, the best aptamer (8Z31) was used for testing the binding ability to a non-modified ZEN in solution. A circular dichroism (CD) spectrum assay was carried out to discern whether the conformation of the aptamer was changed when incubated with free ZEN to prove binding between ZEN and its aptamer. Mixtures of aptamer 8Z31 (2.5 μM) with or without 1 nmol of ZEN (total volumes were 400 μL each) were incubated at RT for 60 min. Subsequently, these samples were analyzed on an MOS-450/AF-CD (BioLogic Science Instruments, Claix, France) instrument ranging from 230 to 320 nm, using 260 nm as the maximum excitation wavelength.

Aptamer-based assay for detecting ZEN

Biotin-labeled aptamer 8Z31 was first coupled with SA-coated magnetic beads and the biotin aptamer/SA-coated beads were incubated with ZEN in BB for 60 min in the dark. After washing with BB, 10 μL of bead suspension was dropped on a thin glass slide and covered with a coverslip. A confocal imaging test was performed to directly observe the binding between aptamer 8Z31 and ZEN on an LSM 700 laser scan confocal microscope.

Then, aptamer 8Z31, immobilized on the magnetic beads, was utilized for the separation and enrichment of ZEN in solution. A series of ZEN solutions ranging in concentration from 7.85 × 10−10 to 3.14 × 10−5 M were prepared using step-by-step dilutions of the high-concentration ZEN solution (7.85 × 10−3 M in methanol) with BB. Subsequently, the aptamer-immobilized magnetic beads were reacted with 6 mL of the aforementioned ZEN solutions at RT for 60 min in the dark. The magnetic beads were separated from the supernatant by an external magnetic field; then, 300 μL of BB was added and the mixtures were heated at 90 °C for 10 min to dissolve the ZEN enriched by the binding of aptamer 8Z31. The fluorescence of the supernatant containing concentrated ZEN, obtained by magnetic separation, was measured by excitation at 335 nm and emission at 450 nm using an F-7000 fluorescence spectrophotometer.

The practicability of the aptamer-based bioassay method to detect ZEN was further validated in beer purchased from a local supermarket. Beer samples (50 mL) were stirred at 60 °C for approximately 60 min until complete degassing. The CO2-free beer samples were sub-packaged in clean tubes and various contents of ZEN were introduced to each sample with the final concentrations ranging from 3.14 × 10−5 to 3.14 × 10−3 M. The samples were diluted with an equal volume of 2× BB when used for the detection using the proposed method. The ZEN contents in the samples were also validated by ELISA analysis with a commercially available ZEN detection kit (Jiangsu Suwei Co. Ltd., Jiangsu, China).

Results and discussion

Immobilization and characterization of zearalenone on magnetic beads

The results of the modification of ZEN to ZENO are shown in ESM Figs. S1 and S2. The coating process involves the covalent coupling of the carboxyl groups of the ZENO with the primary amino groups on the surface of the magnetic beads. Unfortunately, it was not feasible to calculate the bound target concentration from the initial and unbound concentrations using a fluorescence spectrometer because there was unreacted fluorescent EDC remaining in the unbound solution, which influenced the results. Therefore, coupling was confirmed with IR spectra and fluorescence microscopy. Figure 1 compares the IR spectra of the blank magnetic beads and the ZEN-coated beads from the 800- to 1,800-cm−1 wavenumbers. As observed in Fig. 1 (red), the peaks at 1,107 and 1,057 cm−1 were clearly intensified, which corresponded with the absorption of the flavor ester structure of ZEN. The C–N stretching vibrations around 1,100 cm−1 overlapped, indicating that ZEN was immobilized on the magnetic beads through amide bonds. Coupling was further verified by fluorescent imaging of the ZEN-coated beads (see ESM Fig. S3). ZEN on the surface of the magnetic beads exhibited blue fluorescence upon UV excitation.

Fig. 1
figure 1

IR spectra of the blank magnetic beads (blue) and ZEN-coated magnetite beads (red)

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Selection of ZEN-specific aptamers

In our selection, ssDNA aptamers recognizing zearalenone were chosen from a random library containing about 1.2 × 1015 different ssDNA molecules through 14 rounds of SELEX. The conditions of SELEX in each round are shown in ESM Table S1, and there were several key control points. First, it is necessary that the ssDNA library pool be denatured and refolded prior to incubation with the target beads. As a result, the ssDNA can form distinct secondary and tertiary structures, providing the basis for binding to their targets with high affinity and specificity. Second, nonspecific binding sequences were eliminated by negative and counter selections, and the weak binding sequences were removed by an increased number of washings. In addition, the elution steps were increased from three to seven to collect as many aptamers as possible. Third, high-fidelity amplification of the collected ssDNA played a crucial role by preventing undesired deorbit enrichment. As the SELEX progressed, the amount of ssDNA bound to target beads increased, as did the incremental number of dissociated aptamers. Nonspecific amplification products were observed to form if excessive ssDNA templates were added, but this problem was resolved by reducing the number of PCR cycles. Thus, the number of cycles of PCR was optimized in each selection round, decreasing from 25 to 16. Furthermore, free β-ZOL, AFB1, AFB2, FB1, and FB2 were added to the incubation system as competitors to increase the stringency of the selections. Fewer ssDNAs were found to remain on the ZEN-coated beads, as demonstrated by amplification and analysis of both the dissociated ssDNA pool and the supernatant obtained from the incubation system with or without competitors.

The SELEX process was performed until the recovery of ZEN-bound ssDNA reached nearly 80 % of the input ssDNA pool. The selected oligonucleotides from round 14 were amplified by PCR using unmodified primers to enable cloning of the aptamer pool. The variable N40 sequence regions of all 39 aptamer candidates were analyzed and aligned for sequence similarity using DNAMAN software (see ESM Table S2). A and T were the most common bases appearing in the conserved sequence. Twelve sequences were chosen for further screening on the basis of not only their repetitiveness but also their lowest predicted free energy of formation.

Binding assays and determination of K d

Twelve representative sequences were selected and subjected to ZEN affinity analysis. The data points of the eluted ssDNA amounts were plotted against the initial concentration of the ssDNA pool to determine the K d values using nonlinear regression analysis. The analyzed aptamers—1Z22, 2Z14, 3Z35, 4Z12, 5Z28, 6Z7, 7Z32, 8Z31, 9Z29, 10Z21, 11Z1, and 12Z10—have diverse N40 sequence regions and thus have distinct structural features and binding affinities. The R 2 values ranged from 0.90 to 0.97 for the 12 sequences, indicating good fits; thus, the calculated K d values (Table 1) were considered reliable. The K d values of aptamers 5Z28, 8Z31, and 11Z1 were relatively low compared to those of the other aptamers, suggesting that they might have significantly higher affinities for the target. This increased affinity was related to the influence of unique stem and loop structures that induced different binding modes. Figure 2 shows the binding saturation curve of aptamer 8Z31 to illustrate that the amount of bound ssDNA increased with the concentration of aptamer up to the saturation point.

Table 1 Determination of the dissociation constants (K d) of the 12 aptamer candidates
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Fig. 2
figure 2

Binding saturation curve of aptamer 8Z31 to ZEN-coated magnetic beads. The K d values were calculated by nonlinear regression analysis using GraphPad Prism 5.0. Data shown were the means of three replicates; error bars represented the standard errors of the means. Similar experiments were conducted for all of the synthesized candidate aptamers, and the estimated K d values are summarized in Table 1

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Prediction of aptamer structure

The hypothetical secondary structure models of the selected sequences were predicted using RNA structure 3.0 software. The structures of most of the selected aptamers were assembled from stems, loops, bulges, hairpins, triplexes, pseudoknots, and quadruplexes. Three representative sequences—5Z28, 8Z31, and 11Z1—are displayed in ESM Fig. S4a–c. As reported by Stoltenburg et al. [38], the design of the binding domains on aptamers for their targets is based on structural compatibility, aromatic ring stacking, electrostatic and Vander Waals interactions, hydrogen bonding, or a combination of these requirements. After sequence analysis of the consensus sequence motif regions, we found that TAT, TAC, and its mirror image sequence CAT (marked with red and yellow circles, respectively) appeared frequently in the sequences. These sequences might be a component element of potential binding sites. Interestingly, aptamer 8Z31, which exhibited optimal specificity, simultaneously possessed both the TAT sequence found in aptamer 5Z28 and the TAC (CAT) present in aptamer 11Z1. Furthermore, there were three stems consisting of two pairs of A=T in aptamer 8Z31, which were also considered to be possible binding sites.

Determination of specificity

Aptamers 5Z28, 8Z31, and 11Z1 were tested for their specificity via an indirect competitive binding assay against a variety of other mycotoxins, including β-ZOL, AFB1, AFB2, FB1, and FB2. As presented in Fig. 3, the relative fluorescence intensity represented the ability of the mycotoxin molecules to competitively bind the aptamers and change their conformations, thus shedding the 5′-FAM-labeled short strand into the supernatant. All three sequences showed preferential binding to ZEN over the other mycotoxins, with aptamer 8Z31 appearing to possess optimal specificity against ZEN. However, it was determined that there was no necessary causality between the affinity and specificity as the K d value of aptamer 8Z31 was not the lowest among the three tested sequences.

Fig. 3
figure 3

Determination of the specificity of aptamers 5Z28 (a), 8Z31 (b), and 11Z1 (c) by indirect competitive binding. Each data point represents the average ± the standard deviation of three replicates

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Aptamer 8Z31 was determined to be the optimal aptamer candidate for ZEN; thus, further study of the performance of aptamer 8Z31 was conducted using CD analysis. The CD spectrum of the DNA arises from the asymmetric backbone sugars and the helical structures often adopted by nucleic acids. Ligand–DNA interactions, as well as the DNA-binding mode, can be assessed by a comparison of signals of the original DNA bases and the ligand-induced CD spectrum. As shown in Fig. 4, the addition of ZEN caused a clear decrease in the positive elliptic maximum around 275 nm and a slight right shift of the negative minimum around 250 nm, suggesting that the secondary structure of aptamer 8Z31 had been changed upon binding of ZEN.

Fig. 4
figure 4

Circular dichroism spectra of aptamers 8Z31 and 8Z31 in the presence of ZEN

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Quantitative detection of ZEN using the aptasensor based on magnetic separation/preconcentration

Confocal imaging was performed to directly observe the binding between aptamer 8Z31 and ZEN. As shown in Fig. 5, aptamer 8Z31-modified magnetic beads were fluorescent after incubation with ZEN solution, suggesting that ZEN was bound and separated by aptamer 8Z31. It should be noted that no fluorescent magnetic beads were found in the absence of ZEN. Because aptamer 8Z31 showed potential application in the separation of ZEN from free solutions by confocal imaging, an assay based on magnetic separation/preconcentration was first conducted for detecting trace ZEN in the binding buffer. The fluorescence intensity of the dissolved solution was plotted against the concentration of ZEN converted to a logarithmic scale. Figure 6 shows that this assay had high sensitivity and a wide linear range from 3.14 × 10−9 to 3.14 × 10−5 M (y = 291.135 + 24.7851x, R 2 = 0.9975), with a detection limit of 7.85 × 10−10 M.

Fig. 5
figure 5

Confocal imaging of ZEN bound to aptamer 8Z31-coated magnetic beads obtained by an LSM 700 laser scan confocal microscope. a Fluorescence image. b Optical image

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Fig. 6
figure 6

Linear fit of the fluorescence intensity at 450 nm of the solution obtained by an aptasensor based on a magnetic separation/preconcentration procedure versus the concentration of ZEN converted to a logarithmic scale in binding buffer. Error bars represent the standard deviation of triplicate measurements

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The effectiveness of the developed bioassay to detect ZEN levels in real samples was further studied. The beer samples added with different concentrations of ZEN were examined by the developed aptamer-based bioassay and compared with a commercially available ELISA method. The assayed results show that there is no significant difference between the results obtained by the two methods and that they are highly correlated (p < 0.0001), indicating that the aptamer-based method can also effectively detect ZEN in a real sample (see ESM Fig. S5). The accuracy of ZEN detection in beer was also evaluated by determining the recovery of ZEN using a standard recovery method. The recovery rates were between 85.0 and 105.1 % (Table 2), indicating that the aptamer-based method can effectively detect ZEN in a real sample.

Table 2 Recovery results for the added standard ZEN from beer samples obtained using the developed method
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Conclusions

In this study, 14 rounds of increasingly stringent selection conditions were performed to obtain ssDNA aptamers with high binding affinity and specificity to ZEN using the modified magnetic bead-based SELEX technique. Aptamer 8Z31 was determined to be the optimal binding aptamer based on the results of our study and was used for the first quantitative analysis of ZEN using magnetic separation/preconcentration. This aptamer is promising for use in ZEN cleanup and detection systems to analyze low levels of ZEN in food and agricultural products. Future work is to ascertain the shortest aptamers with high affinity and specificity for ZEN to develop low-cost, sensitive detection methods based on the recognition of aptamers to ZEN.