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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction Chengke Wang a , Xiaoya Dong b , Qian Liu b , Kun Wang b, * a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Ochratoxin A can be detected with low limit of detection utilizing the aptasensor. Hybridization chain reaction between DNAs was used to amplify the detection signal. Standard microtiter plates were used to develop high throughput analytical method. The method is label-free, antibodyfree, colorimetric, simple and costsaving.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 October 2014 Received in revised form 14 December 2014 Accepted 15 December 2014 Available online xxx
The combination of high selectivity of aptamer with the peroxidase-mimicking property of DNAzyme has presented considerable opportunities for designing colorimetric aptasensor for detection of ochratoxin A (OTA). The activities of both aptamer (as biorecognition element) and DNAzyme (as signal amplification element) are blocked via base pairing in the hairpin structure. Hybridization chain reaction (HCR) between two hairpin DNAs was employed to further improve the sensitivity of this method. The presence of OTA triggers the opening of the hairpin structure and the beginning of HCR, which results in the release of many DNAzyme, and generates enhanced colorimetric signals, which is correlated to the amounts of OTA with linear range between 0.01 to 0.32 nM, and the limit of detection is 0.01 nM under optimal conditions. OTA in yellow rice wine and wheat flour samples was also detected using this method. We demonstrate that a new colorimetric method for the detection of OTA has been established, which is simple, easy to conduct, label-free, sensitive, high throughput, and cost-saving. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Ochratoxin A Hybridization chain reaction Aptamer Colorimetric method
1. Introduction Ochratoxin A (OTA) is a kind of mycotoxin, which can be found in a large number of food commodities including cereals, wheat, barley, corn, coffee, and wine [1]. As OTA has nephrotoxicity,
* Corresponding author. Tel.: +86 511 88791800; fax: +86 511 88791708. E-mail address: [email protected] (K. Wang).
hepatic toxicity, immune toxicity and mutagenic effects on humans and animals [2,3], it is important to find a method to sensitively detect OTA in agro-foods. Nowadays, high performance liquid chromatography (HPLC) [4], mass spectrometry (MS) [5,6] and antibody based method [7] were employed to detect OTA, despite the accuracy and low limit of detection of these methods, they often require sophisticated equipments, time and laborconsuming and trained operators [8], which hinders their widely usage in high throughput detection. To overcome these drawbacks,
http://dx.doi.org/10.1016/j.aca.2014.12.031 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: C. Wang, et al., Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.12.031
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fluorescent spectroscopy [9,10], electrochemistry [11,12] were employed to detect OTA, although they are easy to conduct, but the sensors used in these experiments often need molecule modification procedure (e.g., fluorescent molecule and enzyme modification process), which is not convenient for OTA detection. So, it is necessary and urgent to develop a simple or no instrumentation yet still providing accurate analysis of OTA in food derivatives [13]. In recent years, aptamer based colorimetric assay were used to detect different kinds of analytes, such as toxin [14], protein [15], cancer cell [16] and heavy metal ion [17]. Due to its inherent selectivity and affinity to detected targets and easy to prepare, aptamer can be used to replace antibodies as recognition elements, which reduce the cost of detection [18]. As an alternative of enzyme, a segment of DNA, termed G-rich horseradish peroxidase (HRP)-mimicking DNAzyme, can form a four-strand structure known as G-quadruplex. After binding with hemin, these hemin–G-quadruplex complexes may display a highly enhanced catalytical activity, which catalyze substrate (e.g., tetramethylbenzidine (TMB)) oxidation and generate a colorimetric signal, providing a label-free mode to amplify the detection signal [19,20]. For example, Yang et al. have developed a DNAzyme based colorimetric method to detect OTA without using expensive enzyme [21]. Recently, hybridization chain reaction (HCR) was applied in many analytical experiments. HCR is based on the chain reaction of recognition and hybridization events between two sets of DNA hairpin molecules, and provide a new route to amplify the intensity of detection signal [22,23]. For example, Liu et al. have detected Escherichia coli uropathogen related DNA using HCR between two auxiliary hairpin DNAs, which is more sensitive than the non-HCR experiments [24]; Zhang et al. have designed two ferrocene modified hairpin structure DNAs to carry out HCR and utilized HCR based electrochemistry to detect human immunoglobulin G (IgG), the more target IgG presented in the solution, the more ferrocenes can be detected, which correlated to stronger electrical signals, the method possesses much higher sensitivity compared with previous experiments [25]. Although aptamer and DNAzyme were used to sensitively detect series of analytes [26,27], and HCR was used to amplify the detection signals in various experiments [24,25,28], to our knowledge, there is no report that combining aptamer and DNAzyme into aptasensors, and using HCR to further amplify the signals to detect OTA in agro-food. As it is necessary and urgent to develop a simple and sensitive method for the detection of OTA, in this regard, a new label-free colorimetric aptasensor for sensitive detection of OTA utilizing HCR has been developed in this experiment. In this aptasensor, aptamer was used as recognition element, and DNAzyme was used as signal amplification element. We believe that this strategy opens up a new avenue for the application of aptamer and DNAzyme in the field of food safety and analysis and food quality screening. 2. Experimental 2.1. Reagents NaCl, CaCl2, KCl, and tris(hydroxymethyl)aminomethane ((HOCH2)3CNH2, (Tris)) were purchased from Dingguo Inc. (Beijing, China). OTA was purchased from Pribolab Inc. (Singapore) 3,30 ,5,50 tetramethylbenzidine (TMB) substrate and CH3COOH (AcOH, acetic acid) were purchased from Aladdin Inc. (Beijing, China). Bovine serum albumin (BSA) was purchased from Sigma–Aldrich Inc. (USA). The ssDNA oligonucleotides and the 4S GreenTM nucleic acid stain were purchased from Sangon Biotech Inc. (Shanghai, China). The sequence of aptamer contained DNA (H1) was 50 -GATCG GGTGT GGGTG GCGTA AAGGG AGCAT CGGAC ACGCC ACCCA CAC30 , the sequence with italic is OTA aptamer, and the sequence with
underline could form hairpin structure. The sequence of the DNAzyme contained DNA (H2) was 30 -GCGGT GGGTG TGGGG AGGGA GGGAG GGTCC TAGCC CACAC C-50 , the sequence with italic could form DNAzyme, and the sequence with underline could form hairpin structure, respectively. The sequence of a random oligonucleotide (DNA3) was 30 -GCGGT GAGTG TGATG TGAGT GGCGA GGAGT GTGGG TATTG G-50 . The DNA stock solution was obtained by dissolving the oligonucleotide in 10 mM Tris–HCl buffer containing 200 mM NaCl (pH 8.4) and was stored at 4 C before use. 96 wells microtiter plates were obtained from Corning Inc. (USA). The water used throughout all experiments was doubledistilled water. All other chemicals were at least of analytical grade and used as received. 2.2. Apparatus Colorimetric measurements were performed with a Labsystems Multiskan MK3 microtiter plate reader (Thermo Life Sciences, USA). Gel electrophoresis experiment was carried out using a DYY6C electrophoresis system (Nanjing, China). The DNA bands were recorded with a Haiqing digital camera system (Nanjing, China). 2.3. Detection of OTA using colorimetric biosensing method The two DNA, H1 and H2 were heated to 95 C for 5 min separately and allowed to cool to room temperature slowly before use otherwise mentioned. In a typical experiment, 80 mL of solution containing 62.5 nM of H1 and different concentrations of analyte (OTA) in 10 mM Tris–HCl buffer (pH 8.4) with 200 mM NaCl, 10 mM CaCl2, 20 mM KCl and 150 nM hemin were added into 96-well microtiter plate and incubated for 10 min at 37 C. After that, 20 mL of 0.25 mM H2 was added, mixed and incubated for 1 h at 37 C, and then 80 mL of TMB substrate (including H2O2) was added and mixed adequately, after incubated for 30 min, 50 mL of 1 M HCl was added and mixed adequately to stop the reaction and make the color of solution turned to yellow. Subsequently, absorbance of the solution was measured at wavelength of 450 nm (OD450 nm). All experiments were performed at room temperature unless otherwise specified. 2.4. OTA detection in yellow rice wine and wheat flour samples The detection solution was composed of 625 mM H1, 100 mM CaCl2, 200 mM KCl and 1.5 mM hemin. 70 mL of OTA contained wine samples or wheat flour samples processed with extraction (the details of sample pretreatment procedure were shown in the Supporting information) were added into the 96-well microtiter plate, and then 10 mL detection solution was added [21,26,29]. After mixing, the microtiter plates were left to react for 10 min. After that, 20 mL of 0.25 mM H2 was added, mixed and incubated for 1 h, and then 80 mL of TMB substrate (including H2O2) was added and mixed, after incubated for 30 min, 50 mL of 1 M HCl was added and mixed adequately. Subsequently, absorbance of the solution was recorded at wavelength of 450 nm. All experiments were performed at room temperature unless otherwise specified. 2.5. Gel electrophoresis experiment Different concentrations of OTA were incubated with H1 at 37 C for 10 min in the presence of 10 mM Ca2+ and 20 mM K+. Then H2 was added and reacted at 37 C for 1 h, the final concentrate of H1 and H2 was 50 nM. A 3.5% (w/v) agarose gel was prepared using a 1 TAE buffer (40 mM Tris AcOH, 2.0 mM EDTA, pH 8.4). The 4S GreenTM nucleic acid stain was used as the oligonucleotide dye and mixed with samples. 10.0 mL samples were introduced into each band using pipette. The gel was run at 80 V for 45 min in a 1 TAE
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buffer and was then scanned using the Haiqing gel image analysis system.
of OTA, from the measurement presented here, 80 mL TMB substrate was used for the next experiments.
3. Results and discussion
3.2.2. Determine the concentration of Ca2+ As previously reported, divalent cations, such as Ca2+ or Mg2+ is essential for the recognition of aptamer with OTA in solution [32,33]. Here we studied the effect of Ca2+ on the detection of OTA. The optimal concentration of Ca2+ is determined by measuring the OD450 nm of the solutions in the absence and presence of 0.5 nM OTA. As shown in Fig. 1A, with increasing the concentration of Ca2+, the OD450 nm of solutions was increased, consequently. However, in the presence of OTA, the enzymatic activity is substantially increased. Similar to our work, the enhanced binding of OTA by aptamer in the presence of Ca2+ was reported in previous reports [11,32]. As shown in Fig. 1B, the differentials of these two measurements (DOD450 nm) resulted in a bell-shape curve with a maximum at about 10 mM Ca2+. So, 10 mM Ca2+ was used in the next experiments.
3.1. Principle of OTA detection The principle of this method is depicted in Scheme 1. Two hairpin oligonucleotides (H1 and H2) were designed to form aptasensor in this proof-of-concept experiment. Especially, aptamer is at the 50 end of H1, and DNAzyme is at the central of H2. Meanwhile, the fragment at the 30 end of H1 is complementary to the 30 end of H2, and the 50 end of H2 is complementary to the 50 end of H1. During the measurement of OTA concentration, after adding OTA into H1 solution in the presence of Ca2+, the OTAaptamer complex is formed and the hairpin of H1 is opened, the newly exposed 30 end of H1 hybridized with 30 end of H2, and opens the hairpin to expose the 50 end of H2, which can in turn hybridized with 50 end of H1; when the hairpin of H2 opened, the DNAzyme segment, upon the addition of K+ and hemin, will form a hemin–Gquadruplex complex which behaves as a kind of HRP-mimicking DNAzyme and displays a highly enhanced catalytic activity compared with hemin itself. In this way, each copy of the OTA can propagate a chain reaction of hybridization events between alternating H1 and H2 hairpins to form a double helix, which contains lots of DNAzyme units. During the measurement, each of the DNAzyme units can catalyze H2O2 oxidize TMB and produce colored products, leading to an amplified signal. As the microtiter plate reader used here cannot maintain the reaction solution at a constant temperature, this will dramatically affect the activity of DNAzyme, and result in the fluctuation of the signals. In addition, for the practical application, to make the measurement of OTA easy to conduct, we chose the absorbance of the solution at 450 nm after adding HCl to determine the concentration of OTA, rather than monitoring the absorbance of the solution at 620 nm for a long time [30,31]. By this means, we can indirectly determine the concentration of OTA with high sensitivity. On the contrary, in the absence of OTA or with other molecules, the hairpins of H1 and H2 cannot open, thus displaying a relatively low background signal. 3.2. Experiments to determine the optimal detection conditions 3.2.1. Determine the amount of substrate As shown in Fig. S1, when the concentration of hemin was determined as 150 nM, the absorbance of solution is increased with increasing the amount of TMB substrate from 0 to 150 mL, and reached maximum above 125 mL. This is because that more substrates are able to produce more colored products in the presence of enzyme. With the aim of detecting low concentrations
3.2.3. Determine the concentration of K+ The stabilization of G-rich sequences of oligonucleotides into quadruplex structures caused by monovalent cations (e.g., K+). Usually DNAzyme fold into G-quadruplex structure before binding to hemin [21,34]. So, for the G-rich DNA sequence used in this experiment, it can be expected that the enzymatic activity will depend on the concentration of K+. As shown in Fig. 1C, the OD450 nm of the solutions in the absence or presence of 0.5 nM OTA were increased with increasing the concentration of K+, similar as Ca2+ determination experiment, in the presence of OTA, the OD450 nm of solution is higher, regardless of the concentration of K+, the differential of OD450 nm (DOD450 nm) of the two measurements also formed a bell-shape curve with a maximum at about 20 mM of K+ (as shown in Fig. 1D). So, 20 mM of K+ was chosen in the next experiments. 3.2.4. Determine the pH value and hybridization time The effects of the solution pH value and hybridization time on the detection of OTA were also examined (as shown in Fig. 1E and F). The experimental results indicate that the detection sensitivity is increased by increasing the solution pH value in the range of 5.4–8.4 (as shown in Fig. 1E). This is due to in the alkaline solution, OTA is negatively charged. The interaction of Ca2+ with negatively charged carboxyl or hydroxyl groups of OTA and negatively charged phosphate backbone of aptamers are dramatically increased [30]. However, Ca2+ might react with OH and form complexes in a more alkaline environment, so pH 8.4 was selected as the optimized solution pH value for detecting OTA. According to previous reports [35–37], the interaction between OTA and aptamer was fulfilled within 3–15 min; therefore, to save the
Scheme 1. Schematic illustration of OTA induced HCR process and the OTA detection mechanism. The illustration is not drawn to scale.
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Fig. 1. The experiment to determine the optimal detection conditions. The OD450 nm of the solution as a function of the concentrations of Ca2+ (A) and K+ (C) in the absence or presence of 0.5 nM OTA, and the differentials of these two measurements (DOD450 nm) as a function of the concentrations of Ca2+ (B) and K+ (D). The OD450 nm of the solutions as a function of the pH values (E) and the hybridization time of H1 and H2 (F) in the presence of 0.5 nM OTA. The experimental conditions: 50 nM H1 and H2, 200 mM Na+, 150 nM hemin, 20 mM K+ and 10 mM Ca2+. The error bars are standard deviations (n = 3).
OTA measurement time, we chose 10 min as the interaction time between OTA and aptamer. Meanwhile, the H1 and H2 hybridization extent was increased with the incubation time increasing from 15 min to 90 min, and reached a maximum at about 60 min (as shown in Fig. 1F). So, 60 min was selected in the next experiments. 3.3. Colorimetric biosensing of OTA By using aptamer and DNAzyme based aptasensor, the signal amplification experiments utilizing HCR were performed to determine the concentration of OTA in solution under the optimized experimental conditions. As can be seen in Fig. 2 and Fig. S2, the color intensity and the OD450 nm of the solution increased with increasing the concentration of OTA from 0.01 to 10 nM, and a linear correlation was obtained between the OD450 nm and the concentration of OTA from 0.01 to 0.32 nM (OD450 nm = 0.023 log [OTA] + 0.205, ([OTA] is the concentration of OTA (nM)), with the correlation coefficient of 0.998). A limit of detection (LOD) of 0.01 nM (3d) was obtained. This LOD is lower or equal to the previous reports (as shown in Table 1), and may satisfy the requirements for quantitative measurement of OTA in agrofood. As the LOD of the aptasensor based method is much lower, so
Fig. 2. OTA detection experiment. The OD450 nm of the solution as a function of the concentrations of OTA. Inset shows the corresponding digital photographs. Other experimental conditions: 50 nM H1 and H2, 200 mM Na+, 150 nM hemin, 20 mM K+ and 10 mM Ca2+. The error bars are standard deviations (n = 5).
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Linear range
Limit of detection
Reference
Fluorescent assay Fluorescent assay Fluorescent assay Fluorescent assay Electrochemistry Electrochemistry Colorimetric assay Colorimetric assay
25–200 nM 0.25–2.5 nM 2.5–250 nM 2.5 nM–250 mM 0.1–3 nM 0.28–37.5 nM 2.5–10 nM 0.01–3.2 nM
24.1 nM 0.05 nM 2 nM 2.5 nM 0.1 nM 0.25 nM 2.5 nM 0.01 nM
[9] [29] [38] [39] [30] [40] [21] This work
when the concentrations of OTA in the real samples are not in the linear range of our method (i.e., much higher than 0.32 nM), we can simply dilute the original solution with buffer, then determine its concentration with a dilution factor. The specificity of the method was also conducted, as shown in Fig. 3, in the absence of H1 or H2,
5
Table 2 Application of HCR based colorimetric method for OTA determination in real samples (n = 3). Sample no. Yellow Yellow Yellow Wheat Wheat Wheat
rice wine 1 rice wine 2 rice wine 3 grain 1 grain 2 grain 3
Added (nM)
Detected (nM)
Recovery (%)
RSD (%)
0.05 0.1 0.2 0.05 0.1 0.2
0.048 0.115 0.225 0.056 0.093 0.212
96 115 113 112 93 106
9.6 5.4 6.7 8.1 7.5 8.9
or replacing H2 with a random oligonucleotide named DNA3, or replacing the target OTA with Tris–HCl buffer or BSA, the OD450 nm of the solutions were relatively lower than the solutions in the presence of H1, H2 and OTA, this experimental result proved that the method is aptamer and DNAzyme based and is specific for OTA detection. At the same time, the HCR between H1 and H2 is essential for the generation of amplified colorimetric signals. 3.4. The electrophoresis experiment
Fig. 3. Selectivity experiment. The concentration of H1, H2 or random oligonucleotide DNA3 is 50 nM, the concentration of OTA is 0.5 nM, and the concentration of BSA is 5 ng mL1. Other experimental conditions: 200 mM Na+, 150 nM hemin, 20 mM K+ and 10 mM Ca2+. The error bars are standard deviations (n = 3).
The HCR between the designed hairpin aptasensors was examined by gel electrophoresis. Electrophoresis gel images showed the products formed by HCR in the presence of various concentrations of OTA. As shown in Fig. 4, from left to right, it could be observed that the molecular weights were similar when H1 or H2 was absent (lanes 1 and 2), indicating that no OTA induced HCR occurs. As a comparison, species with larger molecular weight were appeared when higher concentrations of OTA were mixed with H1 and H2 (lanes 3–5), suggesting that HCR had taken place and formed DNA polymers. In the HCR system, amplification of the initiator recognition event continued until the supply of H1 and H2 was exhausted, and the molecular weight of the resulting products was increased, consequently. The gel electrophoresis experimental results further confirmed the feasibility of the colorimetric method. 3.5. Analytical application in real samples To demonstrate the potential application of the aptamer and DNAzyme based method in real sample detection. The amounts of OTA in yellow rice wine and wheat flour were determined by the standard addition method, the samples were spiked with different concentrations of OTA, then treated using double liquid–liquid extraction and double solvents extraction [26,29]. After the OTA added in the real samples was transferred to buffer solution, the amount of OTA was determined using the linear standard curves obtained in Section 3.3. The recovery of these measurements is in the range of 93–115% (see Table 2), indicating that this method is reliable and practical. 4. Conclusions
Fig. 4. Electrophoresis experiment. The gel electrophoresis images for different concentrations of OTA. From left to right: 50 nM H1 + 0.5 nM OTA (1); 50 nM H2 + 0.5 nM OTA (2); 50 nM H1 + 50 nM H2 + 0 nM (3), 0.02 nM (4) and 0.1 nM (5) OTA, respectively.
In conclusion, combining with the specific recognition ability of aptamer to OTA, the unique catalytical property of HRP mimicking DNAzyme, and the amplification effect of HCR technique, we have developed a colorimetric method for the detection of OTA. Compared with previously reported methods, our approach is simple and has reasonable sensitivity and selectivity (see Table 1). Furthermore, since this experiment is performed in standard 96 wells microtiter plates, it would open up new possibility to develop high throughput technology for the detection of other mycotoxins and hold great potential for the preparation of test kits. Although OTA is chosen here to establish this method by proof-ofprinciple experiment, our approach is readily transferable to real
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analytical problems, such as food safety analysis and quality screening. Meanwhile, it holds promising potential for broad applications in biochemical and biomedical study, environmental monitoring and clinical applications. Acknowledgements The authors would like to thank the NSFC (Nos. 21375050,21305032 and 21305052), China Postdoctoral Science Foundation (No. 2014M551522), Research Foundation of Jiangsu University (No. 13JDG069) and Qing Lan Project for financial supports. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.12.031. References [1] X. Wu, J. Hu, B. Zhu, L. Lu, X. Huang, D. Pang, Aptamer-targeted magnetic nanospheres as a solid-phase extraction sorbent for determination of ochratoxin A in food samples, J. Chromatogr. A 1218 (2011) 7341–7346. [2] A. El Khoury, A. Atoui, Ochratoxin A: general overview and actual molecular status, Toxins 2 (2010) 461–493. [3] A. Pfohl-Leszkowicz, R.A. Manderville, Ochratoxin A: an overview on toxicity and carcinogenicity in animals and humans, Mol. Nutr. Food Res. 51 (2007) 61–99. [4] N.W. Turner, S. Subrahmanyam, S.A. Piletsky, Analytical methods for determination of mycotoxins: a review, Anal. Chim. Acta 632 (2009) 168–180. [5] L.C. Huang, N. Zheng, B.Q. Zheng, F. Wen, J.B. Cheng, R.W. Han, X.M. Xu, S.L. Li, J. Q. Wang, Simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and a-zearalenol in milk by UHPLC-MS/MS, Food Chem. 146 (2014) 242–249. [6] A. Roland, P. Bros, A. Bouisseau, F. Cavelier, R. Schneider, Analysis of ochratoxin A in grapes, musts and wines by LC-MS/MS: first comparison of stable isotope dilution assay and diastereomeric dilution assay methods, Anal. Chim. Acta 818 (2014) 39–45. [7] F. Yu, T. Chi, B. Liu, C. Su, Development of a sensitive enzyme-linked immunosorbent assay for the determination of ochratoxin A, J. Agric. Food Chem. 53 (2005) 6947–6953. [8] P.M. Scott, Methods of Analysis for Ochratoxin A, Mycotoxins and Food Safety, Springer, 2002 pp. 117–134. [9] Z. Guo, J. Ren, J. Wang, E. Wang, Single-walled carbon nanotubes based quenching of free FAM-aptamer for selective determination of ochratoxin A, Talanta 85 (2011) 2517–2521. [10] N. Duan, S.-J. Wu, Z.-P. Wang, An aptamer-based fluorescence assay for ochratoxin A, Chin. J. Anal. Chem. 39 (2011) 300–304. [11] L. Bonel, J.C. Vidal, P. Duato, J.R. Castillo, An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer, Biosens. Bioelectron. 26 (2011) 3254–3259. [12] P. Tong, L. Zhang, J.-J. Xu, H.-Y. Chen, Simply amplified electrochemical aptasensor of ochratoxin A based on exonuclease-catalyzed target recycling, Biosens. Bioelectron. 29 (2011) 97–101. [13] L. Yotova, I. Grabchev, R. Betcheva, D. Marinkova, Smart Biosensors for Determination of Mycotoxines, Detection of Bacteria, Viruses, Parasites and Fungi, Springer, 2010 pp. 389–414. [14] L. Wang, W. Ma, W. Chen, L. Liu, W. Ma, Y. Zhu, L. Xu, H. Kuang, C. Xu, An aptamer-based chromatographic strip assay for sensitive toxin semiquantitative detection, Biosens. Bioelectron. 26 (2011) 3059–3062. [15] B. Deng, Y. Lin, C. Wang, F. Li, Z. Wang, H. Zhang, X.-F. Li, X.C. Le, Aptamer binding assays for proteins: the thrombin example-a review, Anal. Chim. Acta 837 (2014) 1–15. [16] M. Chen, S. Bi, X.Q. Jia, P. He, Aptamer-conjugated bio-bar-code Au-Fe3O4 nanoparticles as amplification station for electrochemiluminescence detection of tumor cells, Anal. Chim. Acta 837 (2014) 44–51.
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Analytica Chimica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction Chengke Wang a , Xiaoya Dong b , Qian Liu b , Kun Wang b, * a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Ochratoxin A can be detected with low limit of detection utilizing the aptasensor. Hybridization chain reaction between DNAs was used to amplify the detection signal. Standard microtiter plates were used to develop high throughput analytical method. The method is label-free, antibodyfree, colorimetric, simple and costsaving.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 October 2014 Received in revised form 14 December 2014 Accepted 15 December 2014 Available online xxx
The combination of high selectivity of aptamer with the peroxidase-mimicking property of DNAzyme has presented considerable opportunities for designing colorimetric aptasensor for detection of ochratoxin A (OTA). The activities of both aptamer (as biorecognition element) and DNAzyme (as signal amplification element) are blocked via base pairing in the hairpin structure. Hybridization chain reaction (HCR) between two hairpin DNAs was employed to further improve the sensitivity of this method. The presence of OTA triggers the opening of the hairpin structure and the beginning of HCR, which results in the release of many DNAzyme, and generates enhanced colorimetric signals, which is correlated to the amounts of OTA with linear range between 0.01 to 0.32 nM, and the limit of detection is 0.01 nM under optimal conditions. OTA in yellow rice wine and wheat flour samples was also detected using this method. We demonstrate that a new colorimetric method for the detection of OTA has been established, which is simple, easy to conduct, label-free, sensitive, high throughput, and cost-saving. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Ochratoxin A Hybridization chain reaction Aptamer Colorimetric method
1. Introduction Ochratoxin A (OTA) is a kind of mycotoxin, which can be found in a large number of food commodities including cereals, wheat, barley, corn, coffee, and wine [1]. As OTA has nephrotoxicity,
* Corresponding author. Tel.: +86 511 88791800; fax: +86 511 88791708. E-mail address: [email protected] (K. Wang).
hepatic toxicity, immune toxicity and mutagenic effects on humans and animals [2,3], it is important to find a method to sensitively detect OTA in agro-foods. Nowadays, high performance liquid chromatography (HPLC) [4], mass spectrometry (MS) [5,6] and antibody based method [7] were employed to detect OTA, despite the accuracy and low limit of detection of these methods, they often require sophisticated equipments, time and laborconsuming and trained operators [8], which hinders their widely usage in high throughput detection. To overcome these drawbacks,
http://dx.doi.org/10.1016/j.aca.2014.12.031 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
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fluorescent spectroscopy [9,10], electrochemistry [11,12] were employed to detect OTA, although they are easy to conduct, but the sensors used in these experiments often need molecule modification procedure (e.g., fluorescent molecule and enzyme modification process), which is not convenient for OTA detection. So, it is necessary and urgent to develop a simple or no instrumentation yet still providing accurate analysis of OTA in food derivatives [13]. In recent years, aptamer based colorimetric assay were used to detect different kinds of analytes, such as toxin [14], protein [15], cancer cell [16] and heavy metal ion [17]. Due to its inherent selectivity and affinity to detected targets and easy to prepare, aptamer can be used to replace antibodies as recognition elements, which reduce the cost of detection [18]. As an alternative of enzyme, a segment of DNA, termed G-rich horseradish peroxidase (HRP)-mimicking DNAzyme, can form a four-strand structure known as G-quadruplex. After binding with hemin, these hemin–G-quadruplex complexes may display a highly enhanced catalytical activity, which catalyze substrate (e.g., tetramethylbenzidine (TMB)) oxidation and generate a colorimetric signal, providing a label-free mode to amplify the detection signal [19,20]. For example, Yang et al. have developed a DNAzyme based colorimetric method to detect OTA without using expensive enzyme [21]. Recently, hybridization chain reaction (HCR) was applied in many analytical experiments. HCR is based on the chain reaction of recognition and hybridization events between two sets of DNA hairpin molecules, and provide a new route to amplify the intensity of detection signal [22,23]. For example, Liu et al. have detected Escherichia coli uropathogen related DNA using HCR between two auxiliary hairpin DNAs, which is more sensitive than the non-HCR experiments [24]; Zhang et al. have designed two ferrocene modified hairpin structure DNAs to carry out HCR and utilized HCR based electrochemistry to detect human immunoglobulin G (IgG), the more target IgG presented in the solution, the more ferrocenes can be detected, which correlated to stronger electrical signals, the method possesses much higher sensitivity compared with previous experiments [25]. Although aptamer and DNAzyme were used to sensitively detect series of analytes [26,27], and HCR was used to amplify the detection signals in various experiments [24,25,28], to our knowledge, there is no report that combining aptamer and DNAzyme into aptasensors, and using HCR to further amplify the signals to detect OTA in agro-food. As it is necessary and urgent to develop a simple and sensitive method for the detection of OTA, in this regard, a new label-free colorimetric aptasensor for sensitive detection of OTA utilizing HCR has been developed in this experiment. In this aptasensor, aptamer was used as recognition element, and DNAzyme was used as signal amplification element. We believe that this strategy opens up a new avenue for the application of aptamer and DNAzyme in the field of food safety and analysis and food quality screening. 2. Experimental 2.1. Reagents NaCl, CaCl2, KCl, and tris(hydroxymethyl)aminomethane ((HOCH2)3CNH2, (Tris)) were purchased from Dingguo Inc. (Beijing, China). OTA was purchased from Pribolab Inc. (Singapore) 3,30 ,5,50 tetramethylbenzidine (TMB) substrate and CH3COOH (AcOH, acetic acid) were purchased from Aladdin Inc. (Beijing, China). Bovine serum albumin (BSA) was purchased from Sigma–Aldrich Inc. (USA). The ssDNA oligonucleotides and the 4S GreenTM nucleic acid stain were purchased from Sangon Biotech Inc. (Shanghai, China). The sequence of aptamer contained DNA (H1) was 50 -GATCG GGTGT GGGTG GCGTA AAGGG AGCAT CGGAC ACGCC ACCCA CAC30 , the sequence with italic is OTA aptamer, and the sequence with
underline could form hairpin structure. The sequence of the DNAzyme contained DNA (H2) was 30 -GCGGT GGGTG TGGGG AGGGA GGGAG GGTCC TAGCC CACAC C-50 , the sequence with italic could form DNAzyme, and the sequence with underline could form hairpin structure, respectively. The sequence of a random oligonucleotide (DNA3) was 30 -GCGGT GAGTG TGATG TGAGT GGCGA GGAGT GTGGG TATTG G-50 . The DNA stock solution was obtained by dissolving the oligonucleotide in 10 mM Tris–HCl buffer containing 200 mM NaCl (pH 8.4) and was stored at 4 C before use. 96 wells microtiter plates were obtained from Corning Inc. (USA). The water used throughout all experiments was doubledistilled water. All other chemicals were at least of analytical grade and used as received. 2.2. Apparatus Colorimetric measurements were performed with a Labsystems Multiskan MK3 microtiter plate reader (Thermo Life Sciences, USA). Gel electrophoresis experiment was carried out using a DYY6C electrophoresis system (Nanjing, China). The DNA bands were recorded with a Haiqing digital camera system (Nanjing, China). 2.3. Detection of OTA using colorimetric biosensing method The two DNA, H1 and H2 were heated to 95 C for 5 min separately and allowed to cool to room temperature slowly before use otherwise mentioned. In a typical experiment, 80 mL of solution containing 62.5 nM of H1 and different concentrations of analyte (OTA) in 10 mM Tris–HCl buffer (pH 8.4) with 200 mM NaCl, 10 mM CaCl2, 20 mM KCl and 150 nM hemin were added into 96-well microtiter plate and incubated for 10 min at 37 C. After that, 20 mL of 0.25 mM H2 was added, mixed and incubated for 1 h at 37 C, and then 80 mL of TMB substrate (including H2O2) was added and mixed adequately, after incubated for 30 min, 50 mL of 1 M HCl was added and mixed adequately to stop the reaction and make the color of solution turned to yellow. Subsequently, absorbance of the solution was measured at wavelength of 450 nm (OD450 nm). All experiments were performed at room temperature unless otherwise specified. 2.4. OTA detection in yellow rice wine and wheat flour samples The detection solution was composed of 625 mM H1, 100 mM CaCl2, 200 mM KCl and 1.5 mM hemin. 70 mL of OTA contained wine samples or wheat flour samples processed with extraction (the details of sample pretreatment procedure were shown in the Supporting information) were added into the 96-well microtiter plate, and then 10 mL detection solution was added [21,26,29]. After mixing, the microtiter plates were left to react for 10 min. After that, 20 mL of 0.25 mM H2 was added, mixed and incubated for 1 h, and then 80 mL of TMB substrate (including H2O2) was added and mixed, after incubated for 30 min, 50 mL of 1 M HCl was added and mixed adequately. Subsequently, absorbance of the solution was recorded at wavelength of 450 nm. All experiments were performed at room temperature unless otherwise specified. 2.5. Gel electrophoresis experiment Different concentrations of OTA were incubated with H1 at 37 C for 10 min in the presence of 10 mM Ca2+ and 20 mM K+. Then H2 was added and reacted at 37 C for 1 h, the final concentrate of H1 and H2 was 50 nM. A 3.5% (w/v) agarose gel was prepared using a 1 TAE buffer (40 mM Tris AcOH, 2.0 mM EDTA, pH 8.4). The 4S GreenTM nucleic acid stain was used as the oligonucleotide dye and mixed with samples. 10.0 mL samples were introduced into each band using pipette. The gel was run at 80 V for 45 min in a 1 TAE
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buffer and was then scanned using the Haiqing gel image analysis system.
of OTA, from the measurement presented here, 80 mL TMB substrate was used for the next experiments.
3. Results and discussion
3.2.2. Determine the concentration of Ca2+ As previously reported, divalent cations, such as Ca2+ or Mg2+ is essential for the recognition of aptamer with OTA in solution [32,33]. Here we studied the effect of Ca2+ on the detection of OTA. The optimal concentration of Ca2+ is determined by measuring the OD450 nm of the solutions in the absence and presence of 0.5 nM OTA. As shown in Fig. 1A, with increasing the concentration of Ca2+, the OD450 nm of solutions was increased, consequently. However, in the presence of OTA, the enzymatic activity is substantially increased. Similar to our work, the enhanced binding of OTA by aptamer in the presence of Ca2+ was reported in previous reports [11,32]. As shown in Fig. 1B, the differentials of these two measurements (DOD450 nm) resulted in a bell-shape curve with a maximum at about 10 mM Ca2+. So, 10 mM Ca2+ was used in the next experiments.
3.1. Principle of OTA detection The principle of this method is depicted in Scheme 1. Two hairpin oligonucleotides (H1 and H2) were designed to form aptasensor in this proof-of-concept experiment. Especially, aptamer is at the 50 end of H1, and DNAzyme is at the central of H2. Meanwhile, the fragment at the 30 end of H1 is complementary to the 30 end of H2, and the 50 end of H2 is complementary to the 50 end of H1. During the measurement of OTA concentration, after adding OTA into H1 solution in the presence of Ca2+, the OTAaptamer complex is formed and the hairpin of H1 is opened, the newly exposed 30 end of H1 hybridized with 30 end of H2, and opens the hairpin to expose the 50 end of H2, which can in turn hybridized with 50 end of H1; when the hairpin of H2 opened, the DNAzyme segment, upon the addition of K+ and hemin, will form a hemin–Gquadruplex complex which behaves as a kind of HRP-mimicking DNAzyme and displays a highly enhanced catalytic activity compared with hemin itself. In this way, each copy of the OTA can propagate a chain reaction of hybridization events between alternating H1 and H2 hairpins to form a double helix, which contains lots of DNAzyme units. During the measurement, each of the DNAzyme units can catalyze H2O2 oxidize TMB and produce colored products, leading to an amplified signal. As the microtiter plate reader used here cannot maintain the reaction solution at a constant temperature, this will dramatically affect the activity of DNAzyme, and result in the fluctuation of the signals. In addition, for the practical application, to make the measurement of OTA easy to conduct, we chose the absorbance of the solution at 450 nm after adding HCl to determine the concentration of OTA, rather than monitoring the absorbance of the solution at 620 nm for a long time [30,31]. By this means, we can indirectly determine the concentration of OTA with high sensitivity. On the contrary, in the absence of OTA or with other molecules, the hairpins of H1 and H2 cannot open, thus displaying a relatively low background signal. 3.2. Experiments to determine the optimal detection conditions 3.2.1. Determine the amount of substrate As shown in Fig. S1, when the concentration of hemin was determined as 150 nM, the absorbance of solution is increased with increasing the amount of TMB substrate from 0 to 150 mL, and reached maximum above 125 mL. This is because that more substrates are able to produce more colored products in the presence of enzyme. With the aim of detecting low concentrations
3.2.3. Determine the concentration of K+ The stabilization of G-rich sequences of oligonucleotides into quadruplex structures caused by monovalent cations (e.g., K+). Usually DNAzyme fold into G-quadruplex structure before binding to hemin [21,34]. So, for the G-rich DNA sequence used in this experiment, it can be expected that the enzymatic activity will depend on the concentration of K+. As shown in Fig. 1C, the OD450 nm of the solutions in the absence or presence of 0.5 nM OTA were increased with increasing the concentration of K+, similar as Ca2+ determination experiment, in the presence of OTA, the OD450 nm of solution is higher, regardless of the concentration of K+, the differential of OD450 nm (DOD450 nm) of the two measurements also formed a bell-shape curve with a maximum at about 20 mM of K+ (as shown in Fig. 1D). So, 20 mM of K+ was chosen in the next experiments. 3.2.4. Determine the pH value and hybridization time The effects of the solution pH value and hybridization time on the detection of OTA were also examined (as shown in Fig. 1E and F). The experimental results indicate that the detection sensitivity is increased by increasing the solution pH value in the range of 5.4–8.4 (as shown in Fig. 1E). This is due to in the alkaline solution, OTA is negatively charged. The interaction of Ca2+ with negatively charged carboxyl or hydroxyl groups of OTA and negatively charged phosphate backbone of aptamers are dramatically increased [30]. However, Ca2+ might react with OH and form complexes in a more alkaline environment, so pH 8.4 was selected as the optimized solution pH value for detecting OTA. According to previous reports [35–37], the interaction between OTA and aptamer was fulfilled within 3–15 min; therefore, to save the
Scheme 1. Schematic illustration of OTA induced HCR process and the OTA detection mechanism. The illustration is not drawn to scale.
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Fig. 1. The experiment to determine the optimal detection conditions. The OD450 nm of the solution as a function of the concentrations of Ca2+ (A) and K+ (C) in the absence or presence of 0.5 nM OTA, and the differentials of these two measurements (DOD450 nm) as a function of the concentrations of Ca2+ (B) and K+ (D). The OD450 nm of the solutions as a function of the pH values (E) and the hybridization time of H1 and H2 (F) in the presence of 0.5 nM OTA. The experimental conditions: 50 nM H1 and H2, 200 mM Na+, 150 nM hemin, 20 mM K+ and 10 mM Ca2+. The error bars are standard deviations (n = 3).
OTA measurement time, we chose 10 min as the interaction time between OTA and aptamer. Meanwhile, the H1 and H2 hybridization extent was increased with the incubation time increasing from 15 min to 90 min, and reached a maximum at about 60 min (as shown in Fig. 1F). So, 60 min was selected in the next experiments. 3.3. Colorimetric biosensing of OTA By using aptamer and DNAzyme based aptasensor, the signal amplification experiments utilizing HCR were performed to determine the concentration of OTA in solution under the optimized experimental conditions. As can be seen in Fig. 2 and Fig. S2, the color intensity and the OD450 nm of the solution increased with increasing the concentration of OTA from 0.01 to 10 nM, and a linear correlation was obtained between the OD450 nm and the concentration of OTA from 0.01 to 0.32 nM (OD450 nm = 0.023 log [OTA] + 0.205, ([OTA] is the concentration of OTA (nM)), with the correlation coefficient of 0.998). A limit of detection (LOD) of 0.01 nM (3d) was obtained. This LOD is lower or equal to the previous reports (as shown in Table 1), and may satisfy the requirements for quantitative measurement of OTA in agrofood. As the LOD of the aptasensor based method is much lower, so
Fig. 2. OTA detection experiment. The OD450 nm of the solution as a function of the concentrations of OTA. Inset shows the corresponding digital photographs. Other experimental conditions: 50 nM H1 and H2, 200 mM Na+, 150 nM hemin, 20 mM K+ and 10 mM Ca2+. The error bars are standard deviations (n = 5).
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C. Wang et al. / Analytica Chimica Acta xxx (2014) xxx–xxx Table 1 Comparison of previous reports and this assay. Analytical methods
Linear range
Limit of detection
Reference
Fluorescent assay Fluorescent assay Fluorescent assay Fluorescent assay Electrochemistry Electrochemistry Colorimetric assay Colorimetric assay
25–200 nM 0.25–2.5 nM 2.5–250 nM 2.5 nM–250 mM 0.1–3 nM 0.28–37.5 nM 2.5–10 nM 0.01–3.2 nM
24.1 nM 0.05 nM 2 nM 2.5 nM 0.1 nM 0.25 nM 2.5 nM 0.01 nM
[9] [29] [38] [39] [30] [40] [21] This work
when the concentrations of OTA in the real samples are not in the linear range of our method (i.e., much higher than 0.32 nM), we can simply dilute the original solution with buffer, then determine its concentration with a dilution factor. The specificity of the method was also conducted, as shown in Fig. 3, in the absence of H1 or H2,
5
Table 2 Application of HCR based colorimetric method for OTA determination in real samples (n = 3). Sample no. Yellow Yellow Yellow Wheat Wheat Wheat
rice wine 1 rice wine 2 rice wine 3 grain 1 grain 2 grain 3
Added (nM)
Detected (nM)
Recovery (%)
RSD (%)
0.05 0.1 0.2 0.05 0.1 0.2
0.048 0.115 0.225 0.056 0.093 0.212
96 115 113 112 93 106
9.6 5.4 6.7 8.1 7.5 8.9
or replacing H2 with a random oligonucleotide named DNA3, or replacing the target OTA with Tris–HCl buffer or BSA, the OD450 nm of the solutions were relatively lower than the solutions in the presence of H1, H2 and OTA, this experimental result proved that the method is aptamer and DNAzyme based and is specific for OTA detection. At the same time, the HCR between H1 and H2 is essential for the generation of amplified colorimetric signals. 3.4. The electrophoresis experiment
Fig. 3. Selectivity experiment. The concentration of H1, H2 or random oligonucleotide DNA3 is 50 nM, the concentration of OTA is 0.5 nM, and the concentration of BSA is 5 ng mL1. Other experimental conditions: 200 mM Na+, 150 nM hemin, 20 mM K+ and 10 mM Ca2+. The error bars are standard deviations (n = 3).
The HCR between the designed hairpin aptasensors was examined by gel electrophoresis. Electrophoresis gel images showed the products formed by HCR in the presence of various concentrations of OTA. As shown in Fig. 4, from left to right, it could be observed that the molecular weights were similar when H1 or H2 was absent (lanes 1 and 2), indicating that no OTA induced HCR occurs. As a comparison, species with larger molecular weight were appeared when higher concentrations of OTA were mixed with H1 and H2 (lanes 3–5), suggesting that HCR had taken place and formed DNA polymers. In the HCR system, amplification of the initiator recognition event continued until the supply of H1 and H2 was exhausted, and the molecular weight of the resulting products was increased, consequently. The gel electrophoresis experimental results further confirmed the feasibility of the colorimetric method. 3.5. Analytical application in real samples To demonstrate the potential application of the aptamer and DNAzyme based method in real sample detection. The amounts of OTA in yellow rice wine and wheat flour were determined by the standard addition method, the samples were spiked with different concentrations of OTA, then treated using double liquid–liquid extraction and double solvents extraction [26,29]. After the OTA added in the real samples was transferred to buffer solution, the amount of OTA was determined using the linear standard curves obtained in Section 3.3. The recovery of these measurements is in the range of 93–115% (see Table 2), indicating that this method is reliable and practical. 4. Conclusions
Fig. 4. Electrophoresis experiment. The gel electrophoresis images for different concentrations of OTA. From left to right: 50 nM H1 + 0.5 nM OTA (1); 50 nM H2 + 0.5 nM OTA (2); 50 nM H1 + 50 nM H2 + 0 nM (3), 0.02 nM (4) and 0.1 nM (5) OTA, respectively.
In conclusion, combining with the specific recognition ability of aptamer to OTA, the unique catalytical property of HRP mimicking DNAzyme, and the amplification effect of HCR technique, we have developed a colorimetric method for the detection of OTA. Compared with previously reported methods, our approach is simple and has reasonable sensitivity and selectivity (see Table 1). Furthermore, since this experiment is performed in standard 96 wells microtiter plates, it would open up new possibility to develop high throughput technology for the detection of other mycotoxins and hold great potential for the preparation of test kits. Although OTA is chosen here to establish this method by proof-ofprinciple experiment, our approach is readily transferable to real
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