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A highly sensitive aptasensor for OTA detection based on hybridization chain reaction and fluorescent perylene probe
Biosensors and Bioelectronics 81 (2016) 125–130
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A highly sensitive aptasensor for OTA detection based on hybridization chain reaction and fluorescent perylene probe Bin Wang a,b,c,n, Yuanya Wu a,c, Yanfen Chen a,c, Bo Weng a,c,n, Liqun Xu a,c, Changming Li a,c a
Faculty of Materials and Energy, Southwest University, Chongqing 400715, China Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Wuhan University, Wuhan 430072, China c Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Chongqing 400715, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 December 2015 Received in revised form 19 February 2016 Accepted 23 February 2016 Available online 24 February 2016
An optical aptasensor was developed for ultrasensitive detection of ochratoxin A (OTA) based on hybridization chain reaction (HCR) amplification strategy and fluorescent perylene probe (PAPDI)/DNA composites. Dendritic DNA concatamers were synthesized by HCR strategy and modified on magnetic nanoparticles through aptamer as medium. A large amount of PAPDI probe aggregated under the induction of DNA concatamers and caused fluorescence quenching. In the presence of OTA, the PAPDI/DNA composites were released from magnetic nanoparticles due to the strong affinity between aptamer and OTA. In ethanol, PAPDI monomers disaggregated and produced strong fluorescence. The present method displays excellent sensitivity and selectivity towards OTA. & 2016 Elsevier B.V. All rights reserved.
Keywords: Optical aptasensor Perylene probe Aptamer OTA Hybridization chain reaction
1. Introduction Ochratoxin A (OTA) has been reported as one of the most toxic and cancerigenic substances to a wide variety of mammalian species (Duarte et al., 2010a). However, it was found that a series of food products (including cereals, wheat, barley, beans, corn, coffee and wine) were susceptible to OTA contamination (Duarte et al., 2010b). OTA contamination in food samples would not cause any observable physical damages (decay, mildew etc.) at the start of the infection. Thus it was hard to diagnose OTA at the initial stage of infection to avoid further damage (McKeague et al., 2014). It is essential to develop a sensitive method for low level OTA detection. Recently aptamer-based detection methods have been rapidly developed due to their noticeable merits such as high binding affinity, ease labeling, remarkable target diversity, convenient automated-synthesis and high stability (Jiang et al., 2014). A variety of colorimetric, electrochemical, fluorescent, and surface plasmon resonance biosensors with high sensitivity (Lee et al., 2014; Rivas et al., 2015; Wang et al., 2015b; Zhu et al., 2015) have been developed for the detection of low concentration of OTA. Furthermore, a series of signal amplification strategies have been developed to realize ultrasensitive and trace determination n Corresponding author at: Faculty of Materials and Energy, Southwest University, Chongqing 400715, China. E-mail addresses: [email protected] (B. Wang), [email protected] (B. Weng).
http://dx.doi.org/10.1016/j.bios.2016.02.062 0956-5663/& 2016 Elsevier B.V. All rights reserved.
of OTA in foods and drinks (Hun et al., 2013; Jiang et al. 2013). The most common strategy to amplify the signals in mycotoxin detection is using catalysts (such as enzyme). For example, rollingcircle amplification (RCA) is an isothermal nucleic acid amplification process that synthesized a long single-stranded nucleic acid product based on DNA polymerase and a circular, single-stranded nucleic acid template (Huang et al., 2013). The electrochemical aptasensor based on RCA displayed super high sensitivity with the limit of detection of 0.065 pg/mL for OTA detection (Huang et al., 2013); Another amplification technique depending on exonucleases catalysed the stepwise removal of mononucleotides from the 3′-end or 5′-end of single-stranded or double-stranded DNA. Yang et al. (2014) constructed OTA aptasensor based on exonuclease-catalysed target recycling amplification. OTA could be sensitively quantified by the recovery of the quantum dot electrochemiluminescence (ECL) with a detection limit of 0.64 pg/mL by this method; Nicking endonuclease, which is an enzyme cutting one strand of a dsDNA at a restriction site, could also be utilized in OTA detection. The OTA aptasensor based on target-induced strand release coupling cleavage of nicking endonuclease was developed by Hun et al. (2013). In this design, DNA polymerase and nicking endonuclease were utilized to extend and cleave DNA strand and produced an amount of targeted ssDNA; Additionally, loop-mediated isothermal amplification (LAMP), as an important nucleic acid amplification technique, has been designed for electrochemically aptasensing of OTA too (Xie et al., 2014). In their design, aptamers were utilized as the forward outer primer to trigger the LAMP reaction, and DNA polymerase was used to catalyse the extension
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of DNA and strand displacement reaction. This strategy displayed excellent sensitivity and selectivity with a detection limit of 0.3 pM. In comparison with above amplification strategies, Hybridization Chain Reaction (HCR) method is the most attractive among them due to its enzyme-free process and kinetics-controlled reaction (Zhao et al., 2015). In HCR, long double-strand DNA structure was constructed by hybridization of different oligonucleotides. HCR amplification strategy has been widely used in the detection of DNA, proteins, biothiols, metal ions, virus, cancer cells, and endonuclease activities (Yang et al., 2015; Ge et al., 2014). However, OTA detection using HCR amplification strategy has not been fully investigated (Wang et al., 2015a) yet. Further optimization and improvement are still required for low level OTA detection. Perylene tetracarboxylic acid diimide (PDI) derivatives have been reported as fluorescence probe due to their high fluorescence quantum yield and photostability (Würthner, 2004; Wang and Yu, 2010). It has been proved that PDI derivatives were remarkable fluorescent probe for optical sensor (Wang et al., 2014; Li et al., 2015; Wang et al., 2011). In this work, a highly sensitive fluorescent aptasensor for OTA determination was designed based on branching-growth of HCR strategy and the fluorescent perylene probe. In this design, positively charged perylene derivative, [(N,N ′-bis(propylenetrimethylammonium)-3,4,9,10-perylenediimide), PAPDI] was synthesized and served as fluorescent probe, while dendritic DNA concatamers synthesized by HCR strategy were utilized to induce the aggregation of PDI probe and further amplify the detection signal. OTA could be detected when it was bound with aptamer and released the DNA/PAPDI composite to produce strong fluorescent signals. This strategy was optimized to achieve highly sensitive detection of OTA and verified in the corn sample for its effectiveness for low level OTA detection in practical applications.
2. Experimental 2.1. Chemicals and apparatus Oligonucleotides used in this study were synthesized and purified with ultra PAGE by Sangon Biotechnology Co., Ltd. (Shanghai, China). The oligonucleotides stock solution was prepared by dissolving the oligonucleotide in 10 mM Tris–HCl buffer (pH 8.4, 200 mM NaCl) and was stored at 4 °C before use. Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), aflatoxin A1 (AFB1), and ochratoxin A (OTA) were purchased from Sigma-Aldrich Chemical Co. (USA). Ochratoxin B (OTB), Zearalenone, and Fumidil were purchased from Leon Technology Co. Ltd. (Beijing). All these mycotoxins including OTA, OTB, AFB1, Zearalenone, and Fumidil were dissolved in 80% methanol for further use. Amino-modified magnetic nanoparticles (AMNPs) were purchased from the Aladdin Reagent Co., Ltd. (Shanghai, China). 3,4,9,10-Perylenetetracarboxylic dianhydride (PDI), N,N′-dimethyl-1,3-propanediamine, methyl iodide and tri (hydroxymethyl) amino methane hydrochloride (Tris–HCl) were obtained from Shanghai Chemical Reagent Co. Perylene probe [(N,N′-bis(propylenetrimethylammonium)3,4,9,10-perylenediimide), PAPDI] was synthesized according to our previous report (Wang and Yu, 2010). All other chemicals were of analytical grade. Deionized water (18.2 MΩ cm at 25 °C) was used throughout the experiments. 2.2. Apparatus All fluorescence measurements were performed on a Shimadzu
RF-5301PC spectro-fluorometer equipped with a 150 W xenon lamp as the excitation source (Ushio Inc., Japan). The Zeta potential measurement was carried out at 25 °C (Zetasizer Nano 90, Malvern), and the surface charge of nanoparticles was determined in 100 mM Tris–HCl (pH 7.4, containing 200 mM NaCl). Scanning electron microscopy (SEM) images were taken by JSM-7800F (Japan). 2.3. Preparation of aptamer modified magnetic nanoparticles The AMNPs were functionalized with the OTA aptamer according to literature (Hua et al., 2013). Briefly, 10 nmol carboxylmodified OTA aptamer was dissolved in 100 μL of MES buffer solution (10 mM, pH 5.2). Then, 50 μL of 2 mM EDC and 50 μL of 2 mM NHS in pH 5.2 MES buffer were mixed with the above aptamer solution and incubated for 30 min to activate the carboxyl group. After that, the mixture was injected into 500 μL of 5 mg/mL AMNPs (100 mM Tris–HCl, pH 8.0) and incubated overnight at room temperature with gentle shaking. Then, the aptamer-modified AMNPs were washed three times with TT buffer (100 mM Tris–HCl, Tween-20 0.01%, pH 7.4). The obtained product was dissolved in 1 mL of Tris–HCl (100 mM, pH 7.4) and stored at 4 °C before use. 2.4. Fluorescence detection of OTA Typically, 10 μL of the as-prepared aptamer-AMNPs suspension was mixed with 100 μL of TS buffer (containing 100 mM, pH 7.4, Tris–HCl and 200 mM NaCl). Then, 2 μL of 10 μM DNA1, DNA2, DNA3, DNA4, DNA5, and DNA6 were added consecutively. The final mixture was incubated at 85 °C for 10 min, naturally cooled to room temperature, and incubated at room temperature for another 30 min. Subsequently, the obtained mixture was magnetically separated and washed with 100 μL of TS buffer solution for three times, and dispersed into 100 μL of TS buffer solution. PAPDI (2 μL, 100 μM) was mixed with the above suspension, followed by the magnetic separation and washed for 3 times using the TS buffer solution. The obtained deposit was dissolved in 100 μL of TS buffer. 10 μL prepared PAPDI/DNA/ AMNPs suspension was taken and mixed with 20 μL Tris–HCl (400 mM, pH 7.4) buffer solution, 10 μL 1 M NaCl solution, and 10 μL OTA with different concentrations (the final concentration of methanol was 16%). After incubation at room temperature for 1 h, the dispersion was magnetically separated. Finally, the supernatant was diluted with 400 μL of ethanol. The fluorescence spectra were recorded on a fluorescence spectrometer and standard curve was calculated based on detection results. The same procedure was performed to estimate the selectivity of the method by replacing OTA with other mycotoxins. 2.5. Assay of gel electrophoresis In order to verify the hybridization of DNA on AMNPs, polyacrylamide gel electrophoresis (PAGE) was performed. Firstly, different concentrations of OTA (0, 20 pM, and 1.0 nM) were incubated with DNA/aptamer/AMNPs nanocomposites for 2 h at room temperature. The released DNA concatamers were used for Gel Electrophoresis analysis. A 12.0% native polyacrylamide gel was prepared by using 1 TBE buffer (100 mM Tris–HCl, 83 mM boric acid, 1 mM EDTA, pH 8.0). The gel was run at 100 V for 60 min with the loading of 5 μL of each sample into the lanes at room temperature. After staining with Goldview Nucleic Acid Gel Stain (Biotium, USA) for 30 min, the gels were scanned using a Gel Imaging System (Bio-Rad, USA).
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2.6. Determination of OTA in corn samples In order to evaluate the feasibility and reliability of the developed method, the application for OTA determination in real samples was performed. Corn samples were purchased from local supermarket and finely grounded. Before characterization, 1 g corn samples were extracted by 1 L 80% methanol. The resulted solution was spiked with different concentrations of OTA, and determination was performed by the proposed method. Typically, 10 μL prepared PAPDI/DNA/AMNPs suspension was mixed with 20 μL Tris–HCl (400 mM, pH 7.4) buffer solution, 10 μL 1 M NaCl solution, and 10 μL of the corn samples with different concentrations of OTA. Then, magnetic separation and fluorescent determination of the supernatant were performed after incubating at room temperature for 2 h. The results of three measurements were listed in Table S2.
3. Results and discussion 3.1. Design principle of OTA aptasensor In aqueous environment, fluorescent probe PAPDI tends to aggregate by aromatic π–π stacking interactions due to its planar aromatic structure (Wang and Yu, 2010). It has been reported that polyanionic DNA could induce the aggregation of PAPDI and lead to the fluorescence quenching by electrostatic interaction. Fig. 1 showed the scheme and emission spectra of PAPDI upon the addition of DNA and ethanol. In aqueous environment, PAPDI showed strong fluorescence. Upon the addition of 5 nM DNA, the fluorescence of PAPDI was quenched completely due to the aggregation of PAPDI. The PAPDI/DNA aggregates were dispersed in ethanol solution, PAPDI monomer was disaggregated. As shown in Fig. 1B, with the increase of ethanol ratio in buffer solution, the fluorescence was recovered gradually. Simultaneously blue-shift of emission spectra was observed due to disaggregation of the fluorescent probe. The PAPDI-DNA system displayed stronger fluorescence intensity in 90% ethanol than that in aqueous solution. Meanwhile, the maximum
Fig. 1. A: Scheme of the aggregation and disaggregation of PAPDI in the presence of DNA1 and ethanol; B: the emission spectra of 0.1 μM PAPDI in 10 mM pH 7.4 Tris– HCl (a), in the presence of 5 nM DNA1 (b), the mixture of 0.1 μM PAPDI and 5 nM DNA1 in 20% (c), 40% (d) 60% (e) 80% (f) 90% ethanol aqueous solution (g).
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emission peak shifted from 548 nm (curve a in Fig. 1B) to 537 nm (curve g in Fig. 1B). Therefore, we would like to design an aptasensor for OTA detection based on the theory discussed above and HCR amplification strategy. The detection scheme is shown in Fig. 2. Firstly, carboxylfunctionalized OTA aptamer was modified on amino-modified magnetic nanoparticles (AMNPs) with the assistance of EDC and NHS. Then, a certain proportion of assistant oligonucleotides (DNA1–DNA6) was added to hybridize with aptamer (the sequences of these oligonucleotides and the hydridization mode were shown in Fig. S1). In order to avoid the influence of different sequences of these oligonucleotides on the target recognition, DNA1 was designed as a medium to combine aptamer and the linear DNA concatamers (by DNA2 and DNA3 hybridization). Both DNA2 and DNA3 contained three fragments, two of which were designed for HCR linear amplification and the third one was dangled to extend DNA side strands. DNA4–DNA6 were designed to extend the side strand of the DNA concatamers. After heating, annealing and magnetic separation, dendritic DNA concatamers were formed along the aptamer. When PAPDI was added into the system, the dye aggregated around the dendritic DNA concatamers. Then, magnetic separation was performed again to remove excessive PAPDI. With the addition of OTA which could bind with aptamer, the composite of PAPDI/DNA concatamers were released from the AMNPs. Finally, magnetic separation was performed and the supernatant was diluted with ethanol to disaggregate the PAPDI/DNA concatamers. The concentration of OTA can be determined by the fluorescence of disaggregated PAPDI monomers. 3.2. Fabrication of OTA aptasensor Amino-modified magnetic nanoparticles (AMNPs) with average diameter of about 100 nm were utilized in this design for magnetic separation (the SEM images of the AMNPs was displayed in Fig. S2). The surface charge of the AMNPs was determined as þ23.6 mV, demonstrating the existence of amino group. When carboxyl-functionalized aptamer was modified on the AMNPs, the surface charge of the nanoparticles decreased to 10.8 mV due to the negatively charged DNA backbone (as shown in Fig. 3B). The surface charge then decreased to 18.6 mV when linear DNA concatamers were formed on the aptamer/AMNPs nanocomposite by HCR strategy and continually decreased to 20.3 mV when dendritic DNA concatamers were modified on aptamer/AMNPs. These results indicated that linear and dendritic DNA concatamers were successfully modified on the aptamer/AMNPs nanocomposite. The HCR effect was explored using PAPDI as fluorescent probe according to the scheme in Fig. 3A. PAPDI was firstly connected with AMNPs, aptamer/AMNPs, linear DNA/aptamer/AMNPs, and dendritic DNA/aptamer/AMNPs before ethanol was added to disaggregate the PAPDI probe for following emission spectra recording. As shown in Fig. 3C, only negligible fluorescence was obtained from AMNPs. In comparison with aptamer/AMNPs, the linear and dendritic DNA/aptamer/AMNPs suspensions exhibited very strong fluorescence. In addition, the signal of dendritic DNA/ aptamer/AMNPs system nearly doubled compared to the linear DNA/aptamer/AMNPs as more PAPDI probe aggregated on the nanocomposites. Therefore, the dendritic-DNA/aptamer/AMNPs composites were adopted to conduct the following measurement. SEM images of AMNPs, dendritic DNA/aptamer/AMNPs and PAPDI/DNA/AMNPs were characterised and shown in Fig. S2 but no obvious differences could be observed from these images. That is probably due to the extraordinarily small size of DNA and PAPDI in comparison with AMNPs making them neglectable from SEM images. The dendritic-DNA concatamers could be released from AMNPs upon the addition of OTA, and gel electrophoresis was performed to characterize the formation of the dendritic-DNA concatamers.
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Fig. 2. The scheme of the OTA aptasensor.
The results in Fig. S3 demonstrated the existence of the DNA concatamers. In addition, the results further confirmed the aptamer was modified on AMNPs which agreed well with zeta potential results.
3.3. Optimization of OTA detection Two factors may significantly affect the sensitivity of OTA detection. One is the density of aptamer assembled on AMNPs surface and the other one is the concentration of oligonucleotides.
Fig. 3. (A) Scheme of DNA hybridization test; (B) the surface charge of AMNPs (a), aptamer/AMNPs (b), linear DNA/aptamer/AMNPs (c), and dendritic DNA/aptamer/AMNPs (d); (C) Corresponding emission spectra of AMNPs (a), aptamer/AMNPs (b), linear DNA/aptamer/AMNPs (c), and dendritic DNA/aptamer/AMNPs (d) upon the addition of PAPDI. (The concentration of DNA1–DNA6 were 0.1, 1.0, 1.0, 1.0, 5.0, and 5.0 μM, respectively). The error bars are standard deviations of three measurement.
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The density of aptamer should be carefully controlled as excessive aptamers modified on AMNPs would affect the growth of dendritic DNA concatamers and the interaction of OTA molecules, while over low aptamer concentration would decrease the sensitivity of OTA determination. The density of aptamer immobilized on the AMNPs was optimized based on the fluorescent response toward OTA detection, when 0.5 nM OTA was mixed with the dendritic DNA/aptamer/ AMNPs composites. After magnetic separation, the fluorescent spectra were recorded to evaluate the detection efficiency. As shown in Fig. S4, in the presence of OTA, strong fluorescence of PAPDI was obtained from all aptamer concentrations. When aptamer concentration changed from 0.2 μM to 10 μM, different fluorescence response signal strengths were detected. The strongest fluorescence response of PAPDI was obtained when the aptamer concentration was 5 μM. In addition, the ratio of these oligonucleotides was also optimized. As shown in Fig. S5, with the increase of DNA concentration, the fluorescent intensity increased gradually. When the concentration of these oligonucleotides (DNA1–DNA6) were beyond 10 nM (DNA1), 150 nM (DNA2), 150 nM (DNA3), 150 nM (DNA4), 600 nM (DNA5) and 600 nM (DNA6) respectively, the detection signal towards OTA was close to equilibration and these optimized concentrations were used for following determination. 3.4. Detection of OTA Under the optimized condition, OTA was highly sensitively determined by the proposed PAPDI–HCR amplification strategy. OTA detection was performed as descried in Section 2.4 in the presence of methanol. As shown in Fig. 4, with the increase of OTA concentration, the fluorescence of PAPDI increased gradually. A well linear correlation between the fluorescence intensity and the concentration of OTA was obtained in the range of 1.0–20 pM. The linear curve fitting equation is F¼32.66C þ 62.86, with a correlation coefficient of R2 ¼0.992, where F is the fluorescence intensity and C is the concentration of OTA (pM). The limit of detection is calculated to be 0.10 pM based on signal to noise ratio of 3 (S/ N ¼3). These results indicated that the present method is more sensitive than most of existing signal amplification methods (as shown in Table 1). The results demonstrated that highly sensitive fluorescence detection of OTA can be realised. According to the requirements of the European Commission, the maximum acceptable OTA level for raw cereal grains, cereal-derived products, and soluble coffee are 5 μg/kg, 3 μg/kg, and 10 μg/kg, respectively (Wei
Fig. 4. The fluorescence response of different concentrations of OTA. Inset: the linear relationship between the fluorescent intensity and the OTA concentration. The error bars are standard deviations (n ¼3).
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Table 1 Determination of OTA in corn samples by the proposed method. Samples Spiked OTA (pM)
Measured OTA (pM)a
Recovery (%)a RSD (%)a,
1 2 3 4 5
0.96 4.85 10.13 14.91 18.23
96.0 97.1 101.3 99.4 101.5
1.0 5.0 10.0 15.0 18.0 a b
b
4.3 3.8 3.2 2.9 3.5
The mean of three experiments. RSD ¼ The relative standard deviation.
et al., 2015). These concentrations could simply be calculated as 2.48 pM, 1.49 pM and 4.96 pM by our extraction method (as described in Section 2.6), which perfectly matched our detection range (1–20 pM). In addition, the concentration of OTA solutes could be easily adjusted by changing the volume of solvent utilized, indicating the suitability of our method in real sample detection. 3.5. Selectivity of the HCR signal-amplified aptasensor The specificity of the developed aptasensor was also tested. Four other mycotoxins including OTB, AFB1, Fumidil and Zearalenone were utilized to verify the anti-interference ability of the strategy. The results were listed in Fig. 5. Only OTA (20 pM) can induce remarkable signal attributed to the inherent specificity of the aptamer toward OTA. The other interfering mycotoxins displayed only negligible fluorescent signal. These results indicated that the developed method possessed sufficient selectivity for OTA detection. 3.6. Determination of OTA in corn samples In order to evaluate the feasibility and reliability of the developed method, the application for OTA determination was performed in corn samples according to the reported method (Hun et al., 2013). Corn samples were purchased from local supermarket and extracted by 80% methanol solution. The accuracy of OTA determination was analysed by a standard addition method. With the addition of known quantities of OTA, the concentration of OTA was detected by the proposed method and the results were listed in Table 1 and Fig. S6. It could be seen that the measured OTA concentration was closed to the spiked values and the recoveries
Fig. 5. Fluorescence spectra of the aptasensor in the presence of 20 pM OTA, OTB, AFB1, Fumidil and Zearalenone (from top to bottom) (all analytes concentration were 20 pM).
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were between 96.0% and 101.5% (n ¼3). The relative standard deviation (RSD) for three measurement was between 2.9% and 4.3%. These results suggested that this method had good accuracy and reliability in agricultural products samples.
4. Conclusion In conclusion, a signal-amplified optical aptasensor was developed for ultrasensitive detection of OTA. The signal amplification technique adopted HCR technique to build dendritic DNA concatemers and induce the aggregation of PAPDI probe. The assembly of PAPDI along DNA concatemers based on the electrostatic interaction between the positively charged PAPDI and negatively charged DNA backbone can further amplify the detection signal by the release of PAPDI probe. The proposed enzyme-free amplification method was simple and sensitive, while exhibited excellent selectivity towards OTA. The detection limit of the proposed aptasensor was 0.10 pM. The results of application in corn samples demonstrated the wonderful feasibility and potentials in the practical applications of agricultural products.
Acknowledgments This work is financially supported by start-up Grant under SWU111071 from Southwest University (Chongqing, China), Ph.D. start-up Grant SWU113111, SWU113112 from Southwest University, National Natural Science Foundation of China (21505108) and Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Wuhan University (ACBM2014004).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.02.062.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A highly sensitive aptasensor for OTA detection based on hybridization chain reaction and fluorescent perylene probe Bin Wang a,b,c,n, Yuanya Wu a,c, Yanfen Chen a,c, Bo Weng a,c,n, Liqun Xu a,c, Changming Li a,c a
Faculty of Materials and Energy, Southwest University, Chongqing 400715, China Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Wuhan University, Wuhan 430072, China c Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Chongqing 400715, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 December 2015 Received in revised form 19 February 2016 Accepted 23 February 2016 Available online 24 February 2016
An optical aptasensor was developed for ultrasensitive detection of ochratoxin A (OTA) based on hybridization chain reaction (HCR) amplification strategy and fluorescent perylene probe (PAPDI)/DNA composites. Dendritic DNA concatamers were synthesized by HCR strategy and modified on magnetic nanoparticles through aptamer as medium. A large amount of PAPDI probe aggregated under the induction of DNA concatamers and caused fluorescence quenching. In the presence of OTA, the PAPDI/DNA composites were released from magnetic nanoparticles due to the strong affinity between aptamer and OTA. In ethanol, PAPDI monomers disaggregated and produced strong fluorescence. The present method displays excellent sensitivity and selectivity towards OTA. & 2016 Elsevier B.V. All rights reserved.
Keywords: Optical aptasensor Perylene probe Aptamer OTA Hybridization chain reaction
1. Introduction Ochratoxin A (OTA) has been reported as one of the most toxic and cancerigenic substances to a wide variety of mammalian species (Duarte et al., 2010a). However, it was found that a series of food products (including cereals, wheat, barley, beans, corn, coffee and wine) were susceptible to OTA contamination (Duarte et al., 2010b). OTA contamination in food samples would not cause any observable physical damages (decay, mildew etc.) at the start of the infection. Thus it was hard to diagnose OTA at the initial stage of infection to avoid further damage (McKeague et al., 2014). It is essential to develop a sensitive method for low level OTA detection. Recently aptamer-based detection methods have been rapidly developed due to their noticeable merits such as high binding affinity, ease labeling, remarkable target diversity, convenient automated-synthesis and high stability (Jiang et al., 2014). A variety of colorimetric, electrochemical, fluorescent, and surface plasmon resonance biosensors with high sensitivity (Lee et al., 2014; Rivas et al., 2015; Wang et al., 2015b; Zhu et al., 2015) have been developed for the detection of low concentration of OTA. Furthermore, a series of signal amplification strategies have been developed to realize ultrasensitive and trace determination n Corresponding author at: Faculty of Materials and Energy, Southwest University, Chongqing 400715, China. E-mail addresses: [email protected] (B. Wang), [email protected] (B. Weng).
http://dx.doi.org/10.1016/j.bios.2016.02.062 0956-5663/& 2016 Elsevier B.V. All rights reserved.
of OTA in foods and drinks (Hun et al., 2013; Jiang et al. 2013). The most common strategy to amplify the signals in mycotoxin detection is using catalysts (such as enzyme). For example, rollingcircle amplification (RCA) is an isothermal nucleic acid amplification process that synthesized a long single-stranded nucleic acid product based on DNA polymerase and a circular, single-stranded nucleic acid template (Huang et al., 2013). The electrochemical aptasensor based on RCA displayed super high sensitivity with the limit of detection of 0.065 pg/mL for OTA detection (Huang et al., 2013); Another amplification technique depending on exonucleases catalysed the stepwise removal of mononucleotides from the 3′-end or 5′-end of single-stranded or double-stranded DNA. Yang et al. (2014) constructed OTA aptasensor based on exonuclease-catalysed target recycling amplification. OTA could be sensitively quantified by the recovery of the quantum dot electrochemiluminescence (ECL) with a detection limit of 0.64 pg/mL by this method; Nicking endonuclease, which is an enzyme cutting one strand of a dsDNA at a restriction site, could also be utilized in OTA detection. The OTA aptasensor based on target-induced strand release coupling cleavage of nicking endonuclease was developed by Hun et al. (2013). In this design, DNA polymerase and nicking endonuclease were utilized to extend and cleave DNA strand and produced an amount of targeted ssDNA; Additionally, loop-mediated isothermal amplification (LAMP), as an important nucleic acid amplification technique, has been designed for electrochemically aptasensing of OTA too (Xie et al., 2014). In their design, aptamers were utilized as the forward outer primer to trigger the LAMP reaction, and DNA polymerase was used to catalyse the extension
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of DNA and strand displacement reaction. This strategy displayed excellent sensitivity and selectivity with a detection limit of 0.3 pM. In comparison with above amplification strategies, Hybridization Chain Reaction (HCR) method is the most attractive among them due to its enzyme-free process and kinetics-controlled reaction (Zhao et al., 2015). In HCR, long double-strand DNA structure was constructed by hybridization of different oligonucleotides. HCR amplification strategy has been widely used in the detection of DNA, proteins, biothiols, metal ions, virus, cancer cells, and endonuclease activities (Yang et al., 2015; Ge et al., 2014). However, OTA detection using HCR amplification strategy has not been fully investigated (Wang et al., 2015a) yet. Further optimization and improvement are still required for low level OTA detection. Perylene tetracarboxylic acid diimide (PDI) derivatives have been reported as fluorescence probe due to their high fluorescence quantum yield and photostability (Würthner, 2004; Wang and Yu, 2010). It has been proved that PDI derivatives were remarkable fluorescent probe for optical sensor (Wang et al., 2014; Li et al., 2015; Wang et al., 2011). In this work, a highly sensitive fluorescent aptasensor for OTA determination was designed based on branching-growth of HCR strategy and the fluorescent perylene probe. In this design, positively charged perylene derivative, [(N,N ′-bis(propylenetrimethylammonium)-3,4,9,10-perylenediimide), PAPDI] was synthesized and served as fluorescent probe, while dendritic DNA concatamers synthesized by HCR strategy were utilized to induce the aggregation of PDI probe and further amplify the detection signal. OTA could be detected when it was bound with aptamer and released the DNA/PAPDI composite to produce strong fluorescent signals. This strategy was optimized to achieve highly sensitive detection of OTA and verified in the corn sample for its effectiveness for low level OTA detection in practical applications.
2. Experimental 2.1. Chemicals and apparatus Oligonucleotides used in this study were synthesized and purified with ultra PAGE by Sangon Biotechnology Co., Ltd. (Shanghai, China). The oligonucleotides stock solution was prepared by dissolving the oligonucleotide in 10 mM Tris–HCl buffer (pH 8.4, 200 mM NaCl) and was stored at 4 °C before use. Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), aflatoxin A1 (AFB1), and ochratoxin A (OTA) were purchased from Sigma-Aldrich Chemical Co. (USA). Ochratoxin B (OTB), Zearalenone, and Fumidil were purchased from Leon Technology Co. Ltd. (Beijing). All these mycotoxins including OTA, OTB, AFB1, Zearalenone, and Fumidil were dissolved in 80% methanol for further use. Amino-modified magnetic nanoparticles (AMNPs) were purchased from the Aladdin Reagent Co., Ltd. (Shanghai, China). 3,4,9,10-Perylenetetracarboxylic dianhydride (PDI), N,N′-dimethyl-1,3-propanediamine, methyl iodide and tri (hydroxymethyl) amino methane hydrochloride (Tris–HCl) were obtained from Shanghai Chemical Reagent Co. Perylene probe [(N,N′-bis(propylenetrimethylammonium)3,4,9,10-perylenediimide), PAPDI] was synthesized according to our previous report (Wang and Yu, 2010). All other chemicals were of analytical grade. Deionized water (18.2 MΩ cm at 25 °C) was used throughout the experiments. 2.2. Apparatus All fluorescence measurements were performed on a Shimadzu
RF-5301PC spectro-fluorometer equipped with a 150 W xenon lamp as the excitation source (Ushio Inc., Japan). The Zeta potential measurement was carried out at 25 °C (Zetasizer Nano 90, Malvern), and the surface charge of nanoparticles was determined in 100 mM Tris–HCl (pH 7.4, containing 200 mM NaCl). Scanning electron microscopy (SEM) images were taken by JSM-7800F (Japan). 2.3. Preparation of aptamer modified magnetic nanoparticles The AMNPs were functionalized with the OTA aptamer according to literature (Hua et al., 2013). Briefly, 10 nmol carboxylmodified OTA aptamer was dissolved in 100 μL of MES buffer solution (10 mM, pH 5.2). Then, 50 μL of 2 mM EDC and 50 μL of 2 mM NHS in pH 5.2 MES buffer were mixed with the above aptamer solution and incubated for 30 min to activate the carboxyl group. After that, the mixture was injected into 500 μL of 5 mg/mL AMNPs (100 mM Tris–HCl, pH 8.0) and incubated overnight at room temperature with gentle shaking. Then, the aptamer-modified AMNPs were washed three times with TT buffer (100 mM Tris–HCl, Tween-20 0.01%, pH 7.4). The obtained product was dissolved in 1 mL of Tris–HCl (100 mM, pH 7.4) and stored at 4 °C before use. 2.4. Fluorescence detection of OTA Typically, 10 μL of the as-prepared aptamer-AMNPs suspension was mixed with 100 μL of TS buffer (containing 100 mM, pH 7.4, Tris–HCl and 200 mM NaCl). Then, 2 μL of 10 μM DNA1, DNA2, DNA3, DNA4, DNA5, and DNA6 were added consecutively. The final mixture was incubated at 85 °C for 10 min, naturally cooled to room temperature, and incubated at room temperature for another 30 min. Subsequently, the obtained mixture was magnetically separated and washed with 100 μL of TS buffer solution for three times, and dispersed into 100 μL of TS buffer solution. PAPDI (2 μL, 100 μM) was mixed with the above suspension, followed by the magnetic separation and washed for 3 times using the TS buffer solution. The obtained deposit was dissolved in 100 μL of TS buffer. 10 μL prepared PAPDI/DNA/ AMNPs suspension was taken and mixed with 20 μL Tris–HCl (400 mM, pH 7.4) buffer solution, 10 μL 1 M NaCl solution, and 10 μL OTA with different concentrations (the final concentration of methanol was 16%). After incubation at room temperature for 1 h, the dispersion was magnetically separated. Finally, the supernatant was diluted with 400 μL of ethanol. The fluorescence spectra were recorded on a fluorescence spectrometer and standard curve was calculated based on detection results. The same procedure was performed to estimate the selectivity of the method by replacing OTA with other mycotoxins. 2.5. Assay of gel electrophoresis In order to verify the hybridization of DNA on AMNPs, polyacrylamide gel electrophoresis (PAGE) was performed. Firstly, different concentrations of OTA (0, 20 pM, and 1.0 nM) were incubated with DNA/aptamer/AMNPs nanocomposites for 2 h at room temperature. The released DNA concatamers were used for Gel Electrophoresis analysis. A 12.0% native polyacrylamide gel was prepared by using 1 TBE buffer (100 mM Tris–HCl, 83 mM boric acid, 1 mM EDTA, pH 8.0). The gel was run at 100 V for 60 min with the loading of 5 μL of each sample into the lanes at room temperature. After staining with Goldview Nucleic Acid Gel Stain (Biotium, USA) for 30 min, the gels were scanned using a Gel Imaging System (Bio-Rad, USA).
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2.6. Determination of OTA in corn samples In order to evaluate the feasibility and reliability of the developed method, the application for OTA determination in real samples was performed. Corn samples were purchased from local supermarket and finely grounded. Before characterization, 1 g corn samples were extracted by 1 L 80% methanol. The resulted solution was spiked with different concentrations of OTA, and determination was performed by the proposed method. Typically, 10 μL prepared PAPDI/DNA/AMNPs suspension was mixed with 20 μL Tris–HCl (400 mM, pH 7.4) buffer solution, 10 μL 1 M NaCl solution, and 10 μL of the corn samples with different concentrations of OTA. Then, magnetic separation and fluorescent determination of the supernatant were performed after incubating at room temperature for 2 h. The results of three measurements were listed in Table S2.
3. Results and discussion 3.1. Design principle of OTA aptasensor In aqueous environment, fluorescent probe PAPDI tends to aggregate by aromatic π–π stacking interactions due to its planar aromatic structure (Wang and Yu, 2010). It has been reported that polyanionic DNA could induce the aggregation of PAPDI and lead to the fluorescence quenching by electrostatic interaction. Fig. 1 showed the scheme and emission spectra of PAPDI upon the addition of DNA and ethanol. In aqueous environment, PAPDI showed strong fluorescence. Upon the addition of 5 nM DNA, the fluorescence of PAPDI was quenched completely due to the aggregation of PAPDI. The PAPDI/DNA aggregates were dispersed in ethanol solution, PAPDI monomer was disaggregated. As shown in Fig. 1B, with the increase of ethanol ratio in buffer solution, the fluorescence was recovered gradually. Simultaneously blue-shift of emission spectra was observed due to disaggregation of the fluorescent probe. The PAPDI-DNA system displayed stronger fluorescence intensity in 90% ethanol than that in aqueous solution. Meanwhile, the maximum
Fig. 1. A: Scheme of the aggregation and disaggregation of PAPDI in the presence of DNA1 and ethanol; B: the emission spectra of 0.1 μM PAPDI in 10 mM pH 7.4 Tris– HCl (a), in the presence of 5 nM DNA1 (b), the mixture of 0.1 μM PAPDI and 5 nM DNA1 in 20% (c), 40% (d) 60% (e) 80% (f) 90% ethanol aqueous solution (g).
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emission peak shifted from 548 nm (curve a in Fig. 1B) to 537 nm (curve g in Fig. 1B). Therefore, we would like to design an aptasensor for OTA detection based on the theory discussed above and HCR amplification strategy. The detection scheme is shown in Fig. 2. Firstly, carboxylfunctionalized OTA aptamer was modified on amino-modified magnetic nanoparticles (AMNPs) with the assistance of EDC and NHS. Then, a certain proportion of assistant oligonucleotides (DNA1–DNA6) was added to hybridize with aptamer (the sequences of these oligonucleotides and the hydridization mode were shown in Fig. S1). In order to avoid the influence of different sequences of these oligonucleotides on the target recognition, DNA1 was designed as a medium to combine aptamer and the linear DNA concatamers (by DNA2 and DNA3 hybridization). Both DNA2 and DNA3 contained three fragments, two of which were designed for HCR linear amplification and the third one was dangled to extend DNA side strands. DNA4–DNA6 were designed to extend the side strand of the DNA concatamers. After heating, annealing and magnetic separation, dendritic DNA concatamers were formed along the aptamer. When PAPDI was added into the system, the dye aggregated around the dendritic DNA concatamers. Then, magnetic separation was performed again to remove excessive PAPDI. With the addition of OTA which could bind with aptamer, the composite of PAPDI/DNA concatamers were released from the AMNPs. Finally, magnetic separation was performed and the supernatant was diluted with ethanol to disaggregate the PAPDI/DNA concatamers. The concentration of OTA can be determined by the fluorescence of disaggregated PAPDI monomers. 3.2. Fabrication of OTA aptasensor Amino-modified magnetic nanoparticles (AMNPs) with average diameter of about 100 nm were utilized in this design for magnetic separation (the SEM images of the AMNPs was displayed in Fig. S2). The surface charge of the AMNPs was determined as þ23.6 mV, demonstrating the existence of amino group. When carboxyl-functionalized aptamer was modified on the AMNPs, the surface charge of the nanoparticles decreased to 10.8 mV due to the negatively charged DNA backbone (as shown in Fig. 3B). The surface charge then decreased to 18.6 mV when linear DNA concatamers were formed on the aptamer/AMNPs nanocomposite by HCR strategy and continually decreased to 20.3 mV when dendritic DNA concatamers were modified on aptamer/AMNPs. These results indicated that linear and dendritic DNA concatamers were successfully modified on the aptamer/AMNPs nanocomposite. The HCR effect was explored using PAPDI as fluorescent probe according to the scheme in Fig. 3A. PAPDI was firstly connected with AMNPs, aptamer/AMNPs, linear DNA/aptamer/AMNPs, and dendritic DNA/aptamer/AMNPs before ethanol was added to disaggregate the PAPDI probe for following emission spectra recording. As shown in Fig. 3C, only negligible fluorescence was obtained from AMNPs. In comparison with aptamer/AMNPs, the linear and dendritic DNA/aptamer/AMNPs suspensions exhibited very strong fluorescence. In addition, the signal of dendritic DNA/ aptamer/AMNPs system nearly doubled compared to the linear DNA/aptamer/AMNPs as more PAPDI probe aggregated on the nanocomposites. Therefore, the dendritic-DNA/aptamer/AMNPs composites were adopted to conduct the following measurement. SEM images of AMNPs, dendritic DNA/aptamer/AMNPs and PAPDI/DNA/AMNPs were characterised and shown in Fig. S2 but no obvious differences could be observed from these images. That is probably due to the extraordinarily small size of DNA and PAPDI in comparison with AMNPs making them neglectable from SEM images. The dendritic-DNA concatamers could be released from AMNPs upon the addition of OTA, and gel electrophoresis was performed to characterize the formation of the dendritic-DNA concatamers.
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Fig. 2. The scheme of the OTA aptasensor.
The results in Fig. S3 demonstrated the existence of the DNA concatamers. In addition, the results further confirmed the aptamer was modified on AMNPs which agreed well with zeta potential results.
3.3. Optimization of OTA detection Two factors may significantly affect the sensitivity of OTA detection. One is the density of aptamer assembled on AMNPs surface and the other one is the concentration of oligonucleotides.
Fig. 3. (A) Scheme of DNA hybridization test; (B) the surface charge of AMNPs (a), aptamer/AMNPs (b), linear DNA/aptamer/AMNPs (c), and dendritic DNA/aptamer/AMNPs (d); (C) Corresponding emission spectra of AMNPs (a), aptamer/AMNPs (b), linear DNA/aptamer/AMNPs (c), and dendritic DNA/aptamer/AMNPs (d) upon the addition of PAPDI. (The concentration of DNA1–DNA6 were 0.1, 1.0, 1.0, 1.0, 5.0, and 5.0 μM, respectively). The error bars are standard deviations of three measurement.
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The density of aptamer should be carefully controlled as excessive aptamers modified on AMNPs would affect the growth of dendritic DNA concatamers and the interaction of OTA molecules, while over low aptamer concentration would decrease the sensitivity of OTA determination. The density of aptamer immobilized on the AMNPs was optimized based on the fluorescent response toward OTA detection, when 0.5 nM OTA was mixed with the dendritic DNA/aptamer/ AMNPs composites. After magnetic separation, the fluorescent spectra were recorded to evaluate the detection efficiency. As shown in Fig. S4, in the presence of OTA, strong fluorescence of PAPDI was obtained from all aptamer concentrations. When aptamer concentration changed from 0.2 μM to 10 μM, different fluorescence response signal strengths were detected. The strongest fluorescence response of PAPDI was obtained when the aptamer concentration was 5 μM. In addition, the ratio of these oligonucleotides was also optimized. As shown in Fig. S5, with the increase of DNA concentration, the fluorescent intensity increased gradually. When the concentration of these oligonucleotides (DNA1–DNA6) were beyond 10 nM (DNA1), 150 nM (DNA2), 150 nM (DNA3), 150 nM (DNA4), 600 nM (DNA5) and 600 nM (DNA6) respectively, the detection signal towards OTA was close to equilibration and these optimized concentrations were used for following determination. 3.4. Detection of OTA Under the optimized condition, OTA was highly sensitively determined by the proposed PAPDI–HCR amplification strategy. OTA detection was performed as descried in Section 2.4 in the presence of methanol. As shown in Fig. 4, with the increase of OTA concentration, the fluorescence of PAPDI increased gradually. A well linear correlation between the fluorescence intensity and the concentration of OTA was obtained in the range of 1.0–20 pM. The linear curve fitting equation is F¼32.66C þ 62.86, with a correlation coefficient of R2 ¼0.992, where F is the fluorescence intensity and C is the concentration of OTA (pM). The limit of detection is calculated to be 0.10 pM based on signal to noise ratio of 3 (S/ N ¼3). These results indicated that the present method is more sensitive than most of existing signal amplification methods (as shown in Table 1). The results demonstrated that highly sensitive fluorescence detection of OTA can be realised. According to the requirements of the European Commission, the maximum acceptable OTA level for raw cereal grains, cereal-derived products, and soluble coffee are 5 μg/kg, 3 μg/kg, and 10 μg/kg, respectively (Wei
Fig. 4. The fluorescence response of different concentrations of OTA. Inset: the linear relationship between the fluorescent intensity and the OTA concentration. The error bars are standard deviations (n ¼3).
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Table 1 Determination of OTA in corn samples by the proposed method. Samples Spiked OTA (pM)
Measured OTA (pM)a
Recovery (%)a RSD (%)a,
1 2 3 4 5
0.96 4.85 10.13 14.91 18.23
96.0 97.1 101.3 99.4 101.5
1.0 5.0 10.0 15.0 18.0 a b
b
4.3 3.8 3.2 2.9 3.5
The mean of three experiments. RSD ¼ The relative standard deviation.
et al., 2015). These concentrations could simply be calculated as 2.48 pM, 1.49 pM and 4.96 pM by our extraction method (as described in Section 2.6), which perfectly matched our detection range (1–20 pM). In addition, the concentration of OTA solutes could be easily adjusted by changing the volume of solvent utilized, indicating the suitability of our method in real sample detection. 3.5. Selectivity of the HCR signal-amplified aptasensor The specificity of the developed aptasensor was also tested. Four other mycotoxins including OTB, AFB1, Fumidil and Zearalenone were utilized to verify the anti-interference ability of the strategy. The results were listed in Fig. 5. Only OTA (20 pM) can induce remarkable signal attributed to the inherent specificity of the aptamer toward OTA. The other interfering mycotoxins displayed only negligible fluorescent signal. These results indicated that the developed method possessed sufficient selectivity for OTA detection. 3.6. Determination of OTA in corn samples In order to evaluate the feasibility and reliability of the developed method, the application for OTA determination was performed in corn samples according to the reported method (Hun et al., 2013). Corn samples were purchased from local supermarket and extracted by 80% methanol solution. The accuracy of OTA determination was analysed by a standard addition method. With the addition of known quantities of OTA, the concentration of OTA was detected by the proposed method and the results were listed in Table 1 and Fig. S6. It could be seen that the measured OTA concentration was closed to the spiked values and the recoveries
Fig. 5. Fluorescence spectra of the aptasensor in the presence of 20 pM OTA, OTB, AFB1, Fumidil and Zearalenone (from top to bottom) (all analytes concentration were 20 pM).
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were between 96.0% and 101.5% (n ¼3). The relative standard deviation (RSD) for three measurement was between 2.9% and 4.3%. These results suggested that this method had good accuracy and reliability in agricultural products samples.
4. Conclusion In conclusion, a signal-amplified optical aptasensor was developed for ultrasensitive detection of OTA. The signal amplification technique adopted HCR technique to build dendritic DNA concatemers and induce the aggregation of PAPDI probe. The assembly of PAPDI along DNA concatemers based on the electrostatic interaction between the positively charged PAPDI and negatively charged DNA backbone can further amplify the detection signal by the release of PAPDI probe. The proposed enzyme-free amplification method was simple and sensitive, while exhibited excellent selectivity towards OTA. The detection limit of the proposed aptasensor was 0.10 pM. The results of application in corn samples demonstrated the wonderful feasibility and potentials in the practical applications of agricultural products.
Acknowledgments This work is financially supported by start-up Grant under SWU111071 from Southwest University (Chongqing, China), Ph.D. start-up Grant SWU113111, SWU113112 from Southwest University, National Natural Science Foundation of China (21505108) and Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Wuhan University (ACBM2014004).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.02.062.
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