Technical Note pubs.acs.org/ac
Detection and Quantitation of Heavy Metal Ions on Bona Fide DVDs Using DNA Molecular Beacon Probes Lingling Zhang,†,§ Jessica X. H. Wong,‡ Xiaochun Li,*,† Yunchao Li,§ and Hua-Zhong Yu*,†,‡ †
Key Laboratory of Advanced Transducers and Intelligent Control Systems (Ministry of Education and Shanxi Province), College of Physics and Optoelectronic Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, China ‡ Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada § Department of Chemistry, Beijing Normal University, Beijing 100875, China S Supporting Information *
ABSTRACT: A sensitive and cost-effective method for the simultaneous quantitation of trace amounts of Hg2+ and Pb2+ in real-world samples has been developed using DNA molecular beacon probes bound to bona fide digital video discs (DVDs). With specially designed T-rich or G-rich loop sequences, the detection is based on the strong T-Hg2+-T coordination chemistry of Hg2+ and the formation of Gquadruplexes induced by Pb2+, respectively. In particular, the presence of metal cations leads to hairpin opening and exposure of the terminal biotin moiety for binding nanogold− streptavidin conjugates. The recognition signal was subsequently enhanced by gold nanoparticle-promoted silver deposition, which leads to quantifiable digital signals upon reading with a standard computer drive. This method exhibits a wide response range and low detection limits for both Hg2+ and Pb2+. In addition, the quantitative determination of heavy metals in food products (e.g., rice samples) has been demonstrated and the method compares favorably with other optical sensors developed recently. lthough most people would choose USB flash drives to store their data and media files nowadays, optical disc technology has been widely explored with the ultimate goal of creating bioanalytical systems for on-site chemical analysis and point-of-care medical diagnostics.1,2 This is because compact discs (CD)/digital video discs (DVDs) can be adapted as inexpensive substrate materials for the preparation of various bioassays,1 and conventional computer drives/disc players are powerful optical devices for biochip signal readout.1,2 Two types of disc-based devices/chips, i.e., lab-on-a-disc microfluidic disks3 and spinning interferometry bioCDs,4 have been the main focus in this field in the past two decades. Conceptually different from hardware modification approaches,5−9 we have been working on the development of digitized molecular analytical systems that are entirely based on bona fide CD/DVD discs and optical drives.1,10−12 Inspired by the delicate data protection and retrieval mechanism inhered in optical disc systems, we developed first a CD-based digital readout protocol for screening biomolecular binding reactions,10 which had been extended to state-of-the-art DVD systems.11 Particularly, the analytical platform based on DVD technology has been demonstrated to be applicable in medical diagnosis (quantitation of human chorionic gonadotropin in urine samples)11 and on-site screening of drugs of abuse.12 In the meantime, other groups have also made significant progress in adapting such digital approaches to create microimmunoassays and cell-counting devices.13−15
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© XXXX American Chemical Society
One of the limitations of DVD-based analytical systems we have developed so far is the assay preparation, i.e., routine adaptation of conventional sandwich-format or competitive immunoassays that rely on antibody−antigen interactions.11,12 Sandwich-type assays generally require multistep surface reactions and additional labeling reagents, while indirect competitive assays would generate “signal-off” responses. By introducing DNA molecular beacon probes (i.e., DNA hairpins) as sensing entities on discs to create “signal-on” responses from analyte-induced conformational changes,16,17 we report herein a DVD-based analytical system for the quantitative detection of heavy metal ions in both standard buffers and real-world samples (extracts from food products). Hg2+ and Pb2+ are the two most widespread heavy metal contaminants with high and persistent toxicity. Human exposure may lead to irreversible damage of the digestive, neurological, and cardiovascular systems.18−21 Conventional methods established for the quantitative analysis of Pb2+ and Hg2+ include atomic absorption spectrometry,22,23 inductively coupled plasma mass spectrometry (ICPMS),24,25 and anodic stripping voltammetry.26 Various novel sensors based on fluorescence,27−33 electrochemical,34−37 and colorimetric readout38−41 have been proposed in the past decade. Our aim to Received: March 6, 2015 Accepted: April 23, 2015
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DOI: 10.1021/acs.analchem.5b00899 Anal. Chem. XXXX, XXX, XXX−XXX
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the free disc-quality diagnostic program (PlexUTILITIES 1.3) was used to read the assay discs at a speed of 6×. After reading, the program can export an error distribution plot and provide statistical results regarding the error numbers and types. Optical images of all assays (the binding strips formed on DVD-Rs) were captured using a flatbed scanner (Microek ScanMaker i700) in the reflective mode and then analyzed in Adobe Photoshop to determine the optical darkness (from the grayscale intensities). The surface topographies of the binding stripes were imaged with an atomic force microscope (Dimension Edge, Bruker Inc.) in tapping mode.
develop DVD assays for heavy metal ion detection is not to compete with the above established methods in terms of sensitivity and accuracy but rather to provide an alternative for on-site detection or screening, particularly in remote locations where advanced instrumentation is not available.
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EXPERIMENTAL SECTION Reagents and Materials. All oligonucleotides were of HPLC-grade and obtained from Shanghai Sangon Biotechnology Inc. (Shanghai, China). The specifically designed sequences of G-rich and T-rich oligonucleotides are 5′-NH2-(CH2)6-TAG CCA ACA AGG TTG GTG TGG TTG GCA T-biotin-3′ (GG probe) and 5′-NH2-(CH2)6-CAA ATG AAC TTT GGT TTC CCT TTT CAT TTT-biotin-3′ (TT probe), respectively.17 DVD-R (4.7G) was ordered from Maxell Taiwan Ltd. (Taipei, Taiwan). Silver acetate, hydroquinone, 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), tris (hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), and bovine serum albumin (BSA, globulin-free, molecular-biology grade) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Nanogold (1.4 nm diameter)− streptavidin conjugates were ordered from Nanoprobes Inc. (New York, USA). Solutions were prepared with deionized water produced from a Barnstead Easypure System (Thermo Scientific Inc., Dubuque, Iowa, USA). Rice samples were immersed in a solution containing 0.7 mM Hg2+ and 5.0 mM Pb2+ for 1 week. The contaminated rice was then washed with water, dried, and ground into a fine powder. A 1.0 g aliquot of the sample was digested with 10 mL of concentrated nitric acid (5 M) and boiled to almost dryness (∼1 mL left). After adding 2−3 mL of water, the mixture was filtered through a membrane filter of 0.2 μm pore size. The filtrate was diluted with water to a total volume of 10 mL. DVD Surface Activation and Assay Preparation. Video files were recorded onto a blank DVD-R, which was then cleaned with ethanol and deionized water. The surface was activated in a UV/ozone system (Model PSD-UV, Novascan Technologies Inc.) for 20−40 min to produce a hydrophilic surface with a high density of carboxylic acid groups. The discs were subsequently immersed in a 0.1 M phosphate buffer at pH 6.0 containing 5 mM EDC and 0.3 mM NHS for 3−5 h to activate the surface-tethered carboxylic acid groups.11,12 A specially designed PDMS (polydimethylsiloxane) plate with two sets of embedded microchannels (0.3 mm × 15 mm × 50 nm, arc shaped) was first put on the activated DVD substrate for the immobilization of 1.5 μL of GG probes (in 10 mM Tris buffer containing 150 mM NaCl and 50 mM MgCl2, pH 7.4) and TT probes (in 10 mM phosphate buffer containing 150 mM NaCl and 50 mM MgCl2, pH 7.4) to form two different testing zones (for Pb2+ and Hg2+). The disc was then kept in a humidity box overnight to form the probe arrays. After blocking with 3% BSA for 1−3 h, 1.5 μL of solution containing different concentrations of Pb2+ and Hg2+ was injected into the channels and kept for 60 min. Nanogold− streptavidin conjugate (0.8 μg/mL) in 20 mM phosphate buffer containing 0.8% BSA and 0.1% gelatin was then injected into each channel and incubated for 30−60 min. The PDMS plate was removed, and the disc was washed thoroughly with water before immersion in a freshly made silver-staining solution (12 mM silver acetate and 45 mM hydroquinone) for 5−20 min, depending on staining conditions, to amplify the signals. Instrumentation and Data Analysis. A Blu-ray/DVD optical drive (PX-LB950UE, Plextor Co.) in conjunction with
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RESULTS AND DISCUSSION Preparation of DNA Hairpin Assays on DVD for Digitized Metal Ion Detection. Different from the traditional sandwich-format or competitive immunoassays, we decided to adapt molecular beacon probes, i.e., DNA hairpins containing Hg2+ or Pb2+-specific sequences in the loop, as sensing entities for the detection of heavy metal cations, a strategy which has been successfully demonstrated for electrochemical and conventional colorimetic analysis.16,17,37 Particularly, two dually modified DNA hairpin probes, one containing guanine (G)-rich fragments (named as GG probe) and the other containing thymine (T)-rich fragments (named as TT probe), were immobilized at different locations on the surface of a DVD (with the aid of a PDMS channel plate) for recognizing Pb2+ and Hg2+, respectively. The immobilized DNA probes, initially in their folded (hairpin-like) forms such that the terminal biotin groups were embedded in the probe layer, prevent the access of nanogold−streptavidin conjugates. In the presence of Pb2+ and Hg2+, hairpin secondary structures were “forced” open and form a stable G-quadruplex and T-Hg2+-T complex, respectively, allowing nanogold−streptavidin conjugates to bind promptly with the biotin groups and to produce signals thereon upon silver enhancement (for details, see the Supporting Information). The strong and specific interaction between the two DNA hairpin probes (GG and TT) and the two metal cations (Pb2+ and Hg2+) have been confirmed previously by using colorimetric assays.17 As shown in Figure 1a, the binding sites on the DVD surface were covered with rather large silver particles; such binding strips disturb the normal disc reading process by blocking the laser beam (reflection and scattering), thus causing significant reading errors (parity inner failure, PIF). We have confirmed previously that the distribution and counts of the resulting PIF peaks have a direct correlation with the physical location and intensity of the binding strips, respectively.12 Figure 1b shows the results of using this assay platform to examine eight solutions containing different concentrations of Hg2+ (from 0 to 50 μM). It was observed that eight binding strips with different darkness appeared on the disc (inset photo), and those strips (except for the blank) indeed induced well-defined error peaks with different heights (PIF counts). To have a better understanding of the performance of this DVD assay, we have integrated the error peaks and normalized them to the area of the binding strips to obtain the corresponding PIF density, which was plotted versus the concentration of Hg2+. Figure 2a reveals that the PIF density (error counts per unit area) increases monotonically with the concentration of Hg2+; even with very low concentrations (0.1−5.0 nM), the signal is significantly higher than that of the blank (an assay strip formed in the absence of Hg2+). Particularly, we have indicated the signal level corresponding B
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Figure 2. (a) The dependence of PIF density and ODR on the concentration of Hg2+; (b) Correlation between the PIF and ODR values obtained for the same set of assays strips (shown in Figure 1b).
show much smaller relative standard deviations and lower blank signals compared to those of ODR readings. In Figure 2b, we plotted the PIF density as a function of the ODR value for each of the standard solutions tested for Hg2+. Unlike the linear relationship observed in our previous studies, the correlation between these two sets of data is rather perplexing. While a general increasing trend in PIF density was observed when the ODR value became larger, a linear relationship is not obvious. A quick rising (titration curve-like) region was observed for a small ODR range (0.20−0.35). It is not clear at this time why the ODR values do not correlate with the PIF densities; however, the AFM (atomic force microscope) images shown in Figure 3 may shed light on the issue. In comparison with the featureless image for a blank sample, nanoparticles ranging from several tens to hundreds of nanometers have been observed for the sample of 0.5 nM Hg2+. The coverage of these particles increased substantially for the sample with 5.0 nM Hg2+, and it seems to become saturated when the concentration is raised to 5.0 μM. At the higher concentrations, the aggregation and multilayer deposition of the particles are evident as well. As illustrated in Figure 1a, the digital reading relies on the disruption of the laser beam (650 nm), which is otherwise reflected by the metal layer in the DVD for data decoding. In contrast, the ODR values are determined from the grayscale values of a scanned image, which is completely dependent on the scanner. We suspect that the unique aggregation of the silver particles in these samples (which are not typical for other assays) may play an important role for the discrepancy mentioned above. Versatility of DVD-Based Metal Detection. We and others have demonstrated that sensitive DNA sensors for Pb2+
Figure 1. (a) Schematic view of the digitized detection of Pb2+ and Hg2+ on a bona fide DVD with a standard optical drive; the Au/silver particles accumulating at the binding site interfere with the reading laser, which creates quantifiable errors. (b) A representative error plot (the PIF distribution) generated upon reading a Hg2+ assay prepared on a DVD; the inset photo shows the eight binding strips corresponding to different concentrations of Hg2+ (as listed).
to the 3δ (standard deviation) in addition to the blank response as the red dash line in Figure 2a; it is clear that a concentration of 0.5 nM (0.1 ppb) can be considered as the limit of detection (LOD) in this experiment. We note that the detectable concentration achieved herein (0.1 ppb) is well below the maximum allowable level of Hg2+ in drinking water (2 ppb) recommended by the United States Environmental Protection Agency.42 To further evaluate the DVD assay performance, we have compared it with the conventional colorimetric protocol, by examining the optical darkness ratio (ODR) of each binding strip with a standard desktop scanner.1,10 As shown in Figure 2a, the ODR values seemingly compare well with the PIF data in terms of the concentration dependence, yet the PIF results C
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Figure 3. Atomic force microscope (AFM) images of the binding strips on a DVD for Hg2+; the assay was prepared following the procedure shown in Scheme S1, Supporting Information, with different concentrations of Hg2+ present.
can be fabricated on the basis of the conformational change from a hairpin DNA to a G-quadruplex to induce either colorimetric or electrochemical signals.17,43,44 Herein, we have immobilized Pb2+-specific DNA probes (GG probe) on a DVDR and adapted the same labeling and signal enhancement protocols. The LOD determined for the detection results of Pb2+ is at least 0.5 nM (see the Supporting Information for details); such a LOD (0.1 ppb) is significantly lower than the allowable level of Pb 2+ in drinking water (15 ppb) recommended by the United States Environmental Protection Agency.42 Besides the detection sensitivity, the specificity of this digital sensing platform was evaluated by testing other common metal ions (such as Na+, Ni2+, Zn2+, Al3+, Fe3+, Li+, Hg2+, Mg2+, Ca2+, Cu2+, Ba2+, and Mn2+). We found that for both Pb2+ and Hg2+ detection of the interfering ions (at 0.1 mM) only caused low levels of reading errors (PIF counts), which are either not detectable or insignificant. However, a few metal cations (e.g., Na+) did produce discernible responses for the Pb2+ sensor, due to the fact that the formation of G-quadruplexes can be induced by other metal cations.45−47 Although the concentrations leading to such a change are much higher (i.e., mM range) in comparison with Pb2+,45 we should consider the limitation of its application in aqueous solutions with high concentrations of alkaline cations. The successful individual detection of Pb2+ and Hg2+ allows us to explore the capability of this digital sensing platform for simultaneous detection of Pb2+ and Hg2+ in binary mixtures. To achieve this goal, the Pb2+- and Hg2+-specific probes (i.e., GG and TT probe) were immobilized at two different sections on the same DVD to form a“Pb2+ testing zone” and a “Hg2+ testing zone”, respectively, by using the specially designed PDMS plate with two sets of microchannels. We hoped that the recognition reactions between the two probes and the two target ions occurring at specified locations could be readily differentiated. With this design, we first analyzed binary aqueous samples containing both Pb2+ (0, 0.05, 50 μM) and Hg2+ (0, 0.05, 50 μM) at different concentrations. Figure 4a shows the PIF responses corresponding to all binding strips as a function of their physical locations (radial distance) on the DVD. To
Figure 4. Simultaneous determination of Hg2+ and Pb2+ on the same disc by immobilizing TT probes and GG probes at defined locations. (a) PIF distribution on the DVD with 16 binding strips including two blanks (SP1, SP2), individual detections of Hg2+ (SP3, SP5) and Pb2+ (SP4, SP6), and simultaneous detections (SP7-SP16); the top inset is an optical image of the binding strips. (b) 3D histogram showing the dependence of PIF density on Hg2+ and Pb2+ concentrations. The error bars represent the standard deviations derived from triplicate measurements.
provide a better overview of the quantitative comparison, we have generated a 3D histogram as shown in Figure 4b. As expected, the blanks in both test zones (SP1 and SP2) caused negligible PIF signals. The samples containing only Hg2+ (SP3, SP4) produced a distinct PIF peak in the Hg2+ test zone (SP3) and low signals in the Pb2+ test zone (SP4) and vice versa (low signal observed for Pb2+-containing samples in the Hg2+ test zone (SP5) but high response in the Pb2+ test zone (SP6)). For the samples containing both Pb2+ and Hg2+, distinct PIF peaks at the corresponding locations in both testing zones (SP7− SP16) were identified. This proof-of-concept experiment not only confirms the selectivity of the digital sensing protocol but also shows its capability of differentiating multiple analytes on a single assay disc. Real-World Sample Testing and Comparison with Other Methods. To minimize the complexity of real-world D
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site screening (when advanced instrumentation and centralized laboratories facilities are not available). A conventional computer optical drive or a stand-alone disc player is the only required instrument; the cost of the analysis is significantly reduced due to its high detection throughput and limited reagent consumption. These features make this digital sensing platform an ideal tool for on-site screening before bringing back samples to a centralized laboratory for further analysis.
sample testing, we have chosen spiked rice as a model system to validate our DVD assay protocol. The samples, together with a series of standard solutions containing known concentrations of Hg2+ and Pb2+, were examined on the DVD platform. Benefitted by the number of samples we can test on a single disc, the set of standard solutions and three sample replicates were examined on the same disc (top insets, Figure 5).
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CONCLUSION
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ASSOCIATED CONTENT
We have developed a DVD-technology-based assay method for the detection of heavy metal ions with high sensitivity and selectivity in both standard buffers and real-world samples. The signal readout is performed with a conventional optical drive, and the assay is prepared on bona fide digital video discs. Particularly, DNA molecular beacon probes (i.e., DNA hairpins) were immobilized on regular DVDs as sensing entities to create “signal-on” responses from analyte-induced conformational changes. Considering the versatility of designing loop sequences in the DNA molecular beacon probes, this approach can be readily extended to the detection of many different analytical targets, other metal cations, proteins, and oligonucleotides.36 In addition, the DVD assay has been demonstrated for the quantitation of Pb2+ and Hg2+ in binary mixtures and in the presence of other interfering metal ions, with LODs down to 0.5 nM and wide linear response ranges (2−500 nM for Pb2+ and 10−1000 nM for Hg2+). These low LODs are adequate for environmental monitoring and food safety tests, which augments its potential application for on-site chemical analysis.
Figure 5. Quantitative detection of (a) Hg2+ and (b) Pb2+ in a contaminated rice sample. The black lines are the best fits to the experimental data within the linear response regions; the red lines show the signal and concentration of the unknown sample. The top insets are the optical images of the assay discs from which the PIF data were obtained.
S Supporting Information *
Additional experimental data including the step-by-step preparation of DVD assays, detection of Pb2+, selectivity tests, and comparison with other methods. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00899.
In Figure 5, we have shown the semilog calibration curves for the detection of Hg2+ (a) and Pb2+ (b), respectively. In both cases, we have obtained satisfactory fitting results in a wide response range (2−500 nM for Hg2+ and 10−1000 nM for Pb2+); PIF(Hg) = 97.7 ± 5.1 × log [Hg2+] + 509 ± 9, R2 = 0.995; PIF(Pb) = 120 ± 1 × log [Pb2+] + 391 ± 1, R2 = 0.999, respectively. On the basis of these calibration equations and the obtained PIF signals (380 ± 2 and 339 ± 14 counts/mm2), the concentrations of Hg2+ and Pb2+ in the unknown samples (i.e., rice extracts) were determined to be 0.046 ± 0.013 and 0.257 ± 0.017 μM, respectively. The rather large relative uncertainty for the Hg2+ concentration can be explained by the nature of error propagation with the semilog calibration curve and its low abundance (nM). The converted abundance of Pb2+ in the original contaminated rice sample was 530 ± 35 ppb, which is much higher than that found in market collected white rice (4− 46 nM), also above the level in samples from known contaminated/mine impacted regions (185 ± 4 ppb).48 Similarly, the concentration of Hg2+ in the contaminated rice sample (92 ± 26 ppb) is comparable to that of rice grown in Hg2+-contaminated soils (10−100 ppb).49 It should be noted that the rice sample testing is a preliminary experiment to show the application potential of the DVD assay; we also acknowledge that the DVD assay may not be able to compete with the conventional ICPMS method and the recently developed fluorescence detection using catalytic and molecular beacon probes in terms of sensitivity and accuracy;50,51 however, it is potentially applicable for on-
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected] (H.-Z.Y.). *E-mail: [email protected] (X.L.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We gratefully acknowledge the financial support from the Natural Science Foundation of China (Grant Nos. 61174010; 21003012; 21233003), Shanxi Provincial International Cooperation Project (Grant No. 2012081043), China Scholarship Council (Grant No. 2013-038), Beijing Science and Technology New Star Project (2010B021), and Scientific Research Foundation for Returned Scholars of Ministry of Education of China. The research was also jointly supported by the Natural Science and Engineering Research Council (NSERC) of Canada.
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(32) Chung, C. H.; Kim, J. H.; Jung, J.; Chung, B. H. Biosens. Bioelectron. 2013, 41, 827−832. (33) Zhu, G.; Li, Y.; Zhang, C.-Y. Chem. Commun. 2014, 50, 572− 574. (34) Shen, L.; Chen, Z.; Li, Y.; He, S.; Xie, S.; Xu, X.; Liang, Z.; Meng, X.; Li, Q.; Zhu, Z.; Li, M.; Le, X. C.; Shao, Y. Anal. Chem. 2008, 80, 6323−6328. (35) Zhang, M.; Ge, L.; Ge, S.; Yan, M.; Yu, J.; Huang, J.; Liu, S. Biosens. Bioelectron. 2013, 41, 544−550. (36) Chen, J.; Tang, J.; Zhou, J.; Zhang, L.; Chen, G.; Tang, D. Anal. Chim. Acta 2014, 810, 10−16. (37) Zhu, Z.; Su, Y.; Li, J.; Li, D.; Zhang, J.; Song, S.; Zhao, Y.; Li, G.; Fan, C. Anal. Chem. 2009, 81, 7660−7666. (38) Lee, J.-S.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 4093−4096. (39) Li, T.; Dong, S.; Wang, E. Anal. Chem. 2009, 81, 2144−2149. (40) Marc, R.; Knecht, M. S. Anal. Bioanal. Chem. 2009, 394, 33−46. (41) Mazumdar, D.; Liu, J.; Lu, G.; Zhou, J.; Lu, Y. Chem. Commun. 2010, 46, 1416−1418. (42) National Primary Drinking Water Regulations: National Report; Publication No. EPA816F090004; United States Environmental Protection Agency, U.S. Government Printing Office: Washington, DC, 2009. (43) Lin, Z.; Li, X.; Kraatz, H.-B. Anal. Chem. 2011, 83, 6896−6901. (44) Li, T.; Wang, E.; Dong, S. Anal. Chem. 2010, 82, 1515−1520. (45) Hai, H.; Yang, F.; Li, J. RSC Adv. 2013, 3, 13144−13148. (46) Chen, X.; Guan, H.; He, Z.; Zhou, X.; Hu, J. Anal. Methods 2012, 4, 1619−1622. (47) Lee, Y.-F.; Nan, F.-H.; Chen, M.-J.; Wu, H.-Y.; Ho, C.-W.; Chen, Y.-Y.; Huang, C.-C. Anal. Methods 2012, 4, 1709−1717. (48) Norton, G. J.; Williams, P. N.; Adomako, E. E.; Price, A. H.; Zhu, Y.; Zhao, F. J.; McGrath, S.; Deacon, C. M.; Villada, A.; Sommella, A.; Lu, Y.; Ming, L.; Mangala, P.; De Silva, C. S.; Brammer, H.; Dasgupta, T.; Islaml, M. R.; Meharg, A. A. Sci. Total Environ. 2014, 485, 428−434. (49) Jackson, B. P.; Punshon, T. Curr. Environ. Health Rep. 2015, DOI: 10.1007/s40572-014-0035-7. (50) Zhang, X. B.; Wang, Z. D.; Xing, H.; Xiang, Y.; Lu, Y. Anal. Chem. 2010, 82, 5005−5011. (51) Qi, L.; Zhao, Y.; Yuan, H.; Bai, K.; Zhao, Y.; Chen, F.; Dong, Y.; Wu, Y. Analyst 2012, 137, 2799−2805.
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DOI: 10.1021/acs.analchem.5b00899 Anal. Chem. XXXX, XXX, XXX−XXX
Detection and Quantitation of Heavy Metal Ions on Bona Fide DVDs Using DNA Molecular Beacon Probes Lingling Zhang,†,§ Jessica X. H. Wong,‡ Xiaochun Li,*,† Yunchao Li,§ and Hua-Zhong Yu*,†,‡ †
Key Laboratory of Advanced Transducers and Intelligent Control Systems (Ministry of Education and Shanxi Province), College of Physics and Optoelectronic Engineering, Taiyuan University of Technology, Taiyuan, Shanxi 030024, China ‡ Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada § Department of Chemistry, Beijing Normal University, Beijing 100875, China S Supporting Information *
ABSTRACT: A sensitive and cost-effective method for the simultaneous quantitation of trace amounts of Hg2+ and Pb2+ in real-world samples has been developed using DNA molecular beacon probes bound to bona fide digital video discs (DVDs). With specially designed T-rich or G-rich loop sequences, the detection is based on the strong T-Hg2+-T coordination chemistry of Hg2+ and the formation of Gquadruplexes induced by Pb2+, respectively. In particular, the presence of metal cations leads to hairpin opening and exposure of the terminal biotin moiety for binding nanogold− streptavidin conjugates. The recognition signal was subsequently enhanced by gold nanoparticle-promoted silver deposition, which leads to quantifiable digital signals upon reading with a standard computer drive. This method exhibits a wide response range and low detection limits for both Hg2+ and Pb2+. In addition, the quantitative determination of heavy metals in food products (e.g., rice samples) has been demonstrated and the method compares favorably with other optical sensors developed recently. lthough most people would choose USB flash drives to store their data and media files nowadays, optical disc technology has been widely explored with the ultimate goal of creating bioanalytical systems for on-site chemical analysis and point-of-care medical diagnostics.1,2 This is because compact discs (CD)/digital video discs (DVDs) can be adapted as inexpensive substrate materials for the preparation of various bioassays,1 and conventional computer drives/disc players are powerful optical devices for biochip signal readout.1,2 Two types of disc-based devices/chips, i.e., lab-on-a-disc microfluidic disks3 and spinning interferometry bioCDs,4 have been the main focus in this field in the past two decades. Conceptually different from hardware modification approaches,5−9 we have been working on the development of digitized molecular analytical systems that are entirely based on bona fide CD/DVD discs and optical drives.1,10−12 Inspired by the delicate data protection and retrieval mechanism inhered in optical disc systems, we developed first a CD-based digital readout protocol for screening biomolecular binding reactions,10 which had been extended to state-of-the-art DVD systems.11 Particularly, the analytical platform based on DVD technology has been demonstrated to be applicable in medical diagnosis (quantitation of human chorionic gonadotropin in urine samples)11 and on-site screening of drugs of abuse.12 In the meantime, other groups have also made significant progress in adapting such digital approaches to create microimmunoassays and cell-counting devices.13−15
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© XXXX American Chemical Society
One of the limitations of DVD-based analytical systems we have developed so far is the assay preparation, i.e., routine adaptation of conventional sandwich-format or competitive immunoassays that rely on antibody−antigen interactions.11,12 Sandwich-type assays generally require multistep surface reactions and additional labeling reagents, while indirect competitive assays would generate “signal-off” responses. By introducing DNA molecular beacon probes (i.e., DNA hairpins) as sensing entities on discs to create “signal-on” responses from analyte-induced conformational changes,16,17 we report herein a DVD-based analytical system for the quantitative detection of heavy metal ions in both standard buffers and real-world samples (extracts from food products). Hg2+ and Pb2+ are the two most widespread heavy metal contaminants with high and persistent toxicity. Human exposure may lead to irreversible damage of the digestive, neurological, and cardiovascular systems.18−21 Conventional methods established for the quantitative analysis of Pb2+ and Hg2+ include atomic absorption spectrometry,22,23 inductively coupled plasma mass spectrometry (ICPMS),24,25 and anodic stripping voltammetry.26 Various novel sensors based on fluorescence,27−33 electrochemical,34−37 and colorimetric readout38−41 have been proposed in the past decade. Our aim to Received: March 6, 2015 Accepted: April 23, 2015
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the free disc-quality diagnostic program (PlexUTILITIES 1.3) was used to read the assay discs at a speed of 6×. After reading, the program can export an error distribution plot and provide statistical results regarding the error numbers and types. Optical images of all assays (the binding strips formed on DVD-Rs) were captured using a flatbed scanner (Microek ScanMaker i700) in the reflective mode and then analyzed in Adobe Photoshop to determine the optical darkness (from the grayscale intensities). The surface topographies of the binding stripes were imaged with an atomic force microscope (Dimension Edge, Bruker Inc.) in tapping mode.
develop DVD assays for heavy metal ion detection is not to compete with the above established methods in terms of sensitivity and accuracy but rather to provide an alternative for on-site detection or screening, particularly in remote locations where advanced instrumentation is not available.
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EXPERIMENTAL SECTION Reagents and Materials. All oligonucleotides were of HPLC-grade and obtained from Shanghai Sangon Biotechnology Inc. (Shanghai, China). The specifically designed sequences of G-rich and T-rich oligonucleotides are 5′-NH2-(CH2)6-TAG CCA ACA AGG TTG GTG TGG TTG GCA T-biotin-3′ (GG probe) and 5′-NH2-(CH2)6-CAA ATG AAC TTT GGT TTC CCT TTT CAT TTT-biotin-3′ (TT probe), respectively.17 DVD-R (4.7G) was ordered from Maxell Taiwan Ltd. (Taipei, Taiwan). Silver acetate, hydroquinone, 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), tris (hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), and bovine serum albumin (BSA, globulin-free, molecular-biology grade) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Nanogold (1.4 nm diameter)− streptavidin conjugates were ordered from Nanoprobes Inc. (New York, USA). Solutions were prepared with deionized water produced from a Barnstead Easypure System (Thermo Scientific Inc., Dubuque, Iowa, USA). Rice samples were immersed in a solution containing 0.7 mM Hg2+ and 5.0 mM Pb2+ for 1 week. The contaminated rice was then washed with water, dried, and ground into a fine powder. A 1.0 g aliquot of the sample was digested with 10 mL of concentrated nitric acid (5 M) and boiled to almost dryness (∼1 mL left). After adding 2−3 mL of water, the mixture was filtered through a membrane filter of 0.2 μm pore size. The filtrate was diluted with water to a total volume of 10 mL. DVD Surface Activation and Assay Preparation. Video files were recorded onto a blank DVD-R, which was then cleaned with ethanol and deionized water. The surface was activated in a UV/ozone system (Model PSD-UV, Novascan Technologies Inc.) for 20−40 min to produce a hydrophilic surface with a high density of carboxylic acid groups. The discs were subsequently immersed in a 0.1 M phosphate buffer at pH 6.0 containing 5 mM EDC and 0.3 mM NHS for 3−5 h to activate the surface-tethered carboxylic acid groups.11,12 A specially designed PDMS (polydimethylsiloxane) plate with two sets of embedded microchannels (0.3 mm × 15 mm × 50 nm, arc shaped) was first put on the activated DVD substrate for the immobilization of 1.5 μL of GG probes (in 10 mM Tris buffer containing 150 mM NaCl and 50 mM MgCl2, pH 7.4) and TT probes (in 10 mM phosphate buffer containing 150 mM NaCl and 50 mM MgCl2, pH 7.4) to form two different testing zones (for Pb2+ and Hg2+). The disc was then kept in a humidity box overnight to form the probe arrays. After blocking with 3% BSA for 1−3 h, 1.5 μL of solution containing different concentrations of Pb2+ and Hg2+ was injected into the channels and kept for 60 min. Nanogold− streptavidin conjugate (0.8 μg/mL) in 20 mM phosphate buffer containing 0.8% BSA and 0.1% gelatin was then injected into each channel and incubated for 30−60 min. The PDMS plate was removed, and the disc was washed thoroughly with water before immersion in a freshly made silver-staining solution (12 mM silver acetate and 45 mM hydroquinone) for 5−20 min, depending on staining conditions, to amplify the signals. Instrumentation and Data Analysis. A Blu-ray/DVD optical drive (PX-LB950UE, Plextor Co.) in conjunction with
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RESULTS AND DISCUSSION Preparation of DNA Hairpin Assays on DVD for Digitized Metal Ion Detection. Different from the traditional sandwich-format or competitive immunoassays, we decided to adapt molecular beacon probes, i.e., DNA hairpins containing Hg2+ or Pb2+-specific sequences in the loop, as sensing entities for the detection of heavy metal cations, a strategy which has been successfully demonstrated for electrochemical and conventional colorimetic analysis.16,17,37 Particularly, two dually modified DNA hairpin probes, one containing guanine (G)-rich fragments (named as GG probe) and the other containing thymine (T)-rich fragments (named as TT probe), were immobilized at different locations on the surface of a DVD (with the aid of a PDMS channel plate) for recognizing Pb2+ and Hg2+, respectively. The immobilized DNA probes, initially in their folded (hairpin-like) forms such that the terminal biotin groups were embedded in the probe layer, prevent the access of nanogold−streptavidin conjugates. In the presence of Pb2+ and Hg2+, hairpin secondary structures were “forced” open and form a stable G-quadruplex and T-Hg2+-T complex, respectively, allowing nanogold−streptavidin conjugates to bind promptly with the biotin groups and to produce signals thereon upon silver enhancement (for details, see the Supporting Information). The strong and specific interaction between the two DNA hairpin probes (GG and TT) and the two metal cations (Pb2+ and Hg2+) have been confirmed previously by using colorimetric assays.17 As shown in Figure 1a, the binding sites on the DVD surface were covered with rather large silver particles; such binding strips disturb the normal disc reading process by blocking the laser beam (reflection and scattering), thus causing significant reading errors (parity inner failure, PIF). We have confirmed previously that the distribution and counts of the resulting PIF peaks have a direct correlation with the physical location and intensity of the binding strips, respectively.12 Figure 1b shows the results of using this assay platform to examine eight solutions containing different concentrations of Hg2+ (from 0 to 50 μM). It was observed that eight binding strips with different darkness appeared on the disc (inset photo), and those strips (except for the blank) indeed induced well-defined error peaks with different heights (PIF counts). To have a better understanding of the performance of this DVD assay, we have integrated the error peaks and normalized them to the area of the binding strips to obtain the corresponding PIF density, which was plotted versus the concentration of Hg2+. Figure 2a reveals that the PIF density (error counts per unit area) increases monotonically with the concentration of Hg2+; even with very low concentrations (0.1−5.0 nM), the signal is significantly higher than that of the blank (an assay strip formed in the absence of Hg2+). Particularly, we have indicated the signal level corresponding B
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Figure 2. (a) The dependence of PIF density and ODR on the concentration of Hg2+; (b) Correlation between the PIF and ODR values obtained for the same set of assays strips (shown in Figure 1b).
show much smaller relative standard deviations and lower blank signals compared to those of ODR readings. In Figure 2b, we plotted the PIF density as a function of the ODR value for each of the standard solutions tested for Hg2+. Unlike the linear relationship observed in our previous studies, the correlation between these two sets of data is rather perplexing. While a general increasing trend in PIF density was observed when the ODR value became larger, a linear relationship is not obvious. A quick rising (titration curve-like) region was observed for a small ODR range (0.20−0.35). It is not clear at this time why the ODR values do not correlate with the PIF densities; however, the AFM (atomic force microscope) images shown in Figure 3 may shed light on the issue. In comparison with the featureless image for a blank sample, nanoparticles ranging from several tens to hundreds of nanometers have been observed for the sample of 0.5 nM Hg2+. The coverage of these particles increased substantially for the sample with 5.0 nM Hg2+, and it seems to become saturated when the concentration is raised to 5.0 μM. At the higher concentrations, the aggregation and multilayer deposition of the particles are evident as well. As illustrated in Figure 1a, the digital reading relies on the disruption of the laser beam (650 nm), which is otherwise reflected by the metal layer in the DVD for data decoding. In contrast, the ODR values are determined from the grayscale values of a scanned image, which is completely dependent on the scanner. We suspect that the unique aggregation of the silver particles in these samples (which are not typical for other assays) may play an important role for the discrepancy mentioned above. Versatility of DVD-Based Metal Detection. We and others have demonstrated that sensitive DNA sensors for Pb2+
Figure 1. (a) Schematic view of the digitized detection of Pb2+ and Hg2+ on a bona fide DVD with a standard optical drive; the Au/silver particles accumulating at the binding site interfere with the reading laser, which creates quantifiable errors. (b) A representative error plot (the PIF distribution) generated upon reading a Hg2+ assay prepared on a DVD; the inset photo shows the eight binding strips corresponding to different concentrations of Hg2+ (as listed).
to the 3δ (standard deviation) in addition to the blank response as the red dash line in Figure 2a; it is clear that a concentration of 0.5 nM (0.1 ppb) can be considered as the limit of detection (LOD) in this experiment. We note that the detectable concentration achieved herein (0.1 ppb) is well below the maximum allowable level of Hg2+ in drinking water (2 ppb) recommended by the United States Environmental Protection Agency.42 To further evaluate the DVD assay performance, we have compared it with the conventional colorimetric protocol, by examining the optical darkness ratio (ODR) of each binding strip with a standard desktop scanner.1,10 As shown in Figure 2a, the ODR values seemingly compare well with the PIF data in terms of the concentration dependence, yet the PIF results C
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Figure 3. Atomic force microscope (AFM) images of the binding strips on a DVD for Hg2+; the assay was prepared following the procedure shown in Scheme S1, Supporting Information, with different concentrations of Hg2+ present.
can be fabricated on the basis of the conformational change from a hairpin DNA to a G-quadruplex to induce either colorimetric or electrochemical signals.17,43,44 Herein, we have immobilized Pb2+-specific DNA probes (GG probe) on a DVDR and adapted the same labeling and signal enhancement protocols. The LOD determined for the detection results of Pb2+ is at least 0.5 nM (see the Supporting Information for details); such a LOD (0.1 ppb) is significantly lower than the allowable level of Pb 2+ in drinking water (15 ppb) recommended by the United States Environmental Protection Agency.42 Besides the detection sensitivity, the specificity of this digital sensing platform was evaluated by testing other common metal ions (such as Na+, Ni2+, Zn2+, Al3+, Fe3+, Li+, Hg2+, Mg2+, Ca2+, Cu2+, Ba2+, and Mn2+). We found that for both Pb2+ and Hg2+ detection of the interfering ions (at 0.1 mM) only caused low levels of reading errors (PIF counts), which are either not detectable or insignificant. However, a few metal cations (e.g., Na+) did produce discernible responses for the Pb2+ sensor, due to the fact that the formation of G-quadruplexes can be induced by other metal cations.45−47 Although the concentrations leading to such a change are much higher (i.e., mM range) in comparison with Pb2+,45 we should consider the limitation of its application in aqueous solutions with high concentrations of alkaline cations. The successful individual detection of Pb2+ and Hg2+ allows us to explore the capability of this digital sensing platform for simultaneous detection of Pb2+ and Hg2+ in binary mixtures. To achieve this goal, the Pb2+- and Hg2+-specific probes (i.e., GG and TT probe) were immobilized at two different sections on the same DVD to form a“Pb2+ testing zone” and a “Hg2+ testing zone”, respectively, by using the specially designed PDMS plate with two sets of microchannels. We hoped that the recognition reactions between the two probes and the two target ions occurring at specified locations could be readily differentiated. With this design, we first analyzed binary aqueous samples containing both Pb2+ (0, 0.05, 50 μM) and Hg2+ (0, 0.05, 50 μM) at different concentrations. Figure 4a shows the PIF responses corresponding to all binding strips as a function of their physical locations (radial distance) on the DVD. To
Figure 4. Simultaneous determination of Hg2+ and Pb2+ on the same disc by immobilizing TT probes and GG probes at defined locations. (a) PIF distribution on the DVD with 16 binding strips including two blanks (SP1, SP2), individual detections of Hg2+ (SP3, SP5) and Pb2+ (SP4, SP6), and simultaneous detections (SP7-SP16); the top inset is an optical image of the binding strips. (b) 3D histogram showing the dependence of PIF density on Hg2+ and Pb2+ concentrations. The error bars represent the standard deviations derived from triplicate measurements.
provide a better overview of the quantitative comparison, we have generated a 3D histogram as shown in Figure 4b. As expected, the blanks in both test zones (SP1 and SP2) caused negligible PIF signals. The samples containing only Hg2+ (SP3, SP4) produced a distinct PIF peak in the Hg2+ test zone (SP3) and low signals in the Pb2+ test zone (SP4) and vice versa (low signal observed for Pb2+-containing samples in the Hg2+ test zone (SP5) but high response in the Pb2+ test zone (SP6)). For the samples containing both Pb2+ and Hg2+, distinct PIF peaks at the corresponding locations in both testing zones (SP7− SP16) were identified. This proof-of-concept experiment not only confirms the selectivity of the digital sensing protocol but also shows its capability of differentiating multiple analytes on a single assay disc. Real-World Sample Testing and Comparison with Other Methods. To minimize the complexity of real-world D
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site screening (when advanced instrumentation and centralized laboratories facilities are not available). A conventional computer optical drive or a stand-alone disc player is the only required instrument; the cost of the analysis is significantly reduced due to its high detection throughput and limited reagent consumption. These features make this digital sensing platform an ideal tool for on-site screening before bringing back samples to a centralized laboratory for further analysis.
sample testing, we have chosen spiked rice as a model system to validate our DVD assay protocol. The samples, together with a series of standard solutions containing known concentrations of Hg2+ and Pb2+, were examined on the DVD platform. Benefitted by the number of samples we can test on a single disc, the set of standard solutions and three sample replicates were examined on the same disc (top insets, Figure 5).
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CONCLUSION
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ASSOCIATED CONTENT
We have developed a DVD-technology-based assay method for the detection of heavy metal ions with high sensitivity and selectivity in both standard buffers and real-world samples. The signal readout is performed with a conventional optical drive, and the assay is prepared on bona fide digital video discs. Particularly, DNA molecular beacon probes (i.e., DNA hairpins) were immobilized on regular DVDs as sensing entities to create “signal-on” responses from analyte-induced conformational changes. Considering the versatility of designing loop sequences in the DNA molecular beacon probes, this approach can be readily extended to the detection of many different analytical targets, other metal cations, proteins, and oligonucleotides.36 In addition, the DVD assay has been demonstrated for the quantitation of Pb2+ and Hg2+ in binary mixtures and in the presence of other interfering metal ions, with LODs down to 0.5 nM and wide linear response ranges (2−500 nM for Pb2+ and 10−1000 nM for Hg2+). These low LODs are adequate for environmental monitoring and food safety tests, which augments its potential application for on-site chemical analysis.
Figure 5. Quantitative detection of (a) Hg2+ and (b) Pb2+ in a contaminated rice sample. The black lines are the best fits to the experimental data within the linear response regions; the red lines show the signal and concentration of the unknown sample. The top insets are the optical images of the assay discs from which the PIF data were obtained.
S Supporting Information *
Additional experimental data including the step-by-step preparation of DVD assays, detection of Pb2+, selectivity tests, and comparison with other methods. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00899.
In Figure 5, we have shown the semilog calibration curves for the detection of Hg2+ (a) and Pb2+ (b), respectively. In both cases, we have obtained satisfactory fitting results in a wide response range (2−500 nM for Hg2+ and 10−1000 nM for Pb2+); PIF(Hg) = 97.7 ± 5.1 × log [Hg2+] + 509 ± 9, R2 = 0.995; PIF(Pb) = 120 ± 1 × log [Pb2+] + 391 ± 1, R2 = 0.999, respectively. On the basis of these calibration equations and the obtained PIF signals (380 ± 2 and 339 ± 14 counts/mm2), the concentrations of Hg2+ and Pb2+ in the unknown samples (i.e., rice extracts) were determined to be 0.046 ± 0.013 and 0.257 ± 0.017 μM, respectively. The rather large relative uncertainty for the Hg2+ concentration can be explained by the nature of error propagation with the semilog calibration curve and its low abundance (nM). The converted abundance of Pb2+ in the original contaminated rice sample was 530 ± 35 ppb, which is much higher than that found in market collected white rice (4− 46 nM), also above the level in samples from known contaminated/mine impacted regions (185 ± 4 ppb).48 Similarly, the concentration of Hg2+ in the contaminated rice sample (92 ± 26 ppb) is comparable to that of rice grown in Hg2+-contaminated soils (10−100 ppb).49 It should be noted that the rice sample testing is a preliminary experiment to show the application potential of the DVD assay; we also acknowledge that the DVD assay may not be able to compete with the conventional ICPMS method and the recently developed fluorescence detection using catalytic and molecular beacon probes in terms of sensitivity and accuracy;50,51 however, it is potentially applicable for on-
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected] (H.-Z.Y.). *E-mail: [email protected] (X.L.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We gratefully acknowledge the financial support from the Natural Science Foundation of China (Grant Nos. 61174010; 21003012; 21233003), Shanxi Provincial International Cooperation Project (Grant No. 2012081043), China Scholarship Council (Grant No. 2013-038), Beijing Science and Technology New Star Project (2010B021), and Scientific Research Foundation for Returned Scholars of Ministry of Education of China. The research was also jointly supported by the Natural Science and Engineering Research Council (NSERC) of Canada.
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