Highly Sensitive Simultaneous Detection of Lead(II) and Barium(II

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Highly Sensitive Simultaneous Detection of Lead(II) and Barium(II) with G‑Quadruplex DNA in α‑Hemolysin Nanopore Chun Yang,†,‡,∥ Lei Liu,†,∥ Tao Zeng,§ Daowu Yang,‡ Zhiyi Yao,† Yuliang Zhao,†,§ and Hai-Chen Wu*,† †

Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China ‡ College of Chemical and Biological Engineering, Changsha University of Science & Technology, Changsha 410004, China § National Center for Nanosciences and Technology of China, Beijing, 100190, China S Supporting Information *

ABSTRACT: Both Pb2+ and Ba2+ can bind with high affinity to some specific DNA sequences, inducing the formation of unimolecular G-quadruplex structures. Translocation of a DNA probe containing such sequences through an αhemolysin nanopore in the presence of Pb2+ or Ba2+ would result in much prolonged DNA translocation events. Quantification of these events can reveal the concentrations of Pb2+ or Ba2+ at as low as 0.8 nM. Besides, Pb2+ and Ba2+ in the solution can be simultaneously detected and individually identified. Furthermore, the probe is highly selective for Pb2+ and Ba2+ detection without interference from other metal ions. This sensing strategy can be extended to many other analytes which can bind to DNA molecules with high affinity. or Ba2+ and employed the complex in the α-hemolysin (αHL) nanopore for the sensitive detection of Pb2+ and Ba2+. Nanopore sensing has proven to be a powerful tool in the detection of various analytes such as metal ions,41,42 proteins,43 DNA,44 RNA,45,46 organic molecules,47−50 etc.51 When an analyte binds within the nanopore, the ionic current will be modulated depending on the size, charged status, and concentration of the analyte. The recorded current fluctuations can be used to discriminate different species. Hitherto, there are few studies dealing with the detection of metal ions using nanopore sensors. Braha and colleagues reported an elegant work of simultaneous detection of Zn2+, Co2+, and Cd2+ with a single sensor element inside an αHL mutant.41 However, it is not straightforward to extend this strategy for the detection of other metal ions. Recently, we have used DNA-based probes in αHL for the detection of Hg2+.42 As part of our interest in developing versatile probes for nanopore sensing, herein we report our effort of using one DNA oligomer for the simultaneous detection of Pb2+ and Ba2+ in the absence of any masking agents.

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eavy metal pollution in the aquatic system and soil has become a serious threat to human health and the environment. Therefore, construction of sensitive and selective sensors for the detection of different heavy metal ions has been a very active research area in recent years.1 Among all the heavy metal ions, the lead ion (Pb2+) is one of the most toxic pollutants in drinking water and surface soil and may be accumulated in the human body as a potential neurotoxin that causes bone and kidney damage.2 So far, several analytical techniques have been developed for Pb2+ detection by employing fluorophores,3−9 chromophores,10,11 electrochemical approaches,12−16 nanoparticles,17−22 DNAzymes,23−30 G-quadruplex DNAs,31−34 etc. Most of the developed methods showed high sensitivity and selectivity for Pb2+ analysis with detection limits typically ranging from several nanomolar to submicromolar. Among the different approaches, Pb2+-induced formation of G-quadruplex structures served as one of the cornerstones in the fabrication of various sensors for Pb2+ detection. Compared with Pb2+, detection of the barium ion (Ba2+) is much less studied.35−39 On one hand, the toxicity of barium might be underestimated since its lighter congeners, magnesium and calcium, are necessary elements for human life, although high doses of soluble barium compounds could affect the nervous system, causing cardiac irregularities, tremors, weakness, anxiety, dyspnea, and paralysis.40 On the other hand, there are very few reports in the literature dealing with any specific barium complexes that can be used for the construction of sensors for Ba2+. In this work, we designed a DNA probe which could form G-quadruplex in the presence of either Pb2+ © 2013 American Chemical Society

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RESULTS AND DISCUSSION Selection of the Electrolyte. It is known that Pb2+ binds with unusually high affinity to some specific DNA sequences, e.g., thrombin binding aptamer (TBA, 5′GGTTGGTGTGGTTGG-3′), inducing the formation of a unimolecular G-quadruplex structure.52 We envisioned that Received: April 22, 2013 Accepted: July 16, 2013 Published: July 16, 2013 7302

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quadruplex DNA (DNA1 and DNA3) translocation was significantly prolonged compared with that of “normal” ssDNA without secondary structures.54 Therefore, we need to find an electrolyte that does not have interactions with Gquadruplex DNAs. Although the ammonium ion (NH4+) is also known to have high affinity with certain G-quadruplex structures,55 we speculate that its analogue tetramethylammonium chloride (TMACl) might be less favorable for binding with G-quadruplex DNAs. After a number of screenings of experimental conditions, we found that using 0.5 M TMACl as an electrolyte solution under the transmembrane potential of 180−200 mV gave the cleanest background in the long dwell time area (100 to 104 ms) which was associated with the unfolding of G-quadruplex structures during DNA translocation and a sufficiently high frequency of DNA translocation events (Figures S1 and S2). Pb2+ Detection. After TMACl was chosen as the electrolyte, we focused on the selection of DNA probes for Pb2+ detection. TBA has been extensively used in Pb2+-related studies in the literature.34 However, when we used TBA as the core sequence in a probe with extended tails, we surprisingly found that the translocation duration of these DNAs (DNA2− 4, Table S1) did not have much difference from that of the control groups (Figure S3). We reasoned this might be because the binding of Pb2+ with TBA is not strong enough to halt the DNA translocation when the complex reached the constriction in αHL (Figure 1B). Thus, we turned our attention to other Gquadruplex DNAs that could bind Pb2+ more tightly. PS2.M is another frequently used G-quadruplex DNA (5′-GTAAG-

when a single-stranded DNA (ssDNA) containing a Gquadruplex sequence was translocated through the αHL nanopore, the presence of Pb2+ would cause the guanine-rich part to fold into a G-quadruplex structure and thus greatly alter the translocation profile of the DNA strand (Figure 1A and B).

Figure 1. The principle of detecting Pb2+ and Ba2+ with G-quadruplex DNA. (A) Translocation of ssDNA through αHL as the control. (B) Illustration of the unfolding and subsequent translocation of Pb2+- or Ba2+-induced G-quadruplexes of DNA1 in αHL. The sequence of the G-quadruplex DNA is marked in red.

To verify this hypothesis, we designed several DNA strands (Table S1) that contain the G-quadruplex sequence for the detection of Pb2+. Nevertheless, we encountered a problem when we chose an electrolyte for single-channel recording experiments. Usually, KCl and NaCl are the most used salts for making electrolyte solutions in the concentration range of 0.5 to 4.0 M. However, K+ and Na+ are also known to be able to stabilize the G-quadruplex structure of certain sequences.53,54 When we used 0.5 M KCl or NaCl as the electrolyte solution for the control experiments, we found that the dwell time of G-

Figure 2. Representation of the translocation of ssDNA (DNA1), Pb2+-induced G-quadruplex (Pb2+-DNA1), and Ba2+-induced G-quadruplex (Ba2+DNA1) through a single αHL nanopore. [A−C] (a) Representative single-channel current traces of the translocation of DNA1, Pb2+-DNA1, and Ba2+-DNA1. [A−C] (b) Expanded view of the events indicated in the current trace with a red star. [A−C] (c) Corresponding 2D event-distribution plots associated with DNA1, Pb2+-DNA1, and Ba2+-DNA1 translocation through the nanopore. The distribution of events is plotted according to current blockage (I/I0 > 0.70). The normalized density of events at each coordinate is indicated by the color code. All experiments were conducted in the buffer of 0.5 M TMACl, 5 mM Tris, pH 8.0, with the transmembrane potential held at +200 mV. DNA1 (final concentration 1.0 μM) was preincubated with Pb2+ or Ba2+ (final concentration 4.0 μM) for 2 h at room temperature before being added into a cis chamber. The number of individual experiments n = 3. 7303

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frequency for high sensitivity.42,45 Here, we used the optimized conditions of 3 M trans/0.5 M cis TMACl with the transmembrane potential held at +200 mV (Figures S5 and S6). For the quantitative analysis of Pb2+ and Ba2+, we adopted the method from an earlier work of our group by drawing a signal oval from the scatter plot data (current blockage vs dwell time) and calculating the probability (PM, M = metal ion) of the events inside the signal oval out of total DNA translocation events (Data Analysis and Figure S7).42 The value of PM is directly associated with the concentration of metal ions. Experiments with various concentrations of Pb2+ and Ba2+ were carried out under otherwise identical conditions, respectively. The results in Figure 4 showed that the detection limits for Pb2+ and Ba2+ were both around 0.8 nM, which is lower than most previously reported values.

TAAAGCGAATTGG-3′) and shown to have strong binding with Pb2+.32,33 When we added a 30-nucleotide tail to both ends of PS2.M (DNA1) and threaded it through αHL in the presence of 4 times the concentration of Pb2+, we observed that the duration of DNA translocation events was prolonged by approximately 3 orders of magnitude compared with the control group (Figure 2Ab and Bb). A new region is generated on the 2D event-distribution contour map, which unambiguously indicates the presence of Pb2+ in solution (Figure 2Bc). The Gaussian fit of the newly appeared region on the 2D plot revealed that the average duration of Pb2+-mediated DNA translocation was 99.7 ± 1.1 ms. To be sure that those long DNA translocation events were caused by Pb2+-stabilized Gquadruplex structures, we conducted another control experiment by threading DNA5, whose sequence is similar to that of DNA1 but incapable of forming G-quadruplex (Table S1), through αHL in the presence of Pb2+. The results showed that the translocation of DNA5 was not affected by the presence of excess Pb2+, which in turn confirmed the important role of the PS2.M sequence in Pb2+ detection (Figure S4). Ba2+ Detection. After the initial success of using PS2.M for sensing Pb2+, we set out to investigate the interference of other metal ions with the detection, as PS2.M is known to have high affinity with several metal ions.33 Most of the commonly studied metal ions did not show any influence on the Pb2+ detection (vide inf ra) except Ba2+, which could also bind with PS2.M to give prolonged DNA translocation events. The dwell time of Ba2+-mediated DNA translocation events is 14.5 ± 1.1 ms (Gaussian fit of the newly appeared region on the 2D plot of Figure 2Cc), about one-seventh of that of Pb2+-DNA1. On the 2D event-distribution contour map, we could see that the new area associated with Ba2+ is to the left of the Pb2+ area with considerable overlapping. To substantiate these results, we carried out circular dichroism (CD) measurements of Gquadruplex structures of DNA1 induced by Pb2+, Ba2+, and their mixture (Figure 3). All three groups gave new peaks in the

Figure 4. Investigation of the detection limit for Pb2+ and Ba2+ detection. (A) Plot of PPb versus Pb2+ concentration. (B) Plot of PBa versus Ba2+ concentration. Illustration of the criteria for quantitation is shown in Figure S7. Experiments were carried out with DNA1 (10 nM) in cis under the conditions of 3 M trans/0.5 M cis TMACl with the transmembrane potential held at +200 mV. The control group value has been offset to zero (number of individual experiments n = 6).

Simultaneous Detection of Pb2+ or Ba2+. As mentioned earlier, the DNA probe (DNA1) can detect both Pb2+ and Ba2+ under the same conditions. This is an interesting finding because in previous studies when G-quadruplex DNAs were used to bind Pb2+ to achieve fluorometric or colorimetric detection, Ba2+ was found to be noninterfering.56 This might be because the Ba2+-mediated G-quadruplex structures are not stable enough to afford positive signals in luminescent sensing, while nanopore analysis possesses sufficiently high sensitivity in distinguishing those metastable structures from the control group. We wondered if the nanopore sensing could distinguish Pb2+ and Ba2+ individually, when they coexisted in the same solution. From the dwell time histogram of DNA1 translocation in the presence of Pb2+ or Ba2+, we could see that there was significant overlapping of log(dwell time) between 0.3 and 2.0 (Figure 5A; for full range data, see Figure S8). Therefore, when

Figure 3. CD spectra of the Pb2+, Ba2+, and mixed-cation-induced Gquadruplexes of DNA1 in the buffer solution (0.5 M TMACl, 5 mM Tris, pH 8.0) at room temperature, DNA1 concentration 2.0 μM.

CD spectra, confirming the formation of G-quadruplex structures in the presence of Pb2+ or Ba2+. The mixed ions group only showed the peak of Pb2+-DNA1, implying that the Pb2+ binding affinity with DNA1 is stronger than that of Ba2+. This result is consistent with G-quadruplex DNA1 translocation results where the dwell time of Pb2+-DNA1 translocation is much larger than that of Ba2+-DNA1. Detection Limit for Pb2+ or Ba2+. Next, we examined the detection limit of this DNA-based nanopore sensing strategy for Pb2+ and Ba2+, respectively. It has been previously reported that asymmetric salt conditions could greatly increase the event 7304

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Figure 5. Effect of different ratios of DNA1 with Pb2+ and Ba2+ for simultaneous determination. (A) Histograms of logarithmically binned dwell times that correspond to the translocation of DNA1 in the presence of 4.0 equivalent Pb2+ or Ba2+. (B−D) Histograms of logarithmically binned dwell times for DNA1 translocation when Pb2+ and Ba2+ coexist in different concentrations: (B) DNA1/Pb2+/Ba2+ = 2:1:1; (C) DNA1/Pb2+/Ba2+ = 1:1:1; (D) DNA1/Pb2+/Ba2+ = 1:2:2. Blue and green solid lines: double Gaussian fitting to the histograms of Pb2+-DNA1 and Ba2+-DNA1, respectively. Black dashed lines: sum of the Gaussian fitting for Pb2+-DNA1 and Ba2+-DNA1. The ratios between the areas under the fitted curves ascribed to the translocation of Pb2+-DNA1 and Ba2+-DNA1 are given: (B) ABa/APb = 0.52; (C) ABa/APb = 0.35; (D) ABa/APb = 0.30. All experiments were performed in 0.5 M TMACl and 5 mM Tris, pH 8.0 at +200 mV (DNA concentration 1.0 μM, number of individual experiments n = 3).

measurement of DNA1 translocation in the presence of an analyte matrix by mixing Pb2+, Ba2+ (1.0 μM each), and other metal ions which were five times more concentrated together with Ca2+ and Mg2+ that were 1.0 mM each (Figure 7). The

the two ions coexisted in the solution, the two peaks would merge to generate a new peak. However, due to the competitive binding of Pb2+ and Ba2+ with DNA1, the ratio of the two peaks might change if metal ions were in excess. Three groups of experiments were conducted by varying the ratio of DNA1 while maintaining Pb2+/Ba2+ to be 1:1. When DNA1 was stoichiometric or in excess, double-Gaussian fits of the peak resembled the superimposition of the separate Pb2+ and Ba2+ peaks (Figure 5B and S8C, Table S2). However, when DNA1 was not sufficient for both ions binding, double-Gaussian fits revealed that the peak of Pb2+ became dominant whereas the peak of Ba2+ decreased accordingly, consistent with the CD measurements (Figures 5C,D and 3). From the above discussion, we can conclude that with the G-quadruplex DNA probe, Pb2+ and Ba2+ can be individually identified without using any masking agents. Selectivity of the DNA Probe for Pb2+ or Ba2+. Finally, we investigated the selectivity of the DNA probe for both Pb2+ and Ba2+. First, we carried out DNA1 translocation experiments in the presence of individual metal ions including Pb2+, Ba2+, K+, Na+, NH4+, Ca2+, Mg2+, Li+, Zn2+, Cd2+, Cu2+, Cr3+, Fe3+, and Hg2+. The results showed that those metal ions did not interfere with the detection of Pb2+ and Ba2+ (Figures 6 and S9). Second, to mimic a realistic sample in which the concentration of other metal ions might be higher than Pb2+ and Ba2+ such as drinking water, we performed another

Figure 7. Simultaneous determination of Pb2+ and Ba2+ in the absence and presence of an analyte matrix. (A) Detection of 1.0 μM Pb2+ and 1.0 μM Ba2+ were carried out in the buffer of 0.5 M TMACl and 5 mM Tris, pH 8.0, at +200 mV. The calculated PPb+Ba is 38.6%. (B) An analyte matrix composed of K+, Na+, NH4+, Li+, Zn2+, Cd2+, Cu2+, Cr3+, Fe3+, Hg2+ (final concentration of each cation: 5.0 μM), and Ca2+, Mg2+ (final concentration 1.0 mM each) was mixed with 1.0 μM Pb2+ and 1.0 μM Ba2+ for the detection experiments. The calculated PPb+Ba is 38.9%. The difference between the two PPb+Ba values is within experimental error range (DNA concentration 1.0 μM for all experiments).

difference between the calculated PPb+Ba of the control group (38.6%) and the analyte matrix group (38.9%) is within experimental error range. Therefore, this DNA-based sensing strategy is highly selective for the detection of Pb2+ and Ba2+.

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Figure 6. Investigation of the selectivity for the detection of Pb2+ and Ba2+. The histogram shows the PM in the presence of individual cations (Pb2+, Ba2+, K+, Na+, NH4+, Ca2+, Mg2+, Li+, Zn2+, Cd2+, Cu2+, Cr3+, Fe3+, and Hg2+). All experiments were performed at +200 mV in 0.5 M TMACl and 5 mM Tris, pH 8.0 (DNA concentration 1.0 μM, concentrations of each cation 4.0 μM, n = 6).

CONCLUSION In summary, we have developed a G-quadruplex DNA-based probe for the detection of Pb2+ and Ba2+ inside an αHL nanopore. There are several distinctive features about this sensing platform. First, TMACl has been used as the electrolyte 7305

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a threshold level that was 30% of the open-pore current. Origin and Clampfit were used for histogram construction, curve fitting and graph presentation. Generation of the Pb2+, Ba2+, Pb2+, and Ba2+ signal oval from raw data is as follows: (1) Extract the events with current blockages larger than 70%. (2) Remove the data points with log (dwell time (ms)) < 0.3 in normal symmetrical salt concentration experiments and log (dwell time (ms)) < 0.8 in salt gradient experiments. (3) Generate the “signal oval” which contains >90% of the translocation events of the metal ion-stabilized DNA1 using Origin 8.5. The function of the oval was fitted by Matlab 7.1. With this function, the oval can be regenerated in the unedited data plot and the control group. Data points inside and outside the oval could be counted using this function.

for single channel recording because high concentrations of KCl and NaCl gave enormous background. Second, the probe can be used for simultaneous sensing of Pb2+ and Ba2+ without using any masking agents. Third, the detection limits for both Pb2+ and Ba2+ are very low (0.8 nM), outcompeting most literature reports. Fourth, the probe has very good selectivity for Pb2+ and Ba2+. In addition, the probe can be made from readily available materials, without the process of probe synthesis, purification, etc. This sensing strategy can be extended to many other analytes which can bind to DNA molecules with high affinity.

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EXPERIMENTAL SECTION Materials. All the ssDNA samples (Table S1) were purchased from Tsingke Technologies (Beijing, China). αHL WT-D8H6 was produced by expression in BL21 (DE3) pLysS Escherichia coli cells and self-assembled into heptamers.42 1,2Diphytanoyl-sn-glycero-3-phosphocholine (DPhPc) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Pentane, hexadecane, tetramethylammonium chloride (BioUltra, for molecular biology, ≥ 99.0%), Tris·HCl, copper(II) sulfate anhydrous, iron(III) nitrate nonahydrate, and chromium(III) perchlorate hexahydrate were purchased from Sigma-Aldrich. Lead(II) nitrate, barium(II) nitrate, calcium(II) nitrate tetrahydrate, mercury(II) perchlorate trihydrate, zinc(II) chloride, magnesium(II) nitrate hexahydrate, cadmium(II) perchlorate hexahydrate, potassium(I) chloride, sodium(I) chloride, and ammonium chloride were obtained from Alfa Aesar (Tianjin). Lithium(I) chloride anhydrous was obtained from Aladdin (Shanghai, China). All reagents were used as received without further purification. CD Measurements. The CD spectra were collected on a JASCO J-815 spectropolarimeter (Tokyo, Japan), of which the lamp was always kept under a stable stream of dry purified nitrogen (99.99%) during experiments. DNA1 (final concentration 2.0 μM) was preincubated with Pb2+ (final concentration 0.2 mM), Ba2+ (final concentration 0.2 mM), and Pb2+ and Ba2+ (final concentration of each cation 0.1 mM) for 2 h at room temperature in the buffer solution (0.5 M TMACl, 5 mM Tris, pH 8) before measurements. Three scans from 260 to 360 nm at 0.1 nm intervals were accumulated and averaged. The background of the buffer solution was subtracted from the CD data. Single-Channel Recording. 1,2-Diphytanoyl-sn-glycero-3phosphocholine was used to form a synthetic lipid bilayer on a 100−150 μm aperture in a 25-μm-thick polytetrafluoroethylene film (Goodfellow, Malvern, PA). Each side of the PTFE film was pretreated with hexadecane (10% (v/v) in pentane) before both compartments were filled with 1 mL of buffer consisting of 0.5 M TMACl, 5 mM Tris, pH 8. Wild type αHL heptamer and the mixture of DNA (final concentration 1.0 μM) and Pb2+, Ba2+, or other ions were added to the grounded cis compartment. For salt gradient experiments, DNA concentration was 10 nM and TMACl concentration was 0.5 M in cis and 3 M in trans. The electrical current was detected with two Ag/AgCl electrodes, recorded with a patch-clamp amplifier (Axopatch 200B; Axon instruments, Foster City, CA), filtered with a low-pass Bessel filter with a corner frequency of 5 kHz and then digitized with a Digidata 1440A A/D converter (Axon Instruments) at a sampling frequency of 100 kHz. The potential was held at +200 mV unless otherwise stated. Data Analysis. Current traces were analyzed with Clampfit 10.2 software (Axon Instruments). Events were detected using

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ∥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (Nos. 21175135, 21205119), National Basic Research program of China (973 program, No. 2010CB933600), and the “100 Talents” program of the Chinese Academy of Sciences.

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dx.doi.org/10.1021/ac401198d | Anal. Chem. 2013, 85, 7302−7307