Conformation-Dependent Exonuclease III Activity Mediated by Metal

Mar 24, 2013 - Our sensing strategy utilizes this conformation-dependent activity of Exo III, which is controlled through the cyclical shuffling of Hg...
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Conformation-Dependent Exonuclease III Activity Mediated by Metal Ions Reshuffling on Thymine-Rich DNA Duplexes for an Ultrasensitive Electrochemical Method for Hg2+ Detection Feng Xuan,† Xiaoteng Luo,†,§ and I-Ming Hsing*,†,‡ †

Department of Chemical and Biomolecular Engineering and ‡Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong ABSTRACT: Hg2+ is known to bind very strongly with T−T mismatches in DNA duplexes to form T−Hg2+−T base pairs, the structure of which is stabilized by covalent N−Hg bonds and exhibits bonding strength higher than hydrogen bonds. In this work, we exploit exonuclease III (Exo III) activity on DNA hybrids containing T−Hg2+−T base pairs and our experiments show that Hg2+ ions could intentionally trigger the activity of Exo III toward a designed thymine-rich DNA oligonucleotide (e-T-rich probe) by the conformational change of the probe. Our sensing strategy utilizes this conformation-dependent activity of Exo III, which is controlled through the cyclical shuffling of Hg2+ ions between the solution phase and the solid DNA hybrid. This interesting attribute has led to the development of an ultrasensitive detection platform for Hg2+ ions with a detection limit of 0.2 nM and a total assay time within minutes. This simple detection strategy could be used for the detection of other metal ions which exhibit specific interactions with natural or synthetic bases.

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chemical sensors22,23 with excellent performance on selectivity against the interferences of other metal ions. Nevertheless, as most of these sensors could hardly provide detection capability in the lower nanomolar range, this limits the technology useful for Hg2+ detection in drinking water that would require the detection limit down to 10 nM or 2 parts per billion (ppb),24 a toxic level of Hg2+ designated by the U.S. Environmental Protection Agency (EPA). More recently, based on the similarity between the secondary structure of a DNA duplex with metal ion-mediated base pairing (M-dsDNA) and the natural double-stranded DNA (dsDNA), several groups have shown that numerous functions and wide applications with regard to normal dsDNA can be extended to M-dsDNA and demonstrated Hg2+ sensors with much improved sensitivity. For example, Liu et al. reported that the catalytic activity of DNAzyme can be switched on through allosteric interactions by the coordination of Hg2+ to the designed T−T mismatches and thus developed a sensing platform for Hg2+ with a detection limit of 2.4 nM.25 Yet, these DNAzyme-based sensors suffer interference from other metal cations. Li et al. revealed that the cleavage activity of a nicking endonuclease toward DNA duplexes with T−T mismatches could be initiated by Hg2+.26 Urata et al. and Park et al. both demonstrated that the DNA polymerase can recognize the metal-coordinated T−Hg2+−T base pairs at the 3′ end of the primer and elongate the primer along the template DNA.27,28

etal ions and nucleic acid bases exhibit interesting and specific-binding interactions. For example, metal ions, such as Ag+, Hg2+, Cu2+, and Ni2+, could bind to specific natural or artificial nucleosides by coordinative forces, forming metal ion-mediated base pairs in which the hydrogen bonds of Watson−Crick (W−C)-type base pairs are replaced by metalbase bonds.1−4 This novel interaction of nucleic acids with metal ions shows promising prospects in a wide range of applications, including constructing DNA-based biosensors, designing molecular-scale logic gates, and modulating DNA functions.5−8 Mercuric ion (Hg2+) is a bioaccumulative and highly toxic environmental pollutant which causes serious human health problems, such as DNA damage, brain damage, organic functions disablement, and immune system homeostasis disruption.9−12 Due to the continuing concern over Hg2+ contamination in the environment and food, sensitive and on-site tracking of Hg2+ in aqueous media is highly desirable in environmental protection and disease prevention. Recently, the coordinative interaction between Hg2+ and bis-thymine demonstrated by Ono and Togashi7 has stimulated new approaches for DNA-based Hg2+ sensors.13,14 To be specific, T−T mismatches in DNA duplexes would attract Hg2+ in aqueous solution to form stable DNA duplexes with Hg2+mediated T−T DNA base-pairing. The binding constant of a T−Hg2+−T base pair is much higher than that of a natural A− T base pair.14 More importantly, this T−Hg2+−T interaction is highly specific, and the T−T base pair can only be stabilized by Hg2+.13 This finding has resulted in development of many interesting fluorescent,15−18 colorimetric,19−21 and electro© XXXX American Chemical Society

Received: January 22, 2013 Accepted: March 24, 2013

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Following these works, Zhu et al. showed a label-free Hg2+ sensor by polymerase-assisted fluorescence amplification with a very low detection limit of 40 pM.29 However, this method requires multistep operation processes and involves sophisticated fluorescent equipment, which may not be suitable for onsite detection. In this paper, we study the exonuclease activity of Exo III on DNA duplexes containing multiple T−Hg2+−T base pairs and demonstrate that the cleavage activity of Exo III can be controlled by T−Hg2+−T base pairing. As Hg2+ could be freed up from the T−Hg2+−T hybrid by Exo III, recycle back to the solution phase, and form new binding with other T-rich oligonucleotides, this conformation-dependent enzymatic activity triggered by metal ions leads to the development of a signal-amplified and immobilization-free electrochemical Hg2+ sensor platform with ultrahigh sensitivity and simple operation.

to be +0.36 V with respect to a Ag/AgCl reference electrode. Before each electrochemical measurement, the detection chip used was sequentially sonicated in an Alconox solution (8 g of Alconox/L of water) for 15 min, propan-2-ol for 15 min, and twice in water for 15 min. After these cleaning procedures, a negatively charged working electrode surface was obtained. Electrochemical measurements were performed with an Autolab PGSTAT30 potentiostat/galvanostat (Eco Chemie) controlled by the General Purpose Electrochemical System (GPES) software (Eco Chemie). Sample incubation was performed with a C1000 thermal cycler (Bio-Rad). For each differential pulse voltammetric (DPV) scanning, 1.5 μL of each of reacted sample solution was pipetted onto the chip to cover the Pt counter electrode, the Pt pseudoreference electrode, and one of the ITO working electrodes. Polyacrylamide Gel Electrophoresis. To prepare the hydrogel, 5 mL 30% gel solution (29:1), 1 mL TBE buffer (10×), 100 μL APS (10%), 10 μL TEMED, and 3.89 mL deionized water were mixed. This mixture contained a final gel percentage of 15%. The gel was polymerized for 1 h at room temperature and then soaked in 1 × TBE buffer (pH = 8.3) for use. 5 μL of each reacted sample was mixed with 1 μL of 6 × loading buffer and was subjected to the 15% nondenaturing polyacrylamide gel electrophoresis (PAGE). The PAGE was carried out in 1 × TBE at a constant voltage of 150 V for about 40 min at room temperature. After staining in diluted Gel-Red solution, the gels were scanned using a Gel Doc XR documentation system (Bio-Rad). Study of Inhibitory Effect of Hg2+ on Exo III. 2 μM refoligo 1 and 2 μM ref-oligo 2 were first hybridized in 1 × NEBuffer 1 at room temperature for 20 min. Different concentrations of Hg2+ (0 to 500 μM) were added and the uniformly mixed solution was incubated at room temperature for another 20 min. 20 units of Exo III was then added to the above solution to a total volume of 50 μL and incubated at 37 °C for 10 min. Finally, this mixture was heated to 80 °C for 10 min to inactivate the Exo III. All the sample solutions were allowed to slowly equilibrate to room temperature for about 20 min and then loaded into lanes for PAGE analysis. Study of Exo III Activity on DNA Duplex with T− Hg2+−T Base Pairs. 2 μM ref-oligo 1 and 2 μM ref-oligo 2, 2 μM ref-oligo 3 and 2 μM ref-oligo 4, 2 μM ref-oligo 5 and 2 μM ref-oligo 6, 2 μM ref-oligo 7 and 2 μM ref-oligo 8, 2 μM refoligo 9 and 2 μM ref-oligo 10 were first mixed, respectively, in 1 × NEBuffer 1 and incubated at room temperature for 20 min. Then, 20 μM Hg2+ was added to the above five mixtures and incubated for another 20 min. 20 units of Exo III was then added to each solution of 50 μL in volume and incubated at 37 °C for 10 min and finally heated to 80 °C for 10 min to inactivate Exo III and stop the digestion reaction. All sample solutions were slowly cooled down to room temperature for about 20 min before PAGE analysis. Amplified Hg2+ Detection. 2 μM e-T-rich probe was first mixed with the Hg2+ sample solution. The mixture was kept at room temperature for 20 min, followed by adding 20 units of Exo III. After homogeneous mixing, the whole 50 μL solution was incubated at 37 or 45 °C for 20 min and then 80 °C for 10 min to inactivate Exo III. Finally, the solution was slowly cooled down to room temperature for about 20 min before PAGE analysis and electrochemical detection was performed on the ITO electrode chip. For the verification of the Hg2+ recycling process and the evaluation of the sensitivity, different concentrations of Hg2+ were added to the system. For the

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EXPERIMENTAL SECTION Reagents. The Exo III and 10 × NEBuffer 1 (1 × NEBuffer, 1:10 mM bis-tris-propane-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.0 at 25 °C) were purchased from New England Biolabs, Inc. and used without further purification. Methylene blue-modified T-rich oligonucleotide (e-T-rich probe) was purchased from Biosearch Technologies, Inc., with the following sequence: 5′-CATTCTTTCTTCCCCTTGTTTGTTT-(methylene blue)-G-3′, with methylene blue modified at the italic T. Reference oligonucleotide 1 (refoligo 1: 5′-GTAGCGGTCTTGCGG-3′), reference oligonucleotide 2 (ref-oligo 2: 5′-CCGCAAGACCGCTACAAAAA3′), reference oligonucleotide 3 (ref-oligo 3: 5′-GTAGCGGTCTTGCGG-3′), reference oligonucleotide 4 (ref-oligo 4: 5′-CCGCTAGACCGCTACAAAAA-3′), reference oligonucleotide 5 (ref-oligo 5: 5′-GTAGCGGTCTTGCGG-3′), reference oligonucleotide 6 (ref-oligo 6: 5′-CCGCTAGTCCGCTACAAAAA-3′), reference oligonucleotide 7 (ref-oligo 7: 5′-GTAGCGGTCTTGCGG-3′), reference oligonucleotide 8 (ref-oligo 8: 5′-CCGCTTGACCGCTACAAAAA-3′), reference oligonucleotide 9 (ref-oligo 9: 5′-GTAGCGGCTTTGCGG-3′) and reference oligonucleotide 10 (ref-oligo 10: 5′CCGCTTTGCCGCTACAAAAA-3′) were purchased from Invitrogen and purified by standard desalting. 10 × TBE (Tris-borate-EDTA) buffer was purchased from Fermentas. GelRed Nucleic Acid Gel Stain (10000× in water) was purchased from Biotium. 29:1 acrylamide/bis-acrylamide 30% gel stock solution was purchased from Bio-Rad. Ammonium persulfate (APS) was purchased from USB Corporation. N,N,N′,N′-tetramethylethylenediamine (TEMED), metal salts [Hg(NO3)2, Pb(NO3)2, Ni(NO3)2, Cd(NO3)2, FeCl2, FeCl3, ZnCl2, CuCl2, and MnCl2] were purchased from Sigma-Aldrich (St. Louis, MO, USA). All aqueous solutions were prepared with deionized water (specific resistance >18.2 MΩ/cm) obtained with a Milli-Q reagent grade water system (Millipore). ITO Electrode Chip Fabrication and Electrochemical Measurements. The ITO-coated glass chip used for electrochemical measurement was fabricated in the Nanoelectronics Fabrication Facility (NFF) of our University. The design is similar to the microchip previously reported by our group for immobilization-free electrochemical DNA detection.30 It consists of one Pt counter electrode, one Pt pseudoreference electrode, and four patterned circular ITO spots as working electrodes. The active surface area of each ITO working electrode spot is 7.85 × 10−3 cm2. The potential of the Pt pseudoreference electrode in 1 × NEBuffer 1 was determined B

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Figure 1. (A) Illustration showing the digestion of ref-oligo 1 in the ref-oligo 1/ref-oligo 2 duplex by Exo III. (B) PAGE analysis of Hg2+ inhibition effect on the activity of Exo III: a, 2 μM ref-oligo 1 + 2 μM ref-oligo 2; b, 2 μM ref-oligo 2; c, 2 μM ref-oligo 1 + 2 μM ref-oligo 2 + Exo III; d, 2 μM ref-oligo 1 + 2 μM ref-oligo 2 + Exo III + 10 μM Hg2+; e, 2 μM ref-oligo 1 + 2 μM ref-oligo 2 + Exo III + 100 μM Hg2+; f, 2 μM ref-oligo 1 + 2 μM ref-oligo 2 + Exo III + 200 μM Hg2+; g, 2 μM ref-oligo 1 + 2 μM ref-oligo 2 + Exo III + 500 μM Hg2+.

was not affected by Hg2+ at concentrations lower than 100 μM. When the concentration of Hg2+ was increased to 200 μM, the band representing the ref-oligo 1/ref-oligo 2 duplex appeared in lane f, indicating an inhibiting effect of Hg2+ on Exo III activity as the ref-oligo 1 in the ref-oligo 1/ref-oligo 2 duplex was not completely digested. When the Hg2+ concentration was further increased to 500 μM, the activity of Exo III was completely blocked, such that no band representing the ref-oligo 2 was observed. This result confirms that Hg2+ has an inhibiting effect on Exo III activity. However, it only emerges at a relatively high concentration of Hg2+, under our experimental condition. Exo III Activity on DNA Duplex with T−Hg2+−T Base Pairs. In the above investigation of the inhibition effect on Exo III activity, the Hg2+ is not incorporated within the DNA duplex. Another possible way that Hg2+ may inhibit Exo III activity is by forming T−Hg2+−T base pairs in the dsDNA. There is no previous evidence on whether the T−Hg2+−T base pair would block the digestion of DNA by Exo III. In the T− Hg2+−T base pair, the Hg2+ could be directly incorporated to the N3 position of the thymidine, replacing the imino proton and forming a pair bonding with two oppositely oriented thymidine residues. The van der Waals radius of Hg2+ is about 1.44 Å, and the distance between W−C-type base pairs in the DNA duplex is about 3.4 Å; therefore, the incorporation of Hg2+ into the T−T mismatched pair could hardly affect the rigidity of the double helix structure and its association with Exo III. On the other hand, as the cleavage of nucleotides in dsDNA by Exo III is a consecutive and base-by-base process, any blockage of the involved steps (e.g., dissociation of the cleaved nucleotide) could result in the reduction of Exo III activity. The thymine bases in T−Hg2+−T base pairs bind to Hg2+ through covalent N−Hg bonds, the bonding strength of which is stronger than that of hydrogen bonds in A−T and G− C base pairing.14 Thus, the dissociation of the cleaved T base from the T−Hg2+−T base pair would likely play a role in determining the activity of Exo III on the DNA duplex containing T−Hg2+−T base pairs. Thermally induced dissociation and association processes of DNA duplexes containing T−Hg2+−T base pairs reported by Ono’s group suggest that

evaluation of selectivity, the electrochemical responses of 10 nM, 50 nM, and 500 nM Hg2+ were compared with those of 8 types of interferential metal ions (Pb2+, Mn2+, Ni2+, Cu2+, Fe2+, Fe3+, and Cd2+) at high concentrations (1, 5, and 10 μM, respectively).

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RESULTS AND DISCUSSION Inhibiting Effect of Hg2+ on Exo III Activity. Exo III is an enzyme which has a specific exodeoxyribonuclease activity for dsDNA in the direction from 3′ to 5′ and the digestion products are normally single-stranded DNA (ssDNA) and 5′-P mononucleotides. More specifically, this enzyme is able to digest from the blunt end, recessed 3′ end, and nicked sites, while its activity on the protruding 3′ end of more than 4 nt is limited.31 The activity of Exo III can be affected by certain cations. For instance, Mg2+ and Co2+ support the activity of Exo III as essential metal cofactors,32 while Na+, Pb2+, Fe2+, and Cd2+ have an inhibiting effect on the Exo III activity.33,34 Recently, the possible inhibiting effect of Hg2+ on Exo III activity was reported by Yuan et al.35 To eliminate the possibility of hindrance brought by Hg2+ itself to the activity of the enzyme in our Exo III-based Hg2+ detection, the digestion of dsDNA by Exo III under various Hg2+ concentrations was first investigated. As illustrated in Figure 1A, the sequence of ref-oligo 1 was designed to be complementary to the 5′ part of ref-oligo 2, forming a blunt end. Therefore, the Exo III would digest ref-oligo 1 from its 3′ terminus. 5′-P mononucleotides were cleaved off successively, and the digestion continued up to the end of ref-oligo 1 or until the residue of ref-oligo 1 dissociated from ref-oligo 2. Ref-oligo 2 would be intact due to the protection by its protruding 3′ terminus of 5 nt. As shown in Figure 1B, the bands in lanes a and b correspond to the refoligo 1/ref-oligo 2 duplex and ref-oligo 2, respectively. The band in lane c appears at the same position as the band in lane b, indicating that Exo III could completely digest ref-oligo 1 when Hg2+ was absent in the sample. In the presence of Hg2+ at concentrations of 10 or 100 μM, the bands in lanes d and e also appear at the same position as the band in lane b, indicating the complete digestion of ref-oligo 1 and that the activity of Exo III C

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Figure 2. (A) Sequences of DNA duplexes containing different numbers of T−Hg2+−T base pairs. (B) PAGE analysis of the digestion products of the DNA duplexes in (A). a, 2 μM ref-oligo 1 + 2 μM ref-oligo 2; b, 2 μM ref-oligo 2; c, 2 μM ref-oligo 1 + 2 μM ref-oligo 2 + Exo III + 20 μM Hg2+; d, 2 μM ref-oligo 3 + 2 μM ref-oligo 4 + Exo III + 20 μM Hg2+; e, 2 μM ref-oligo 5 + 2 μM ref-oligo 6 + Exo III + 20 μM Hg2+; f, 2 μM ref-oligo 7 + 2 μM ref-oligo 8 + Exo III + 20 μM Hg2+; g, 2 μM ref-oligo 9 + 2 μM ref-oligo 10 + Exo III + 20 μM Hg2+.

the dissociation of cleaved T base from the T−Hg2+−T base pairs during the Exo III digestion process is possible.14 To investigate the influence of T−Hg2+−T base pairs on the activity of Exo III, the sequences of ref-oligo 1 and ref-oligo 2 were modified to introduce different numbers of T−T mismatches for the construction of five different T−Hg2+−Tcontaining duplex sequences, as shown in Figure 2A. Each of these DNA duplexes was incubated with an excessive amount of Hg2+ to ensure that each T−T mismatched pair has one Hg2+ ion incorporated. In addition, the concentration of Hg2+ was set at 20 μM, much lower than 100 μM, such that there would be no inhibitory effect on Exo III activity (refer to Figure 1). The Exo III digestion of these DNA duplexes was analyzed by PAGE, as shown in Figure 2B. In accordance with the results of the previous experiments, in the presence of 20 μM Hg2+, the Exo III was active and the ref-oligo 1/ref-oligo 2 duplexes (without T−T mismatch) were completely digested into refoligo 2 (lane c). When there were one or two separate T− Hg2+−T base pairs present in the duplex, no inhibiting effect on exonuclease activity was observed, and the duplexes were digested into ssDNAs, as can be seen from lanes d and e. When there were two or three consecutive T−Hg2+−T base pairs, the digestion of the duplexes by Exo III was still efficient, as there were no bands representing the undigested duplexes in lanes f and g. These results show that Exo III can function just as well on DNA duplexes with up to T−Hg2+−T base pairs as on normal DNA duplexes and suggest that the binding of N−Hg bonds does not prevent the digestion by Exo III or the dissociation of the cleaved T base from the T−Hg2+−T base pair. A very interesting observation of two lower gel bands is shown in lanes f and g (pointed by an arrow in Figure 2B), indicating that part of the intact ref-oligo 8 released after digestion of ref-oligo 7 and all of the intact ref-oligo 10 released after digestion of ref-oligo 9 were further digested into a shorter oligonucleotide. This was probably caused by the internal basepairing of ref-oligo 8 and 10 (indicated in red characters in Figure 2A). The melting temperature of the self-primed refoligo 10 with four complementary bases was 40 °C. After refoligo 9 was digested, at the reaction temperature of 37 °C, the

released ref-oligo 10 was self-primed to form a 3′ end with less than 4 protruding bases and was further digested by Exo III. Notably, the melting temperature of self-primed ref-oligo 8 with three complementary bases was about 25 °C, which was much lower than the reaction temperature (37 °C). Theoretically, few released ref-oligo 8 would be self-primed to form a 3′ end with less than 4 protruding bases and further digested. However, it turned out that nearly half of the released ref-oligo 8 was further digested, as shown in lane f of Figure 2B. We believe that this interesting experimental observation could be adequately explained after taking into account both of the kinetics of Exo III enzymatic reaction and thermodynamic equilibrium between ref-oligo 8 and its corresponding selfprimed strand. Thermodynamically, at the reaction temperature of 37 °C, there will still be a trace of self-primed ref-oligo 8 in equilibrium with the single-stranded ref-oligo 8 due to the probable internal base-pairing, although the high temperature (37 °C) would favor a much higher probability of singlestranded ref-oligo 8 than that of the self-primed ref-oligo 8 of low melting temperature (25 °C). Nevertheless, in the presence of Exo III, as soon as the self-primed ref-oligo 8 shows up in the solution, the double-stranded structure with a 3′ end with less than 4 protruding bases would induce Exo III activity, leading to the further digestion of the self-primed ref-oligo 8. This enzymatic excision process breaks the thermodynamic equilibrium and shifts the process toward more self-primed ref-oligo 8 available for further enzymatic digestion. This might also be the reason why Exo III in reported literature occasionally shows the digestion activity on ss DNAs. Hg2+-Triggered Digestion of DNA by Exo III. As show in the above study that DNA duplexes with T−Hg2+−T base pairs can be digested by Exo III as efficiently as normal DNA duplexes, a thymine-rich DNA oligonucleotide was designed such that the digestion of this oligonucleotide can be triggered by the presence of Hg2+. The triggered digestion by Exo III was monitored by an electrochemical platform, requiring no DNA probe immobilization, as illustrated in Figure 3. This protocol takes advantage of the different diffusivity between oligonucleotides and mononucleotides toward a negatively charged ITO electrode surface. The designed thymine-rich DNA oligonuD

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Figure 3. Illustration of the Hg2+-triggered digestion of the designed eT-rich probe by Exo III monitored by the immobilization-free electrochemical monitoring protocol.

cleotide (e-T-rich probe) consists of a 26-nucleotide DNA strand and a methylene blue (MB) tag labeled at an internal T base close to the 3′ terminus. In the absence of Hg2+, the e-Trich probe adopts a random coil structure and this single-strand state is resistant to the digestion of Exo III. The undigested e-Trich probe, due to the electrostatic repulsion between its negatively charged backbone and the negative ITO electrode surface, can hardly get into contact with the electrode, therefore a negligible electrochemical response is observed. In the presence of Hg2+, because of its interaction with the T bases, T−Hg2+−T base pairs would be formed, folding the linear e-Trich probe into a hairpin structure consisting of a 4-nt loop (CCCC) and an 11-bp stem with a blunt 3′ terminus. This conformational switch converts the e-T-rich probe into an Exo III-preferred substrate and therefore activates the enzyme, resulting in the stepwise removal of mononucleotides from the 3′ terminus of the e-T-rich probe and the release of a thymidine monophosphate with the MB tag (eNT) before ultimately deforming the hairpin structure. The released eNT, due to its less negative charge and smaller size, possesses much a higher diffusivity toward the negatively charged ITO electrode than that of the e-T-rich probe, leading to a distinct increase in the electrochemical signal. In this way, the increment of the MB signal would well-indicate the triggered activity of Exo III. Moreover, we hypothesize that the Hg2+ could be released from the T−Hg2+−T base pair during the digestion process and fold a new undigested e-T-rich probe, starting a new cycle of digestion. In this way, Hg2+ could cyclically shuffle between the solution phase and the e-T-rich probes, serving as a “catalytic trigger”, which can amplify the digestion of a large amount of eT-rich probes. As can be seen in the experimental results shown in Figure 4A, a readily detectable gain of electrochemical signal was observed when Hg2+ was introduced to the system, indicating the successful triggering of the digestion activity of Exo III. However, it should be noted that, even when Hg2+ was absent, a non-negligible signal (green peak) was also observed, which was higher than the signal without Exo III (red peak). This implies that some of the e-T-rich probes were digested, even without the triggering of Hg2+, due to the residual exonuclease activity of Exo III against unfolded e-T-rich probes. Moreover, a small signal (light blue) was observed in the presence of Hg2+

Figure 4. DPV scans of different reaction mixtures after incubation at (A) 37 °C or (B) 45 °C for 20 min, respectively. Probe stands for e-Trich probe in the figures.

without Exo III. This could be explained by reduction of electrostatic repulsion between the T−Hg2+−T self-coordinated DNA duplex and ITO electrode, as a result of the shield effect of the positive Hg2+ ion incorporation. However, this signal gain is far smaller than the electrochemical signal in the presence of Exo III and Hg2+ (dark blue). Although some groups have demonstrated that the specificity of Exo III was much better at 4 °C than at 37 °C,31 lowering the temperature would largely increase the required incubation time (24 h at 4 °C in some cases). From the results of the previous experiment, we assumed that the nonspecific digestion of e-T-rich probes by Exo III might be caused by intramolecule base pairing. Therefore, it was expected that decreasing the possibility of intramolecule base pairing by properly increasing the reaction temperature would reduce the nonspecific signal in the absence of Hg2+. Figure 4B shows the result when the reaction was conducted at 45 °C. In comparison with the case at 37 °C, the signal without Hg2+ (green peak) was significantly decreased, which indicates that much less e-T-rich probes were digested. Meanwhile, the signal with Hg2+ (dark blue peak) increased slightly due to the enhanced enzyme activity at 45 °C. Thus, a better signal-to-background ratio was obtained without extending the reaction time. This result also further supports our previous assumption that the nonspecific digestion of ssDNA by Exo III could be caused by intramolecule base paring. To confirm the hypothesized mechanism of Hg2+ releasing and cyclical shuffling between the solution phase and the e-Trich probes, a time-course experiment was conducted. A 50 μL mixture containing 2 μM e-T-rich probes and 20 units of Exo III was incubated with Hg2+ at a concentration of 20 or 0.1 μM E

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Figure 5. (A) DPV peak currents plotted against incubation time in the presence of 20 μM or 0.1 μM Hg2+, respectively. The reaction mixtures each contained 2 μM e-T-rich probe and 20 units of Exo III and were incubated at 45 °C. (B) PAGE analysis of the reacted mixtures in (A). a, 2 μM e-Trich probe; b, 2 μM e-T-rich probe + Exo III; c, 2 μM e-T-rich probe + Exo III + 0.1 μM Hg2+; d, 2 μM e-T-rich probe + Exo III + 20 μM Hg2+.

for a certain period of time, and the electrochemical signal was measured and plotted against the time of incubation. As shown in Figure 5A, the signal with 20 μM (excessive) Hg2+ saturated rapidly, indicating that all the e-T-rich probes were folded and digested as soon as the Hg2+ was introduced. Meanwhile, the signal with 0.1 μM (inadequate) Hg2+ showed a continuous increase at a relatively slower rate, before finally reaching the same plateau. As the concentration of e-T-rich probes was 20 times higher than that of Hg2+, the complete digestion of the eT-rich probes implies that Hg2+ releasing and cyclical shuffling did occur. The PAGE analysis of the reaction products is shown in Figure 5B. As anticipated, all of the e-T-rich probes were digested into shorter oligonucleotides by Exo III, even when as little as 0.1 μM Hg2+ was added. Sensing Performance as an Amplified Hg2+ Detector. The observations and findings described above suggest that the “triggered” activity of the Exo III could be utilized for amplified Hg2+ detection with ultrahigh sensitivity. We challenged the system with Hg2+ of different concentrations. The previous time-course with 0.1 μM Hg2+ showed a roughly linear increase within the first 20 min, followed by a plateau stage after 30 min of incubation. Thus, all samples were incubated at 45 °C for 20 min before subjected to electrochemical scans. As Figure 6A shows, without any background subtraction, a detectable signal increase could be readily achieved with Hg2+ concentrations as low as 0.5 nM which is 20 times lower than the EPA standard (10 nM of Hg2+ for drinkable water). Figure 6B depicts the relationship between peak currents and the Hg2+ concentrations, which showed a sigmoid shape. We believe this could be attributed to the fact that the e-T-rich probe could be folded after binding two or three Hg2+ ions, although there were seven pairs of T−T mismatches on each e-T-rich probe. The peak current has a linear correlation with the Hg2+ concentration in the lower concentration range, as shown in the inset of Figure 6B. The calibration equation is determined to be peak value (nA) = 1.38C + 1.26 with a correlation coefficient of 0.97426, where C is the concentration of Hg2+. The detection limit is estimated to be 0.2 nM, which produces a signal equal to the background signal plus three times the standard deviation. To the best of our knowledge, this is the best detection limit achieved among all the reported electrochemical Hg2+ sensors and it is even lower than most of the existing fluorescencebased Hg2+ detectors.

Figure 6. Sensitivity of the amplified Hg2+ detector. (A) DPV scans of reaction mixtures containing a 2 μM e-T-rich probe, 20 units of Exo III, and varying concentrations of Hg2+ after incubation at 45 °C for 20 min. (B) DPV peak currents plotted against concentrations of Hg2+. Inset: DPV peak currents plotted against lower concentrations of Hg2+. Mean values and standard deviations are obtained from three independent experiments.

The selectivity of the detection was tested by substituting the Hg2+ in the system with various metal ions, which are commonly present in real samples, such as Pb2+, Mn2+, Zn2+, Ni2+, Cu2+, Fe2+, Fe3+, and Cd2+. As shown in Figure 7, each F

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electronics Fabrication Facility and Division of Biomedical Engineering of HKUST is also acknowledged.

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Figure 7. Selectivity of the amplified Hg2+ detector. All competing metal ions were tested at 1, 5, and 10 μM. For comparison, signal responses to 10, 50, and 500 nM of Hg2+ ions are also presented. Each of the reaction mixtures contained a 2 μM e-T-rich probe and 20 units of Exo III and was incubated at 45 °C for 20 min before DPV scans.

metal was tested at three relatively high concentrations (1, 5, and 10 μM) under the same experimental conditions. None of the tested metal ions gave peak currents higher than half of that produced by 10 nM Hg2+ ions. Such excellent selectivity is attributed to the specific T−Hg2+−T base pairing and the preferential activity of Exo III.

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CONCLUSIONS In summary, the study described above has demonstrated that Hg2+ which specifically interacts with T−T mismatched base pairs can be employed to intentionally trigger the activity of Exo III, which is well-monitored by an immobilization-free electrochemical approach. Furthermore, Hg2+ is demonstrated to be able to be released from the T−Hg2+−T base pairs during the probe digestion and cyclically shuffle between the solution phase and the solid DNA hybrid. These findings lead to a novel system for electrochemical Hg2+ detection with ultrahigh sensitivity and excellent selectivity. In addition to the convenience in operation, the system is operated with low cost and portable equipment and provides a practical solution for rapid screening of Hg2+ contamination, especially in remote areas. Moreover, this system can be further rationalized to detect other metal ions by utilizing other metal ion-interacting bases.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (852) 23587131. Fax: (852) 31064857. Present Address §

Shenzhen Unimedica Technology Company, Ltd., 2nd Floor, Building No. 1, Sangtai Science Park, Xili Town, Nanshan District, Shenzhen, China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the funding support from the Research Grants Council of the Hong Kong SAR Government (Grant 602111). The laboratory facility provided by the NanoG

dx.doi.org/10.1021/ac400228q | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

(35) Yuan, M.; Zhu, Y. G.; Lou, X. H.; Chen, C.; Wei, G.; Lan, M. B.; Zhao, J. L. Anal. Methods 2012, 4, 2846−2851.

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dx.doi.org/10.1021/ac400228q | Anal. Chem. XXXX, XXX, XXX−XXX