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Kinetic Discrimination of Metal Ions Using DNA for Highly Sensitive and Selective Cr3+ Detection Wang Li,*,†,§ Zijie Zhang,§ Wenhu Zhou,‡,§ and Juewen Liu*,§ †

College of Food Science & Engineering, Central South University of Forestry & Technology, Changsha, Hunan 410004, China Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, China § Department of Chemistry, Water Institute and Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada ‡

S Supporting Information *

ABSTRACT: Most metal sensors are designed for a strong binding affinity toward target metal ions, and the underlying principle relies on binding thermodynamics. The kinetic aspect of binding, however, was rarely explored for sensing. In this work, the binding kinetics of 19 common or toxic metal ions are compared based on a fluorescence quenching assay using DNA oligonucleotides as ligands. Among these metals, Cr3+ shows uniquely slow fluorescence quenching kinetics, and the quenched fluorescence cannot be recovered by EDTA or sulfide. Most other metals quenched fluorescence instantaneously and can be fully recovered by these metal chelators. Various factors such as DNA sequence and length, chelating agent, pH, and fluorophore type were studied to understand the binding mechanism, leading to a unique two-stage binding model for Cr3+. This system has a wide dynamic range of up to 50 μM Cr3+ and a low limit of detection of 80 nM. It is also useful for measuring Cr3+ in lake water. This work proposes a new metal sensor design by monitoring binding kinetics with Cr3+ being a primary example. KEYWORDS: biosensors, metal ions, aptamers, ligand exchange, fluorescence mammals as enzyme cofactors.14 However, excessive levels of Cr3+ are toxic, which is attributed to DNA damage and adverse effects on cellular structures and components.15 Cr(VI) cannot damage DNA if it is not reduced under physiological conditions.16,17 As Cr3+ might be the ultimate reason for toxicity, it is important to detect it. Instrumentation methods such as inductively coupled plasma mass spectrometry were developed for chromium analysis, but cannot determine the oxidation state.18−20 Recently, organic molecule probes and nanomaterials21−25 were also employed for Cr3+ detection. However, its reliable sensing remains a challenge in the sensor field. DNA has emerged as a powerful platform for metal analysis in the past two decades. Several metal ions (such as Ag+ and Hg2+) have rationally designed aptamers for binding.26−28 Many more metals can act as cofactors for DNAzymes (such as Na+,29,30 Ag+,31 Ca2+,32 Pb2+,33 Cd2+,34 Cu2+,35 UO22+,36 and Ce3+).37,38 We recently found that Cr3+ can also serve as a cofactor for a DNAzyme, but this DNAzyme is more active with many other metals.39 For all these sensors, metal recognition is all based on binding thermodynamics, while the kinetic concept has not been explored.

A critical aspect of managing the risk of metal exposure is to develop sensors. Sensors complement analytical instruments by providing fast, on-site results.1−3 For this purpose, various selective metal ligands have been developed including small molecules, peptides, proteins, and DNA.4−7 Most sensors rely on binding affinity or thermodynamics for recognizing target metals. Each metal is different in size, charge, thiophilicity, and coordination geometry preference, which is the basis of such sensors (Scheme 1A).8 A parallel aspect is binding kinetics, which, however, has rarely been explored for sensing. We reason that it might be possible to discriminate metal ions even without using highly specific ligands, but just by examining their binding kinetic signatures (Scheme 1B). When a metal ion inner sphere coordinates with a ligand in water, its hydration shell needs to be partially displaced. Thus, the kinetics of binding is highly related to the ligand exchange rate, which is much faster than 1 s−1 for most common metal ions (Scheme 1C).9,10 Since these are faster than the human time scale of operation, we typically do not notice it. A few metals, however, have extremely slow ligand exchange rates ( Eu3+ > Cr3+ > Pb2+ > Fe2+ > Co2+ > Al3+ > Ni2+ > Cd2+ in 11 min. As will be shown later, Cr3+ can also reach full quenching given enough reaction time. This simple experiment already shows the possibility of using binding kinetics to detect Cr3+, even with a random sequence. We chose these metals since they are common in nature. After quenching, we added 1 mM EDTA at 16 min to each sample to chelate the metal ions. The fluorescence immediately recovered to almost 100% by EDTA44 in most cases (Figure 1C), suggesting that their binding to DNA is relatively weak. Cr3+ and Fe3+ were the two exceptions (Figure 1B), whose fluorescence could not be recovered at all, although EDTA stopped further quenching by Cr3+. It is unclear to us which ligand has a stronger thermodynamic affinity for Cr3+: EDTA or the DNA. These two molecules, however, provide an interesting kinetics signature for Cr3+. Slow Cr3+ Binding Is General to All DNA Sequences. Using a random DNA sequence, we already demonstrated the unique kinetics of Cr3+ binding. Since different DNA bases have

a Other metals such as Pt2+, Pd2+, and Ir3+ are not listed since they are rare in environmental water samples.

By examining binding kinetics, we may not need highly specific ligands. Metal ions can bind to DNA (and nucleobases) in general.40,41 Herein, we use random DNA as a ligand to study its binding and dissociation kinetics for Cr3+ detection. At the same time, Cr3+ can efficiently quench adjacent fluorophores.16 With these properties, Cr3+ was efficiently and selectively detected.

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MATERIALS AND METHODS

Chemicals. The DNA oligonucleotides included FAM-labeled DNAs, were purchased from Integrated DNA Technologies (Coralville, IA) and Eurofins (Huntsville, AL). See Table 1 for their sequences and modifications. All metal salts used in this work were obtained from Sigma-Aldrich. All solutions were prepared using Milli-Q water. Fluorescence Quenching Assay. In a typical quenching system, FAM-labeled DNA (100 nM) and a metal ion (100 μM) were mixed in MES buffer (50 mM MES, 25 mM NaCl, pH 6.5). Then the fluorescence of the sample was recorded with the excitation wavelength of 485 nm and the emission wavelength of 535 nm using Infinite F200

Table 1. DNA Oligonucleotide Sequencea

a

DNA names

sequences and modifications (from 5′ to 3′ terminal)

FAM-rndDNA FAM-A5 FAM-A10 FAM-A15 FAM-A30 FAM-C5 FAM-C10 FAM-C15 FAM-C30 FAM-T15 FAM-G15

FAM-ACGCAACGACAAAAAAAAAAA FAM-AAAAA FAM-AAAAAAAAAA FAM-AAAAAAAAAAAAAAA FAM-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA FAM-CCCCC FAM-CCCCCCCCCC FAM-CCCCCCCCCCCCCCC FAM-CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC FAM-TTTTTTTTTTTTTTT FAM-GGGGGGGGGGGGGGG

FAM represents the label of carboxyfluorescein. 664

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Figure 1. Reaction kinetics of FAM-labeled random DNA sequences (100 nM) in the presence of various metal ion (100 μM) in MES buffer, pH 6.5. The arrowheads at 5 min indicate the time point of adding metal ions and at 16 min indicate adding EDTA. The metal ions are grouped to be (A) no quenching, (B) with quenching but no recovery by EDTA, and (C) fast quenching and full recovery by EDTA.

For FAM-C15, a few different stages of kinetics were observed and the reaction became slower and slower at the later stages (the fluorescence quenching efficiency is ∼90% in 60 min). FAM-G15 has much slower overall kinetics and the final fluorescence for FAM-G15 was only 60%. This might be related to their tendency to form various secondary structures such as the i-motif and Gquadruplex, to hide the DNA bases and the fluorophore, so that Cr3+ cannot get within a close enough distance to the fluorophore for efficient quenching. The slow binding of Cr3+ and DNA was also confirmed by isothermal titration calorimetry (ITC, Figure S2). It took about 10 min for the system to reach thermal equilibrium, similar to the fluorescence based measurement. Likewise, we also observed more quenching with FAMA15 and T15 with Fe3+ (Figure 2B), although the kinetics were quite similar in this case. Fluorescence Recovery Using Sulfide. Since EDTA failed to recover the fluorescence for Fe3+ and Cr3+, we added a different ligand, sulfide, after 60 min of reaction (Figure 2). Since sulfide can strongly precipitate many metal ions, we suspect that it might be more effective than EDTA for Cr3+ and Fe3+. Indeed, the fluorescence of the Fe3+ added samples was restored to some degree, whereas the FAM-C15 and FAM-G15 recovered more fluorescence than the FAM-A15 and FAM-T15 did. Still, no recovery was seen in the Cr3+ samples, implying that the binding of Cr3+ by DNA is very tight. Longer DNA Has Faster Quenching Kinetics. Next, we studied the effect of DNA length using poly-C and poly-A DNA, since poly-A has high quenching efficiency and poly-C has fast quenching kinetics. As shown in Figure 3A, the kinetics is faster with longer DNA. For the poly-C DNA, we clearly observed two distinct kinetic stages. First, right after adding Cr3+, a very quick drop in fluorescence was observed (e.g., within 1 min), and the extent of the drop was proportional to the length of the DNA. Then, a slower stage of quenching followed. For this slower stage, the rate was independent of DNA length since the traces were parallel to each other. Once a final value was achieved, no more quenching occurred. Note that C5 and C10 can reach 100% quenching, while C15 and C30 were quenched only 90%, despite that they had a faster initial quenching rate. This suggests that internal secondary structures such as the i-motif (which are favored by long poly-C DNA) is important in affecting the final quenching efficiency. For the poly-A DNAs, we can still divide the quenching into two stages, but the initial fast stage is not very obvious (Figure

quite different metal binding properties, we next used the four 15-mer DNA homopolymers to understand the effect of DNA sequence. While the exact kinetics of quenching are different after adding Cr3+ (Figure 2A), all of them are slow. The

Figure 2. Quenching kinetics of the four 15-mer FAM modified DNA homopolymers (100 nM) by (A) Cr3+ and (B) Fe3+. Arrows on the left and right of each figure indicate the time point of adding the metal ion (100 μM) and adding Na2S (800 μM), respectively.

fluorescence of FAM-A15 and FAM-T15 decreased linearly to almost zero within 30 min. This is a very intriguing kinetic profile. If fluorescence quenching is linearly proportional to the amount of Cr3+ binding, then as more Cr3+ binds to DNA and the available binding sites decrease, one should expect slower and slower quenching over time. The fact that a linear quenching profile is observed indicates that Cr3+ binding is accelerated as more Cr3+ ions are associated with these two DNAs. 665

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Figure 3. Quenching kinetics of FAM-modified (A) poly-C DNA and (B) poly-A DNA of different lengths by Cr3+. The concentration of total bases was constant (1.5 μM). Cr3+ was added at 0 min. (C) Initial rate of quenching by Cr3+ of poly-A and poly-C DNA.

Figure 4. Quenching kinetics of FAM-A15 (A) and FAM-C15 (B) by different concentrations of Cr3+. Cr3+ was added at 0 min. (C) Extent of fluorescence quenching at 60 min.

3B). We calculated the rate of quenching of the first stage for all the DNAs (Figure 3C), although both sequences have a similar trend, the quenching by poly-C is much faster. For the second stage, longer poly-A still quenched fluorescence faster. A longer DNA sequence has more reaction sites for Cr3+ binding and thus it is reasonable for its faster quench. Overall, to achieve faster quenching, long poly-C DNA can be used. Optimization of pH and Fluorophore. pH is an important factor governing metal/DNA interactions. In particular, a higher pH value is often associated with metal hydrolysis and precipitation. We measured the quenching kinetics of Cr3+ in different pH (Figure S3). As expected, the quenching kinetics is quite sensitive to pH. Both FAM-A15 and FAM-C15 have a similar dependency on pH. For both quenching speed and quenching efficiency, the optimal order is pH 5.0 < pH 5.5 < pH 6.0 < pH 6.5 ≈ pH 7.0 > pH 7.5. Therefore, the optimal pH is 6.5 to 7.0. These results indicate that the effect of pH mainly acts on Cr3+ rather than on DNA. Otherwise, we expect to see different trends for different DNA sequences. While a higher pH is beneficial for Cr3+/DNA interaction, if the pH passes neutral, the reaction is likely inhibited by hydrolysis of Cr3+. In addition, we also found that the quenching performance is independent of fluorophore species (Figure S4). All of the four tested fluorophores, FAM, Cy3, TYE665, and AlexaFluor 647, showed a similar performance in the presence of Cr3+. At least 96% fluorescence of these dyes linked to DNA were quenched by Cr3+. These fluorophores cover emission from green to far red,

and suggest that the mechanism of quenching is not energy transfer, but electron transfer. Detection of Cr3+. Since unique fluorescence quenching kinetics can be used to tell the difference between Cr3+ and the rest of the common metal ions we tested, this system might be useful as a sensor for Cr3+ because of its significance in agriculture and the environment. After identifying Cr3+ based on its kinetic signature, we next tested the sensitivity of using this method. We added a series of Cr3+ concentrations (from 0 to 125 μM) into the solution containing a FAM-labeled DNA. Cr3+ has very characteristic slow quenching kinetics for both FAM-A15 (Figure 4A) and FAM-C15 (Figure 4B). The increase in Cr3+ concentration results in an accelerated decline of fluorescence kinetics. The plot of the remaining fluorescent signal as a function of Cr3+ concentration is illustrated in Figure 4C. The remaining fluorescence is linearly related to Cr3+ concentration in a certain range. For FAM-A15, the calibration curve has a linear range from 0 to 50 μM and a limit of detection (LOD) of 0.2 μM Cr3+ based on the signal being higher than three times the background variation. For FAM-C15, the linear range is to 30 μM with a LOD of 0.08 μM Cr3+. Such LODs compare favorably with those reported with other fluorescent methods based on binding affinity.45−47 The limits of chromium in drinking water recommended by the U.S. Environmental Protection Agency (EPA), European Community (EC), and World Health Organization (WHO) are 100 μg/L (1.92 μM) (EPA 822-R06-013), 50 μg/L (0.96 μM) (98/83/EC), and 50 μg/L (WHO 666

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Figure 5. (A) Influence of adenine and its derivatives (500 μM) on the binding of Cr3+ (50 μM) with FAM-A15. The incubation time of Cr3+ with DNA is 60 or 150 min (inset) before the addition of adenine or its derivatives. (B) Influence of ATP on the restoration of fluorescence after the addition of AMP. AMP (500 μM) was added at the time point of 0 min for all samples. Arrows represent the time point of ATP addition (500 μM). (C) Proposed mechanism of interaction of Cr3+ with DNA.

ISBN: 9241546743), respectively.22,48 Therefore, both probes tested here can meet the requirement of detection in drinking water. In all of the above work, the FAM-labeled DNAs were exposed only to a single metal ion. It is quite likely that real water samples may contain a few different metals. To test this quantitatively, we challenged our sensor with a few other metal ions added together with 1 μM Cr3+ (Figure S5). Metals that are commonly found in environmental water samples, such as 2 mM K+ or Ca2+, did not have much effect on the quenching property of Cr3+, and Cr3+ could be detected normally. Pb2+ at 100 μM quenched more efficiently, since Pb2+ alone is a quencher. However, it is unlikely to have such high Pb2+ concentrations in water. For example, the toxic limit of Pb2+ in drinking water is only 70 nM as defined by the US Environmental Protection Agency (EPA), and we tested 100 μM Pb2+ here. The iron content in water can be higher (1− 30 μM),49 and the Canadian guideline for drinking water is ∼5 μM iron. The presence of iron can be detected through fluorescence recovery by sulfide, but not by EDTA. If a sample contains too many interfering ions, such as iron, immediate and full quenching of sensor fluorescence occurs. Then, the sensor cannot work since the quenching kinetics information is lost. In that case, dilution or other operations need to be performed to lower the interfering metal concentrations. To evaluate its practical applications, this method was further tested in real environmental water. The recovery experiment of different Cr3+ concentrations was carried out in local lake water. Three spiked Cr3+ samples were prepared and the results are shown in Table S1. Satisfactory recoveries (91−110%) were attained, revealing the excellent performance of the present strategy beyond simple buffer solutions. Binding Mechanism Studies. After demonstrating its analytical potential, we aim to gain further insights into the Cr3+ binding mechanism. Our data in Figure 1B showed that while EDTA cannot recover the fluorescence quenched by Cr3+, it can arrest the reaction and prevent further quenching. This indicates a much quicker binding kinetics between Cr3+ and EDTA compared to Cr3+ to DNA. However, it might be that EDTA was added at a much higher concentration of 1 mM versus 100 nM of the DNA. Here, we studied the effect of EDTA more carefully. Fluorescence quenching by Cr3+ can be arrested by EDTA at any time (10, 20, 30 min, Figure S6A). Nevertheless, EDTA cannot restore the fluorescence, even a little. This suggests that EDTA only binds to the free Cr3+ in solution, while it cannot extract Cr3+ from the complex of Cr3+-DNA.

Furthermore, when Cr3+ was mixed with EDTA, even as briefly as 0.5 min, it could no longer bind DNA (Figure S6B). The same phenomenon also can be seen when the Cr3+ is replaced by Pb2+ (Figure S6C). Therefore, DNA is kinetically at a disadvantage compared to EDTA (much more EDTA was added here) for the reaction. Since fluorescent quenching is caused by the interaction of Cr3+ and DNA, adenine and its derivatives (adenosine, AMP, and ATP) were used next to see if they could extract Cr3+ from FAMA15 (Figure 5A). These compounds were added at 0 min to the Cr3+/FAM-A15 mixture already reaching ∼75% quenching. Adenine and adenosine did not affect the binding of Cr3+ with DNA, and the fluorescence continued to drop. However, AMP arrested the fluorescence quenching, similar to EDTA. It is interesting to note that ATP even partially recovered the fluorescence from 25.8% to 33.9%. This implies that ATP can extract Cr3+ from the complex of Cr3+/DNA. The difference between ATP and AMP is the number of phosphates, and we reason that the extracted Cr3+ was associated with the phosphate group on the DNA. To further confirm this mechanism, we designed the following experiment. We added AMP and ATP simultaneously to a sample already quenched by ∼55%; 4.0% recovery was achieved in 15 min (Figure 5B). We then added AMP first and waited 15 min before adding ATP; the recovery was only 2.1% in 15 min. Finally, after waiting 90 min, ATP could not restore fluorescence at all. This experiment indicates that a slow transition of the binding state of Cr3+ on DNA (instead of with the Cr3+ in solution) happened over these 90 min. Indeed, ATP was just like other adenine derivatives, which cannot do anything after full quenching has been reached (inset of Figure 5A), suggesting that the binding converted from phosphate binding to other binding sites. Based on the above data, we reason that there are two forms of complexes of Cr3+/DNA (termed Form I and Form II). The Form I binding is quick, but relatively weak, and it can be gradually transited to the much more stable Form II (Figure 5B). ATP can compete with Form I binding, but it cannot compete with Form II. Both Form I and Form II binding can quench fluorescence. It is interesting to note that the addition of excess adenine or adenosine has no effect on DNA binding, suggesting that direct binding of Cr3+ to nucleobase or nucleoside is unfavorable, at least kinetically unfavorable (Figure 5A). Therefore, the Form I binding likely involves the phosphate backbone, and the 667

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ACS Sensors potential structure of Form I is either [P···Cr···Base] or [P···Cr] (P represents the phosphate in DNA). Binding of DNA phosphate to Cr3+ is largely electrostatically driven and, thus, is a fast process. In particular, FAM-C15 and FAM-G15 both showed a very fast initial quenching (Figure 2A), likely due to their i-motif and G-quadruplex structures, exposing the phosphate backbone to facilitate quick Cr3+ binding. For comparison, poly-A and poly-T DNAs have fewer internal secondary structures and their phosphate groups are less exposed to the solvent, showing slower initial fluorescence quenching. Moreover, DNAs with different bases show different kinetic profiles (Figure 2A), such as quenching speed and final quenching efficiency, indicating the bases participated in Form II binding. The very stable Form II binding suggests its innersphere coordination nature. We believe the lack of binding for adenosine and adenine to Cr3+ is due to kinetic effects. Based on the above discussion, we proposed the binding mechanism in Figure 5C. Further Discussion. Measuring reaction kinetics is a very common way of sensing, especially for those involving enzymes for target recognition. Even for DNA, catalytic DNA or DNAzyme based sensors are often characterized by their reaction kinetics.3−5,50,51 Here, however, the detection of Cr3+ is very different, since no catalysis is involved, and it is a pure binding reaction. The discrimination of Cr3+ is based on its water or chloride ligand exchange rate with the nucleobases on DNA.52 In this work, Cr3+ is the only special metal in terms of its slow DNA binding kinetics, while other common metals are quite fast on the human time scale (Scheme 1). However, this does not mean that this cannot be a more general method for other metal ions. Note that the rate of ligand exchange on metals can differ by 16 orders of magnitude,10 which leaves a very large room for discriminating different metal ions kinetically. For this method to be applied to other metals, we need to slow down the ligand exchange rate, or increase the time resolution of our instrument. This can be achieved by changing the solvent or ligand to allow for their sensing. For example, Co3+ is exchanged inert with amine, while Co2+ is quite labile with water.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Fax: (+1) 519-746-0435. Phone: (+1) 519-888-4567 extension 38919. ORCID

Zijie Zhang: 0000-0003-2757-3071 Juewen Liu: 0000-0001-5918-9336 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding of this is from The Natural Sciences and Engineering Research Council of Canada (NSERC) and National Natural Science Foundation of China (21305037). W.L. is supported by China Scholarship Council (CSC, 201606135019) to visit Prof. Liu's lab.

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CONCLUSIONS In summary, we have demonstrated the concept of metal sensing based on its binding kinetics. Taking advantage of the unique fluorescence quenching kinetic profile of labeled DNA by Cr3+, we developed a simple and efficient strategy for its detection with high sensitivity and low LOD. This was achieved with random DNA sequences. Compared to other metal ions, quenching by Cr3+ is not only slower, but also irreversible, as fluorescence cannot be recovered after stable binding is established. A twostage binding model was proposed by first a quick electrostatic phosphate binding followed by a slow, but tighter base coordination. There is still a lot of room for improving the Cr3+ sensor. For example, the current design is a signal-off sensor, and signal-on sensors are more desirable analytically. We also want to expand the method to other important metal ions. Overall, kinetic discrimination provides an additional dimension for metal sensing and sensor development in general.

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Effect of free fluorescein fluorophore, pH value, various modified dyes, and EDTA, ITC, the recovery of spiked Cr3+ in lake water (PDF)

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DOI: 10.1021/acssensors.7b00115 ACS Sens. 2017, 2, 663−669