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Nanopore Single-Molecule Analysis of Metal Ion-Chelator Chemical Reaction Lin Wang, Fujun Yao, and Xiaofeng Kang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01119 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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Analytical Chemistry

Nanopore Single-Molecule Analysis of Metal Ion-Chelator Chemical Reaction

Linlin Wang,┼ Fujun Yao┼ and Xiao-feng Kang┼,**

┼

Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College

of Chemistry & Materials Science, Northwest University, Xi’an 710069, P. R. China

Corresponding author: Prof. Xiao-feng Kang

∗

College of Chemistry & Materials Science Northwest University, Xi'an 710069, P. R. China Fax: +86-029-88302604; Tel: +86-029-88302604; E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Metal ions play critical roles in wide range of biochemical and physiological processes, but they can cause toxicity if excessive ingestion or misregulation. Chelating agents offer an efficient mean for metal ions intoxication and therapeutics of diseases. Studies on metal ion-chelator interactions are important for understanding the reaction mechanism and developing new specific metal chelator drugs. However, it remains a significant challenge to detect the metal ion-chelator interactions at molecular level. Here, we report a label-free nanopore sensing approach that enables single-molecule investigation of the complexation process. We demonstrate that the chemical reaction between Cu2+ and carboxymethyl-β-cyclodextrin (CMβCD) in nanoreactor is completedly different from in the bulk solution. The formation constant (Kf = 4.70 × 104 M-1) increases 14,417-fold in the nanopore than that in the bulk solution (Kf = 3.26 M-1). The bioavailable CMβCD as a natural derivative with higher affinity for Cu2+ could be used in the safe medicinal removal of toxic metal. Based on the different ionic current signatures across an α-hemolysin (α-HL) mutant (M113N)7 nanopore lodged with a CMβCD adaptor in the presence and absence of Cu2+, the reversible molecular binding events to CMβCD can be in-situ recorded and the single-molecule thermodynamic and kinetic information can be obtained. Interestly, we found that the Cu2+ binding leads to the increase of channel current, rather than the blocking as usual nanopore experiment. The uncommon (on/off) characteristic could be very useful for fabricating nanodevice. Furthermore, the unique nanopore sensor can provide a highly sensitive approach for detecting metal ions.

INTRODUCTION Metal ions can be beneficial or hazardous to human health and enviromental stability. On the one hand, trace metals play important roles in numerous biological processes including signal


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transduction, gene expression, biosynthesis and regulation of enzymes.1 On the other hand, elevated levels of metal ions in humans have been implicated in a variety of diseases, from antibiotic resistance and cancer to metabolic disorders and mental retardation.2 For example, many evidence suggested that metals overload such as copper, zinc and iron are closed related with neurodegenerative diseases.3 To prevent potential human health problems from toxic metal ions, methods to remove them are highly required. Various techniques have been developed for heavy metals removal, such as chemical precipitation,4 adsorption,5 membrance,6 ion-exchange.7 Among these methods, chelating agents are the most versatile and effective approach used due to its capability of binding to metal ions and form complex structures. Recently, a host of chelators have been used for heavy metal intoxication8 and chelation therapy is now recognized as a potential treatment for related diseases.9 For example, metal chelators can reduce the metal-mediated Aβ aggregation, ROS (Reactive Oxygen Species) formation and neurotoxicity.10 However, the efficacy of the chelation therapy depends on the specific of the chelators, as well as the relative stability of the chelator-metal complexes. Therefore, the binding studies of chelators with metals are useful for designing new specific metal chelator drugs, and also provide an increased understranding of the biological roles of metal ions in complex living system. Traditional studies involving metal-chelator interactions include UV-visible spectrometry,11 computational chemistry,12 NMR spectrum,13 and ESI-MS.14 Although many reports deal with metal-chelator interaction, the results have mostly been based on the ensemble averaging. Therefore, it is neccessry to analyze the formation and dissociation process of an individual metal complexes at molecular level. Herein, we employed α-HL (M113N)7 nanopore to investigate the interactions of Cu2+ with CMβCD. Our results suggest that the nanopore sensors can be used for the direct observation of single-molecule chemistry between metal ion Cu2+



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and chelator CMβCD. The nanopore technique is a highly sensitive, stochastic analytical technology. Its inherent merits, such as simplicity, label-free and easy genetic and chemical modification, make it a powerful tool in the detection of various analytes.15 Biological nanopore such as the α-HL has been widely used as a single-molecule identifier and detector for small organic molecules,16 metal ions,17 DNA,18 enzymes,19 proteins20 and biomolecular complexes.21 Furthermore, the nanopore has also been successfully applied to study G-quadruplex structures,22 DNA oxidative damage products,23 RNA secondary structures,24 unfolding of proteins,25 as well as rapid and low-cost DNA sequencing.26 In the following work, we will illustrate the establishment of the nanopore platform and the details regarding single-molecule interaction between Cu2+ and CMβCD. Cu2+ and CMβCD are selected as a model system due to two main reasons: i. Cu2+ is widely present in environment as well as physiologically important in living organisms; ii. It is well established that βCD can be used as a molecular adaptor in nanopore sensing.27 Thus CMβCD with highly charged density should has the ability to chelate metal ions through its -COOH functional groups. Besides, CD complexes with metal ions could have a range of applications in the field of chiral recognition and of metalloenzymatic mimicking.28 In this work, we noted that Cu2+-CMβCD complexes generated distinctive electronic signatures, which were different from the original βCD and its derivatives as reported previously in the nanopore. We further demonstrated that the thermodynamic and kinetic constants of Cu2+-CMβCD complexes can be influenced by varying experimental conditions, i.e., pH, transmembrane voltage, temperature. This work provides an effective approach capable of both analyzing metal ion-chelator interactions and detecting metal ions.


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EXPERIMENTAL SECTION Reagents and Materials. Carboxymethyl-β-cyclodextrin (CMβCD) was purchased from Aladdin，and its average degree of carboxymethyl substitution is 5.5. CuCl2 (>99.0%) was bought from Sigma-Aldrich. 2-Diphytanoylphosphatidylcholine (DPhPC) lipid was obtained from Avanti Polar Lipids. The thickness of Teflon film (Goodfellow) was 25 µm. The mutant of α-HL, M113N protein monomer was prepared by expressing in Escherichia coli BL-21 (DE3) pLysS and purified by size exclusion chromatography. The assembly and purification of heptametrical protein pore (M113N)7 were carried out as reported previously.29 Single-Channel Recording. A bilayer of DPhPC was formed over a 120−160 µm orifice in a Teflon septum that divided a planar bilayer chamber into cis and trans compartments. The formation of the bilayer was achieved using the Montal-Mueller method.30 The solutions in the compartments contained 2 M NaCl, which buffered with PBS at pH 3.0 and 8.5, 50 mM MOPS at pH 6.5, respectively. The (M113N)7 mutant protein was added to the cis compartment, which was connected to a “ground”. The final concentration of the α-HL proteins used for the single-channel insertion was 0.05−0.2 ng/mL. CMβCD was added to the trans compartment and metal ions were added to the cis compartment. Current was recorded with a patch clamp amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, CA), filtered with a built-in four-pole Bessel filter at 5 kHz, sampled at 20 kHz by a computer equipped with a Digidata 1440 A/D converter (Molecular Devices). Data Analysis. Single-channel event amplitude and duration were analyzed using Clampfit 10.3 (Molecular Devices) and Origin 8.0 (Microcal, Northampton, MA) software. Mean dwell time values (τoff) and mean interevent interval values (τon) were obtained from the dwell histograms and the interevent interval histograms after the peaks were fitted to single exponential functions. The



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standard deviation of open pore current (I0) was obtained from single channel current baseline histograms by fitting the distributions to Gaussian functions. The values of mean signal amplitude and current blocking rate (I/I0) were obtained from signal amplitude and I/I0 histograms by fitting the distributions to Gaussian functions.

RESULTS AND DISCUSSION Reversible Interaction Between the CMβCD and Cu2+. In a first set of experiments, we studied the interaction between CMβCD and an engineered α-HL (M113N)7 protein nanopore. Single-channel recordings were made in solutions containing 2 M NaCl, buffered with 50 mM MOPS, at pH 6.5. As shown in Figure 1a, without CMβCD, no interference signal was generated in the open pore current at different voltages. Then when the CMβCD was added to the cis chamber, it was found that they did not bind to the engineered α-HL (M113N)7 pore at negative potential (Figure 1b). Although CMβCD could generate current blocking signals at positive high voltages, these events are short-lived. The mean duration was still less than 1 ms even if at a voltage of + 160 mV (Figure 1b, S1). These events probably generated from the weak binding of CMβCD with the inner wall of the vestibule. Next, We further investigated the binding of the (M113N)7 pore to CMβCD from the trans chamber (Figure 1c). After adding 80 µM CMβCD to the trans chamber, the binding of CMβCD to (M113N)7 induces reversible blocks of the ionic current. Typical ionic current traces are presented in Figure 1d. The channel current dropped from –83.2 ± 2.5 pA to –6.0 ± 1.7 pA (n = 5) with a blockage amplitude (I1/I0) 92.9 ± 1.8 % (Figure 1e), and the mean duration of these blocks τoff was 626 ± 12 ms (Figure 1f). The long dwell time of the events revealed tightly binding of CMβCD to (M113N)7 pore from the trans chamber, which would enhance sensing with molecular adapters.31


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Hence we selected adding CMβCD to the trans chamber in subsequent work. Previous studies indicated that the channel current was partially blocked with an average current blockage of 65% when βCD was bound to the α-HL pore.27,

32

In contrast, the CMβCD reduced the pore current to a

much larger extent in the present work. A plausible explanation for larger current blocking may be due to the entering -COOH groups into the cavity of the CD under the action of electric field force, and thus leading to reduction of the ions current flow. This interpretation is supported by previous studies regarding the electrolyte ions translocating through the center of CD molecular adapter.32,33

Figure 1

In the next step, we probed the interactions between Cu2+ and CMβCD. Firstly, a control experiment demonstrated Cu2+ did not affect the open pore current even its concentration reached 120 µM,34 and no detectable signal produced in the (M113N)7 pore without the presence of CMβCD (Figure S2). However, when Cu2+ was being added to the cis chamber, a new secondary current level was generated with the presence of CMβCD in the trans chamber (Figure 1d). The new generated signals should be ascribed to Cu2+-CMβCD complexes. To be sure the secondary signals shown in Figure 1d are exclusively generated by Cu2+-CMβCD complexes. We performed another control experiment with βCD, which has no -COOH functional groups to complex metal ions. The interactions of βCD with (M113N)7 was conducted in the presence of Cu2+ under the same conditions. The secondary current level was not observed (data not shown), confirming the role that -COOH groups play in the interactions of Cu2+ with CMβCD (Figure 2a), the reason why we added the Cu2+ and the CMβCD to opposite sides of the pore is because they are oppositely chared. Therefore, at



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negative potential, the positively charged Cu2+ is driven from the cis to the trans chamber, while CMβCD can be tightly bound to the pore from the trans chamber under the action of the electrophoretic force. Thus, the electrical-driven flow would overcome the diffusion-based transport limit and enhance the single-molecule chemical reaction.35 In addition, we also investigated the interaction between Cu2+ and the CMβCD with both of them in the trans chamber. As shown in Figure 1g, the event frequency of Cu2+-CMβCD complexes decreased compared with those of Cu2+ in the cis chamber. One possible reason for our observation that addition of Cu2+ to the trans chamber produced less frequent Cu2+-CMβCD complex events is because the electric field led to the dissociation of Cu2+-CMβCD complex as it entered the pore. This is in agreement with previous results which suggested that the electric field has the potential to cause the dissociation of the drug-peptide complex when the small drug molecule and peptide were added to the same side of the pore.36 The average current level (I2/I0) of the Cu2+-CMβCD complexes is displayed in Figure 1e. The value was 76.6 ± 1.3% (n = 6), which increased about 16.3% (I2/I0 – I1/I0) compared with the events of free CMβCD. Figure 1f shows the mean dwell time of the complexes, with a value of 1.10 ± 0.12 ms. The above results testify that Cu2+ does interact with the CMβCD, leading to the fluctuation of the ionic current between I1 and I2 separated by ∆I = 13.6 ± 0.4 pA. The reason why Cu2+ binding leads to the increase of channel current, rather than the blocking as usual nanopore experiment may be due to the come out of the carboxymethyl chain from the CD cavity resulting from the complexation. The unoccupied CMβCD cavity will leave room for the electrolyte ions such as Na+ and Cl- to translocate through. The conclusion is in line with previous reports which demonstrated that the oxygen ligand -COOH groups were very effective for Cu2+ with high specificities and they could complex Cu2+ outside the CMβCD cavity.37 The unique phenomenon is


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different from usual nanopores, which could be very useful for constructing nanodevice switch. Besides Cu2+, we also studied the interaction of CMβCD with a variety of other divalent and trivalent metal ions such as Hg2+, Zn2+, Ni2+, Sr2+ Co2+, Pb2+, Mn2+, Sn2+, Ba2+, Ca2+, Mg2+, Fe3+ and Al3+ (data not shown). Since the aim of this paper was single-molecule analysis of CMβCD interactions with Cu2+, a detail discussion regarding CMβCD binding with other metal ions will be carried out in another separate manuscript.

Figure 2

By the detail analysis of the current signatures associated with the uncomplexed or Cu2+-complexed CMβCD interactions with (M113N)7 pore, four different types of current-time (I–t) traces were observed, which we refer to as type 1, type 2, type 3 and type 4 events (Figure 2b). These events demonstrated the existence of three states (pore, pore-CMβCD and pore-CMβCD-Cu2+) that are correlated with the processes of association and dissociation between them. Type I and type II events both started with the current switch from level I0 to level I2 between state 1 and state 2, indicating direct formation of CMβCD-Cu2+ complexes in the pore. Then, a series of association and dissociation events between pore-CMβCD and pore-CMβCD-Cu2+ were observed. Finally, the events ended with returning to open base current (state 1) through state 3 for type 1 and state 2 for type 2, respectively. However, type 3 and type 4 events began from state 1 to state 3, and lastly came back to state 1 after state 2. We can observe through these events that the reversible conversion of three states, which forms a close loop. By the conversion between state 1 and state 2, we can directly monitor single-molecule chemical reaction between the Cu2+ from cis chamber and the CMβCD



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from trans chamber in the pore. Whereas the two-step reversible reaction process can be observed by the path between state 1 and state 3, then between state 3 and state 2.

Figure 3

The effect of Cu2+ concentration on reversible current events of the Cu2+-CMβCD complexes was carried out. As shown in Figure 3a, the binding frequency of CMβCD with Cu2+ increased with an increase in Cu2+ concentration. In the kinetic analysis of the events between state 2 and state 3, we assumed that the CMβCD and Cu2+ reversibly form a binary complex (Figure 3b) and that the Cu2+ concentration inside the pore equals that in solution.38 The reciprocal of the mean τoff versus the Cu2+ concentration has a near zero slope (Figure 3c), which is consistent with a unimolecular dissociation mechanism (1/τoff = koff, koff is the dissociation constant). In contrast, a plot of the reciprocal of the mean τon is proportional to the Cu2+ concentration (Figure 3d), which is consistent with a bimolecular interaction for which 1/τon = kon[Cu2+] (kon is the association constant). The forward and reverse rate constants were derived from the τ values. The association constant kon2-3 was (3.26 ± 0.18) × 107 M-1 s-1, the dissociation constant koff2-3 was 917 ± 18 s-1, and thus the formation constant of Kf2-3 was (3.56 ± 0.25) × 104 M-1 (Kf2-3 = kon2-3/koff2-3) (n = 6). The formation constant of CMβCD for Cu2+ at the single-molecule level is four orders of magnitude higher than that in the bulk solutions (Kf = 3.26 M-1).37 The greater Kf could be ascribled to two possible reasons: i. The nanopore confined Cu2+ and CMβCD in the nanoscale-confined spaces so that they can encounter each other more frequently; ii. The electrophoresis and electroosmotic driving forces overcame the diffusion-based transport limit in the bulk, and enhanced mass transport rate of these two oppositely charged reaction substrates, and


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thus shortened the reaction time.35 Our results suggest that complexation kinetic in nanochannels is different from that in bulk solutions, which could be due to the unique reaction environment such as nanocavity structure, etc. Similarly, kinetic analyses between state 1 and state 3 as well as between state 1 and state 2 were carried out. τon1-3 = 1870.5 ± 98.1 ms, τoff1-3 = 626 ± 12 ms, kon1-3 = (6.69 ± 0.15) × 103 M-1 s-1, koff1-3 = 1.60 ± 0.03 s-1, Kf1-3 = (4.18 ± 0.18) × 103 M-1. Interestedly, Kf1-3 × Kf2-3 = (1.49 ± 0.17) × 108 M-1, which is approximately equal to the Kf1-2 value [(3.95 ± 0.30) × 108 M-1] directly obtained from the traces of state 1 and state 2. The result indicates that the single-molecule kinetic analysis regarding metal ion-chelator interactions can be done in various ways based on the events of distinct states. Effect of pH on the Interaction of Cu2+ with CMβCD. The pH dependence experiments of CMβCD binding with Cu2+ were conducted at different pH values of 3.0, 6.5 and 8.5. As shown in Figure S3, the pH of the electrolyte solution plays an important role in the binding process of Cu2+ and CMβCD. At pH 3.0, no current modulation of level I1 was detected. When the pH increased to 6.5, the current signals of level I2 appeared. In addition, our experimental results showed that, as the pH value of the electrolyte increased from pH 6.5 to pH 8.5, the ∆I2/I0 value and the mean dwell time τoff of Cu2+-CMβCD complexes events remain almost unchanged, but the binding frequency (1/τon) of Cu2+ to CMβCD was reduced 4.72-fold from 326 s-1 to 69 s-1. The results were not unreasonable considering the charge state of CMβCD and the exiting forms of Cu2+ species in aqueous solution. It is well known that Cu2+ species can be present in aqueous solution in the forms of Cu2+, Cu(OH)+, Cu(OH)2, Cu(OH)3-, Cu(OH)42-,39 and the predominant species is Cu2+ at pH

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