Determining the Physical Properties of Molecules with Nanometer

Review Cite This: ACS Sens. XXXX, XXX, XXX−XXX

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Determining the Physical Properties of Molecules with NanometerScale Pores Haiyan Wang,†,‡ Jessica Ettedgui,†,§ Jacob Forstater,†,§ Joseph W. F. Robertson,† Joseph E. Reiner,∥ Huisheng Zhang,‡ Siping Chen,‡ and John J. Kasianowicz*,†,⊥ †

National Institute of Standards and Technology Physical Measurement Laboratory, Gaithersburg, Maryland 20899, United States Shenzhen Key Laboratory of Biomedical Engineering, School of Medicine, Shenzhen University, 3688 Nanhai Road, Shenzhen 508060, China § Department of Chemical Engineering, Columbia University New York, New York 10027, United States ∥ Department of Physics, Virginia Commonwealth University, Richmond, Virginia 23284, United States ⊥ Department of Applied Physics Applied Mathematics, Columbia University New York, New York 10027, United States ‡

ABSTRACT: Nanometer-scale pores have been developed for the detection, characterization, and quantification of a wide range of analytes (e.g., ions, polymers, proteins, anthrax toxins, neurotransmitters, and synthetic nanoparticles) and for DNA sequencing. We describe the key requirements that made this method possible and how the technique evolved. Finally, we show that, despite sound theoretical work, which advanced both the conceptual framework and quantitative capability of the method, there are still unresolved questions that need to be addressed to further improve the technique.

KEYWORDS: nanopores, ion channels, sensors, analyte detection, DNA sequencing, microRNA, protein−DNA interactions, chemically modified nanopore, gold nanoclusters, metallonanoclusters

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receptor channels as biosensors. For example, designing nanopores that bind specific analytes and convert the interaction into predictable channel gating events is a daunting task. Another seemingly simple method for nanopore-based sensing of analytes was to measure the ionic current in response to the reversible transport of molecules into and out ̈ of the channel pore. This concept, while compelling, is naive. First, any spontaneous gating that occurs in the absence of the target analyte would complicate the use of channels as sensors. This problem was overcome when it was shown that the voltage-dependent gating in the αHL channel could be essentially eliminated.11−13 Second, many channels are so narrow that only water and small ions can enter them. However, Krasilnikov and colleagues demonstrated that the αHL channel was sufficiently wide that polymers of ethylene glycol with molecular mass ≤ 2000 g mol−1 could partition into the pore.14,15 Third, assuming one-dimensional Brownian motion16 of a polymer through the channel and a channel length ca. 4 nm (i.e., the width of a lipid bilayer membrane), the

he ability to detect and characterize single biological molecules provides insight into their physical properties and molecular mechanisms of action that cannot be gained from ensemble measurements. Conventional single molecule spectroscopy methods are based on the measurement of mechanical forces (AFM)1,2 and light (fluorescence).3,4 However, it was shown that individual molecules could also be probed by measuring their effect on the ionic current that otherwise flows freely through single ion channels.5 The conceptual basis for this method was partially founded in the ability of receptor channels at neural synapses to discriminate between different neurotransmitters. These channels, and others that are the basis for nerve and muscle activity6−10 function by gating (i.e., switching) between discrete conducting states, open and close in response to a physical stimulus (e.g., the binding of a neurotransmitter) in a dose-dependent manner. Experimental evidence suggested that sensing individual analytes based on how the target molecules influence the “gating” behavior of channels was possible.11,12 For example, it was shown that hydronium and deuterium ions in aqueous solution could be discriminated based on the ionic current fluctuations they caused in the channel formed by Staphylococcus aureus alpha-hemolysin (αHL).11,12 Those positive results notwithstanding, there were major hurdles to mimicking © XXXX American Chemical Society

Received: September 12, 2017 Accepted: January 8, 2018

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DOI: 10.1021/acssensors.7b00680 ACS Sens. XXXX, XXX, XXX−XXX

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DETECTION AND CHARACTERIZATION OF INDIVIDUAL RNA AND DNA OLIGONUCLEOTIDES The detection of analytes with a nanopore was first done with the channel formed by Staphylococcus aureus toxin α-hemolysin (αHL).11 The membrane-spanning segment of the heptameric pore is ca. 2.6 nm wide and 5 nm long, and a 1.4 nm diameter constriction separates it from a 3.6 nm diameter vestibule region in the channel’s extramembranous cap domain.43,44 A cartoon illustration of the αHL channel in cross section is shown in Figure 1. Early results demonstrated the ability of the αHL channel to detect single-stranded RNA and DNA oligonucleotides20 and distinguish between polydeoxyadenylic acid and polydeoxycytidylic acid molecules.28 Because the oligos were transported through the pore in a linear fashion, it was suggested that nanopores might eventually prove useful in sequencing DNA directly by reading the ionic current time series (Figure 2).20

mean time an individual molecule would take to traverse the pore is ca. 80 ns, i.e., orders of magnitude less than the bandwidth of a conventional patch clamp apparatus,17 making it seemingly impossible to directly detect the molecules via a Coulter counter effect.18 This issue was also resolved experimentally when it was found that some molecules bind to the pore walls long enough to allow their detection.11,12,19,20 Since then, protein and solid-state nanopore-based sensors were developed for a wide range of biotechnological applications.5,21 They have been used to detect analytes as diverse in their chemistry as ions,11,12,22,23 polymers,24−27 nucleic acids,20,28 and metallic nanoparticles.29−32 Refined analysis of the methods have enabled nanopores to sequence DNA,5,20,28,33−35 provide mass distributions of polymers complementary to mass spectrometry,24,25,27,36 monitor the conformations (folded versus unfolded) of single proteins,37,38 and perform single molecule force spectroscopy.39−42 The principle of nanopore-based sensing of individual molecules is illustrated in Figure 1. The entry of a single

Figure 2. Nanopore-based DNA sequencing. It was shown that individual molecules of single-stranded DNA thread through the S. aureus α-hemolysin channel pore in a linear fashion. If each type of base caused a unique reduction in the ionic current, the DNA sequence could be read directly from current time series in a tickertape-like fashion.20

With this system, single nucleotide discrimination could not be easily achieved for several reasons. First, up to 10 to 15 nucleotides can occupy the relatively narrow β-barrel pore at any given time45−47 and there are three distinct base “sensing” regions within this 5 nm long pore.48 Second, the translocation rate of nucleic acids through the pore (1−20 μs/nucleotide for homopolymers) is several orders of magnitude slower than diffusive motion, but still too fast to accurately record the effects of individual bases on the channel conductance.28 Third, the rate at which each base passes a constriction in the pore is uncontrolled due to Brownian motion. Enzymes that process DNA were used to address the latter two issues by acting as a molecular brake for oligonucleotide transport through the pore. As a proof of concept, DNA replication catalyzed by a bacteriophage T7 DNA polymerase (DNAP) and by the Klenow fragment of DNA polymerase I (KF) was observed in real time with an αHL nanopore (e.g., see Figure 2 in ref 49). A DNA template bound to a DNAP and KF complex was held at the αHL orifice, where its replication was initiated and measured.50 The nucleotide additions to the synthetic complementary DNA strand progressed through the pore against an opposing applied voltage. To improve the temporal resolution, bacteriophage phi29 DNA polymerase (DNAP) was used to ratchet DNA templates through the

Figure 1. Schematic illustration for nanopore-based analyte detection. Individual molecules that enter the pore (embedded in a phospholipid bilayer membrane) from an aqueous solution reduce the ionic current i (measured with a high impedance amplifier, A) that otherwise flows freely in response to an applied electrostatic potential V. The physical properties of the molecules are estimated from both the magnitude of the current decrease and the distribution of residence times of the molecules in the pore.

molecule into the pore reduces the latter’s ionic conductance. As we describe below, both the magnitude of the conductance changes, and the lifetimes of the current blockade events provide information about pore-permeant species. For this class of sensor to operate effectively, the analytes must interact with the pore, such that the pore conductance is interrupted long enough to characterize the event, but short enough that many molecules can be counted in a given experiment. B


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ACS Sensors channel.51 DNA replication by phi29 DNAP controlled sequential movement of more than 50 bases through the αHL pore from a precise starting point on a DNA primer strand, without active voltage control, at a rate of 2.5−40 nucleotides s−1. Beyond controlling the motion and rate of nucleotide translocation, the geometry of the pore also needed to be optimized over that of αHL to improve the resolution for DNA sequencing. With a wide mouth52 and smaller constriction (1.2 nm diameter, 0.6 nm long), the MspA protein from Mycobacterium smegmatis53,54 appeared to be more appropriate for sequencing applications (Figure 3).52 However, extensive mutations of the protein were needed to improve the electrostatic interactions of the pore with DNA (see below) and allow DNA to be driven into and through the pore.55−57 Improved stabilization of this system was achieved by anchoring the phi 29 DNAP to the membrane via a cholesterol anchor.58 Using the nanopore chemistry embodied in the β-

amyloid secretion channel CsgG,59 single-base accuracy in direct sequencing was estimated to be 90−95%.60

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DETECTION AND CHARACTERIZATION OF SMALL MOLECULES The next step was to discover whether nanopores could be used to determine the physical properties of molecules and whether they could discriminate between different molecules in the same chemical class. Synthetic Poly(ethylene glycol) Polymers. In the late 1980s, little was known about the structures of membrane proteins. To partially address this issue, Krasilnikov and colleagues used water-soluble polymers of ethylene glycol (PEG) to estimate the limiting pore diameter of ion channels. PEGs, which are commercially available in a wide range of mean molecular masses, reduce the bulk ionic conductivity of aqueous electrolyte solutions. Krasilnikov and colleagues correctly reasoned that only PEGs with radii of gyration smaller than the pore radius would enter the pore and thereby reduce the mean channel conductance.14,61,62 Thus, these polymers can be used as a probe to determine, at low resolution, the inner geometry of a channel.14,61−64 Krasilnikov’s group and ours demonstrated that the opposite held true as well: nanopores can accurately and precisely determine the size of polymers at high resolution.24,25,65 Figure 4A illustrates a histogram of αHL ion channel current blockades caused by a polydisperse sample of PEG 1500 and a 95% pure sample of a PEG 29-mer. The number of peaks present is identical to those obtained with a MALDI-TOF mass spectrometer. Each peak in the plot corresponds to a given size of PEG, and the method can easily resolve the polymers to better than the monomer limit.24,25,27 The residence times for events in each of the peaks are distributed exponentially (three of which are illustrated in Figure 4B), which suggests the barrier for polymers to exit the pore is analogous to that of a simple first order reversible chemical reaction. A fit to these distributions is used to estimate the mean residence time for each size polymer in the pore.24,25 The results demonstrate that larger polymers reduce the pore conductance more and spend a greater time in the pore than do smaller ones. Furthermore, both the pore conductance and residence time depend on the applied potential, despite the nonelectrolyte nature of PEG. We developed a simple physical and chemical theory that accounts for these results.25 The magnitude of PEG-induced current blockades is attributed to both volume exclusion and the binding of cations to the polymer. The latter effect might seem surprising because the binding constant of K+ ions to PEG is extremely weak (i.e., ca. 0.1 to 1 M). However, the resultant reduction in the mobility of the cations in the pore increases their mean transit time and thus contributes to the decrease in the observed current.66 Why does the mean residence time depend on the polymer size? Other investigations of PEGs with the αHL channel only studied the effects of the mean polymer size on the pore conductance.14,62,67 Using the ability to study the interaction of each size PEG with the pore, it was shown that both the polymer-induced blockade depths and the mean residence times of the polymers in the pore depend on the applied potential,25 but the effect is much greater for the latter. Additional work is needed to better understand this effect. The ability to discriminate between different-sized polymers (Figure 4) provided the basis for another single molecule nanopore-based DNA sequencing method.33,68,69 This ap-

Figure 3. High resolution DNA sequencing with the Mycobacterium smegmatis MspA channel. (A) A cross section of the channel shows that the pore has a wider vestibule and a shorter constriction than those in the αHL nanopore (Figure 2). (B) A cartoon illustrating the method. The target DNA (black) bound to a processivity enzyme (green) is captured in the pore (i, ii). A blocking oligomer (red) prevents the enzyme from processing the DNA until it is in the pore and a sufficiently large electrical force is applied to the DNA to remove it (ii, iii), which allows sequencing to commence (iv). Then, the enzyme pulls the target DNA through the pore constriction as it builds the complementary strand and the current levels caused by each nucleotide in the constriction correspond to the oligo’s sequence (iv). The gray arrows indicate the direction of the DNA’s motion through the pore. (C) The ionic current that corresponds to events (i) through (iv). Adapted from ref 57 with permission. C


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latter effect is crucial because, without such interactions, molecules that partition into the pore would diffuse through it much faster than the system’s time resolution.5 Since the advent of nanopore-based detection of molecules,11,12 other nanopores have been studied for their potential in this arena. Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila.70 Proaerolysin is a water-soluble inactive protoxin, which after activation by nicking the protein near its C-terminus, spontaneously forms ion channels in lipid bilayers.71,72 The channel is slightly anion selective, has a single channel conductance of 420 pS in 1 M KCl,73 and a limiting pore diameter between 1.3674 and 1.7 nm.71 Like the αHL ion channel, aerolysin forms a heptameric pore from seven identical monomers,75 has a cylindrical component that spans the membrane, and has a wheel-like cap domain that lies outside the membrane.74,76 It was determined experimentally that the residence time of a given size PEG polymer in the aerolysin nanopore is ca. 10-fold longer27 than that in the αHL channel.24,25,65 More recently, it was shown that α-helix polypeptides also spend more time on average in the aerolysin nanopore than in αHLs.77 Long and colleagues took advantage of this capability to demonstrate that the aerolysin nanopore could be used to detect and discriminate between different length short oligonucleotides36,78 (between 2 and 10 bases per oligo). Interestingly, the mean residence time of those small dA homopolymers is nearly 3 orders of magnitude longer than that in αHL.28,36,78 The results in Figure 5 show that in the well-resolved current blockades aerolysin yielded clearly distinguishable Gaussian peak values in the current levels that were assigned to different length short dA oligomers. Bacillus anthracis secretes three toxins that lead to anthraxinduced cell death. One of those is protective antigen (PA), which binds to cell membranes and is cleaved into PA63, which

Figure 4. Sizing molecules with a single nanopore (single molecule “mass spectrometry”). (A) The inset shows a typical blockade event and the mean current values of the unoccupied and occupied pore (⟨iopen⟩ and ⟨i⟩, respectively). The full plot is a histogram of the relative ionic current blockade depth ratio ⟨i⟩/⟨iopen⟩, where the values of 0 and 1 correspond to a fully blocked and unoccupied pore, respectively, for a polydisperse mixture of PEG1500 (black) and a highly purified sample of PEG (n = 29) (orange), where n is the number of monomers in the polymer. (B) The residence time distributions for three different size PEG molecules, n = 29 (orange), 38 (green), and 48 (blue). The distributions are well-described by single exponentials, which suggests the interaction between the PEG and the pore is a simple reversible reaction. Larger polymers block the pore more and spend more time there (on average) than do smaller ones.24 Adapted from ref 24 with permission.

proach is like the conventional sequencing-by-synthesis (SBS) method, but reads surrogates (i.e., tags) for each base electrically, not via fluorescence measurements. The DNA to be read is captured by a DNA polymerase molecule bound to the outside of the pore. A complementary strand is synthesized by adding one base (with a unique polymer tag attached to it) at a time and adding it to the strand being extended. As the base is incorporated into the DNA, the tag is captured by the pore, released from the nucleic acid by polymerase, and read by the pore. By using slowly diffusing polymers as the tags (either synthetic33 or natural68) and attaching the polymerase directly to the pore, the probability of correctly identifying the tags can approach 100%.31,34 This optimized strategy provided a highly scalable, potentially low-cost single molecule electronic DNA sequencing platform (see Figure 7 in ref 21 and data in refs 68 and 69). Other Nanopores Interact Differently with Individual Molecules. The αHL channel has proven to be useful for nanopore-based sensing because it remains open over a wide range of applied potentials,13 and molecules that enter it apparently bind reversibly to its pore walls.5,11,12,19,24,25 The

Figure 5. Discrimination of extremely short DNA homopolymers with the Aeromonas hydrophila aerolysin nanopore. (A) Cartoon representation of the nanopore’s putative structure. (B) Histograms of the relative ionic current blockade ratio show that aerolysin can easily discriminate between adenosine homopolymers that contain between 2 and 10 monomers, which are barely detectable by the αHL nanopore.36 Part (B) adapted from ref 78 with permission. D


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MicroRNAs. As discussed above, the ability to detect individual molecules of single stranded RNA (ca. 150−430 nucleotides long) and DNA with a nanopore provided the basis for a novel sequencing method.20 Because the polymers transport through the pore in a linear fashion, there is essentially no practical upper limit on the length of oligonucleotides that can be studied with that method. However, the ability to detect very small fragments of nucleic acids would be beneficial, because microRNAs (ca. 18−21 nucleotides long) are implicated in disease.112,113 It was shown that a 30 nucleotide-long segment of polyA in a diblock copolymer with 70 cytosines could be detected.28 However, that capability was not sufficient for microRNA detection and discrimination. Gu and colleagues therefore designed a series of oligonucleotide probes that interact with specific target microRNAs and that produce long-lived and multilevel ionic current states (Figure 6A).114 The unresolved

forms a heptameric pore. The PA63 pore conductance in the presence of different size ions and polymers suggests the pore is ca. 1.2 nm in diameter.79,80 Site directed mutagenesis studies,81,82 molecular models,83 and cryo-electron microscopy (at 2.9 Å resolution)84 suggest the β-sheet stem pore domain extends significantly past one side of the membrane and the channel has a larger cap domain83 than that in either the αHL44 or aerolysin74 channels. The channel is highly cation selective85 and binds cationic polypeptides,86 which is consistent with structural studies that suggest the pore is lined extensively with negative charges.83 PA63 has been proven useful for both the detection of two other anthrax toxins (Lethal Factor and Edema Factor) with high sensitivity (ca. 40 pM) and screening for therapeutic agents against them.87,88 Many studies have demonstrated reversible interactions between the N-terminus of Lethal Factor and Edema factor,89−91 but under certain conditions the interaction becomes irreversible.86 The MspA channel had to be modified for DNA sequencing applications (described above) because the native channel produces spontaneous ionic current blockades in the absence of single-stranded DNA, which would confound the characterization of nucleic acids.55 In addition, WT MspA has a high density of negative charge in the pore,52 which would prohibit oligonucleotide translocation.55 Replacing the aspartate residues in the narrowest constriction with asparagines removed the negative charge in a critical region inside the pore, thereby allowing the channel to detect nucleic acids.56 Interestingly, in the absence of the phi29 DNAP, the translocation rate of nucleic acids through the mutant MspA nanopore is between 2 and 10 times faster than for unmodified αHL,55 which may be attributed to a combination of the geometry of and electrostatics within the pore.55,92 While the β-barrel pore forming toxins have been invaluable for the analysis of DNA, polymers, and small molecules, they have several limitations for the study of larger molecules. For example, the relatively large hydrodynamic radii of proteins prevents their detection by a nanopore, unless the proteins are denatured.37,38,93 To analyze intact proteins (other than the anthrax toxins), two promising strategies have emerged.94 One is to select pore-forming proteins with large cavities such as Cytolysin A, which has a ca. 5.5 nm diameter vestibule and a 3.3 nm constriction.95−98 The second is to use proteins with a flexible, conical structure such as Fragaceatoxin C,94,99 which can capture and characterize both single and double stranded DNA.100 Other channels have been used to detect analytes of interest. For example, OmpF secreted by Escherichia coli,101−106 which allows the passage of ions107 and small neutral solutes up to ca. 650 g mol−1,103 was used to detect different antibiotics.108 In addition, the maltoporin bacterial outer membrane channel can discriminate different types of sugar molecules109,110 and detect single lambda phage particles.111 The Use of Probes to Aid the Detection of Molecules. Often, the direct detection of an analyte is difficult or impossible because the duration of the ionic current blockade is too short for the system bandwidth or the magnitude of the blockade is not deep enough to distinguish itself from the background. Under these conditions, it is advantageous to utilize chemical probes, which can interact with an analyte either inside the pore or form complexes outside the pore.42 The following section provides some recent highlights from the use of this approach.

Figure 6. Probe-assisted detection of individual small microRNA molecules with a single nanopore. (A) A cartoon illustration of the microRNA (red) complexed to a probe molecule (green). (B) The ionic current blockades caused by either the free microRNA or the free probes are markedly shorter in duration than those caused by the complex. (C) Closer examination of a single event shows the capture of the complex and subsequent unbinding via a multilevel current trace. (D) A cartoon that illustrates the events that give rise to the signals in (B) and (C): the capture of the complex up to the release of the signal tag (level 1), the current increases due to the loss of the signal tag from the pore (level 2), and the microRNA translocates through the constriction (level 3).114 Adapted from ref 114 with permission.

transient signals in Figure 6B are caused by microRNA and probe molecules themselves. The longer-lived blockades (Figure 6B) are due to individual probe:microRNA complexes. A typical event caused by the complex has three well-defined current levels (Figure 6C). Levels 1, 2, and 3 correspond to the complex, probe, and microRNA interacting with the pore, respectively, as illustrated in the cartoon in Figure 6D. Importantly, the method was used to detect lung cancerrelated microRNAs in patients’ plasma samples compared to a control group of healthy individuals.114 The results suggest that nanopore-based single molecule detection method might be useful for noninvasive screening and possibly early stage cancer diagnosis. E


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ACS Sensors Protein−DNA Interactions. Over the past two decades, protein nanopores have been used to detect, characterize, and discriminate between different molecules.20,24,115,116 This capability was made possible because some analytes bind reversibly inside the pore and reside there for orders of magnitude longer than predicted from the diffusion limit.5,19 In an effort to not depend on natural pores (which have a limited range of sizes and chemistries), Li and others demonstrated that an argon ion beam could drill nanometer-scale holes in ultrathin silicon nitride membranes117−122 or other solid-state materials.123 Early work showed that the interactions between analytes of interest (e.g., DNA) were relatively weak, which meant that only long strands of duplex DNA could be detected.121,124 In addition, solid-state nanopores exhibited 2 orders of magnitude more current noise than their biological counterparts.125−127 However, adding a layer of polydimethylsiloxane to the surface outside the pore reduced the excess noise by 10-fold.125 Several methods were developed to detect relatively small molecules with solid-state nanopores.117,128 Dekker and colleagues demonstrated that the recombination protein A bound to lambda DNA (ca. 48,000 base pairs) gave rise to deeper current blockades in 20 nm diameter silicon nitride nanopores.129 Wanunu and colleagues showed that the binding of low molecular mass intercalators (e.g., ethidium bromide) to 400 base pair duplex DNA altered the ionic current blockade depth in a 3 nm diameter nanopore.130 Because native solid-state nanopores have no significant interactions with single-stranded or duplex DNA, the prospects for identifying short nucleic acids or other small molecules seemed doubtful. However, several recent experimental findings dispelled that concern. In one instance, small solid-state pores stalled the translocation of a bent short duplex RNA motif from the hepatitis C virus. The interaction of an antiviral RNA drug straightened the duplex RNA and allowed it to translocate through the pore ca. 100-fold faster.131 In another example, Hall and colleagues demonstrated that neither streptavidin nor 90 base pair duplex DNA caused ionic current blockades in an 8 nm diameter solid-state nanopore (Figures 7A and B, left and center, respectively).132 However, when the protein was bound to a single biotinylation site on the DNA, voltage dependent current blockades were readily detected (Figure 7B, right). Most importantly, the method was further developed to enable the sequence-specific recognition of microRNAs and other short nucleic acids.133 Chemically Modified Nanopores. The relatively slow development of solid-state nanopore technology can be attributed in part to the difficulty in controlling the geometry and chemistry to the level of precision offered by proteinaceous pores. Three critical problems with this technology had to be solved: eliminate fouling that severely limits how long a pore can be used, increase the pore’s aqueous wettability, and increasing the residence times for molecules in the pore. These issues were addressed in part by modifying solid-state nanopores with lipid bilayer membranes (Figure 8A).135,136 Specifically, the lipid is fused to the hydrophobic support structure and the bilayer structure retains the fluidity of a bilayer membrane.64 Apparently, biorecognition sites anchored to the membrane surface slow the translocation rate of target protein analytes through the pore. In addition, controlling the geometry of attachment allows the distribution of current blockade depths to help estimate the hydrodynamic radius of the target proteins (Figure 8B−D).135 Furthermore, by altering

Figure 7. Potential for biomarker detection with a solid-state nanopore via the specific interaction of a protein with DNA synthesized with a recognition element. (A) Schematic of the experiment. Monomeric streptavidin (MS) binds to biotin, b, on the DNA giving rise to ionic current blockades as the complex is driven through a solid-state pore. (B) Ionic current time series for MS, biotinylated DNA, and the MS:DNA complex. Reproduced from ref 134 with permission.

the nature of the linker (e.g., its flexibility), a nanopore ionic conductance measurement can be used to estimate the electrostatic shape of the protein by modeling the tumbling dynamics of the protein dipole and its effect on the electric field (and thus the ionic flux) through the pore.136 Analyte Residence Time in a Nanopore Altered by Another Molecule. The residence time of an analyte in the nanopore is influenced by several factors including interactions of the molecule with the pore wall,25,63,66 the magnitude of the applied potential,39 and molecules (either free to enter and leave the pore or that are bound to the pore) that reversibly bind to the analyte.25,137−141 The residence time can also be altered by another molecule that does not bind to the analyte, as was shown for the metallic nanocluster-enhanced increase in PEG residence time in the αHL pore (Figure 9).30,142 Negatively charged gold nanoclusters stabilized with glutathione (Au25SG18)143 and PEG weakly bound with cations25 were added to the cis and trans sides of the phospholipid membrane, respectively. A negative applied electrostatic potential (with respect to the trans side) forces both analytes into the pore. In the absence of Au (Figure 9A, left), PEG causes transient blockades, as described above. When Au is present (Figure 9A, right), the mean PEG-induced current blockade durations are more than an order of magnitude longer than the Au-free case (not shown). While the exact mechanism for this effect is unknown, this method leads to ca. 3-fold improvement in the resolution of the single molecule nanopore spectroscopy (Figure 9B). Mathematical Methods and Algorithms for Small Molecule Characterization. As described above, several molecular- and chemical-based methods were developed to F


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Figure 9. Metallic clusters enhance the ability of nanopores to detect oppositely charged polymers. (A) PEG causes deep, short blockades under an applied electric field (left), and glutathione-protected gold clusters (Au25SG18,) cause the polymer-induced blockades to become longer lived (right). (B) Nanopore spectroscopy of a polydisperse PEG sample with (orange) and without (blue) Au25SG18 in the pore and 60 mV applied potential. The tallest peaks in the spectra were due to a spike calibration with monodisperse PEG. Adapted with permission from refs 30 and 142.

A molecule that enters a nanopore instantaneously changes the latter’s resistance. Because it takes a finite time to equilibrate the charge on the capacitance elements, the ionic current relaxes to a steady state value with a measurable time constant. As a result, it is not possible to directly observe the steady-state blockade level for events that last less than approximately five times the characteristic charging and discharging times. Fortunately, the use of a simple lumped electrical circuit model (Figure 10, left) permits the extraction of information about the analytes from relatively short-lived blockades, even from those that do not reach their steady-state values.144,145 Specifically, the observed transient response can be calculated analytically using an equivalent circuit model, which accounts for the bilayer capacitance (Cm), the change in the pore resistance (ΔRp), and other resistive elements (the series resistance, Rs and the open pore resistance, Rp) in the system.

Figure 8. Modified solid-state nanopore for protein detection. (A) A lipid-coated nanopore with a bound receptor is used to orient a protein analyte for detection with ionic conductance measurements. The magnitude of the current blockade for streptavidin (B), antibiotin Fab fragments (C), and antibiotin antibodies (D) demonstrates effects from both volume (V) and shape factor (γ) of the proteins. Reproduced from ref 135 with permission.

increase the residence time of individual analytes in the pore. More recently, a theoretical analysis of the analyte-induced ionic current blockades, which we discuss below, markedly increased the amount of data that could be used to detect and characterize target molecules. G


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Figure 10. Analytical method for making use of unresolved events in nanopore-based sensing. (Left) The electrical circuit model that assumes the partitioning of a molecule into the pore increases the pore resistance by ΔRp. (Center) Conventional threshold-based analysis only computes the mean value of the steady-state segment in the blockade. (Right) Applying a time-dependent solution to the circuit model for the reversible partitioning of molecules into the pore (ADEPT) provides an accurate estimate of the steady-state current value for events that do not reach the steady-state.144,145

changes as a function of pH. That result was confirmed with nanopores (Figure 11), but with 70-fold lower nanoparticle concentration due to the better sensitivity of the nanopore method.32

The system response to a step change in the pore resistance caused by the entry of a single molecule is V i0 = R +a R , where i(t ) = i0 − β(1 − e−t / τ ), p

β=

Va ΔR p (R p + R s + ΔR p)(R p + R s)

s

is the amount by which the ionic

current decreases in the steady state, and τ =

CmR s(R p + ΔR p) R p + R s + ΔR p

is

the characteristic time constant of the response. By fitting the transient current blockade data with these equations, the projected steady-state values can be estimated. Because the relatively short-lived events are generally far more numerous in the exponentially distributed residence time blockades, this method provides more useful data per unit time, which improves the limit of detection of smaller molecules that otherwise would not be characterized. This approach has been integrated into an open-source analytical software toolkit for single molecule analysis.145

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Figure 11. Nanopore-based characterization of metallic nanoparticles. At pH 5.5 (blue) and pH 7.5 (orange), two principal species of 12phosphotungstic acid were detected by the αHL channel, and the relative amount of each species depended on the pH. The applied potential was 120 mV.32 Adapted from ref 32 with permission.

CONCLUSIONS, STATE-OF-THE-ART, AND CURRENT LIMITATIONS OF THE METHOD Despite the success of nanopore-based spectroscopy in the detection, characterization, and discrimination of a wide range of molecules,5,21 there are still several points that need to be better understood to more fully exploit the technique. For example, although a detailed physical-chemical theory was developed to describe the ionic current blockade depth and event duration,25 that model does not address the method’s resolution (and does not yet account for results obtained with channels other than the one formed by αHL). Below are two examples of how well a nanopore can discriminate between different physical particles, for reasons that are not yet clear. Metallic nanoclusters are typically analyzed with bulk analytical methods (e.g., analytical ultracentrifugation, NMR, and dynamic light scattering).146 Single molecule analysis using nanopores allows the characterization of metallic clusters on an individual basis.29,30,147 In principle, for each cluster type, a characteristic range of current blockades and dwell times can be observed. More recently, pH-dependent structural changes in metallic nanoclusters (i.e., discrete metal oxygen clusters known as polyoxometalates) in solution were monitored with an αHL nanopore. 31P NMR measurements showed that the relative concentrations of the two-principal nanoparticle species ([PW11O39]7− and another species (presumably [P2W5O23]6−)

More surprising was the ability of the nanopore to discriminate between structural isomers of metallic nanoclusters (Figure 12). The removal of three adjacent WO6 fragments from the phosphotungstic acid structure by hydrolytic cleavage of the metal−oxygen bonds results in a trivacant lacunary anion (i.e., ions with fragments missing) [PW9O34]9−, which leaves the central PO4 tetrahedron exposed either at its base (A-type) or apex (B-type) (Figure 12A). IR spectroscopy confirmed the separate syntheses of the A- and B-type isomers (Figure 12B). When added to an aqueous solution, the pure A-type species partially converts to the B-type, and vice versa, which was confirmed using the αHL nanopore (Figure 12C). Specifically, the predominately A- and B-type isomers are easily separated in terms of their ionic current blockade depths (Figure 12C) and the residence time distributions (Figure 12D). The orientation of the exposed phosphorus,148 and therefore the reactivities,148−150 of the two species differ. Conceivably, the latter might cause the different residence time distributions of the two species in the pore (Figure 12D). However, it is not yet clear what causes the markedly different blockade depths (Figure 12C). H


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ACS Sensors

document in order to describe an experimental procedure or concept adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the entities, materials, or equipment are necessarily the best available for the purpose.

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Figure 12. Nanopore-based detection and characterization of metallic nanocluster isomers. (A) The polyoxometalate Keggin ion [PW12O40]12‑ can be structurally altered to one of two isomeric forms. (B) The two isomer samples are readily distinguished with IR spectroscopy. (C) In aqueous solution, the highly purified isomers partially revert to the other isoform. A histogram of the relative current blockade ratio shows that a single αHL ion channel can easily discriminate between the two isomers. (D) The residence time distributions of the two isomers are also markedly different. Reproduced from ref 32 with permission.

These uncertainties notwithstanding, nanometer-scaled pores have proven to be valuable tools for the detection and characterization of a wide range of molecules. In addition to providing the basis for sequencing long strands of DNA, the method has demonstrated better accuracy and precision in the separation of molecules based on size than gel electrophoresis. In addition, the method works at the single molecule limit and can often work without the use of labels.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Joseph W. F. Robertson: 0000-0002-2087-3939 Joseph E. Reiner: 0000-0002-1056-8703 John J. Kasianowicz: 0000-0001-5106-7326 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Institutes of Health NHGRI (Grant R01 (HG007415), a European Molecular Biology Organization Fellowship (to J.E.), the National Science Foundation of China (Grants 61427806, 61372006), Shenzhen Science Plan (Grant JSGG20130411160504896), Shenzhen Dedicated Funding of Strategic Emerging Industry Development Program (Grant JCYJ20130408173226864), and China Postdoctoral Science Foundation (Grant 2014 M560670). Certain commercial entities, equipment, or materials may be identified in this I


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