Selective Multidetection Using Nanopores - Analytical Chemistry (ACS

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Selective Multi-detection Using Nanopores Masateru Taniguchi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504186m • Publication Date (Web): 11 Nov 2014 Downloaded from http://pubs.acs.org on November 17, 2014

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Analytical Chemistry

Selective Multi-detection Using Nanopores Masateru Taniguchi The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047 Japan

Recent developments in nanopore devices are re-examined from the perspective of their selectivity for detecting single or multiple analytes. Although nanopore devices have been developed for label-free, high-throughput, and low-cost DNA sequencers, the use of these devices has been widely expanded to several other applications, because some nanopore devices, unlike biosensors that perform molecular recognition via intermolecular interactions, can perform selective multidetection using single-molecule measurements. Nanopore devices can be classified into three types: bionanopores, solid-state nanopores, and nanogap nanopores. Bionanopores and solid-state nanopores can detect selective translocation events of single biomolecules by detecting variations in ionic currents. Furthermore, bionanopores have been successfully employed for identifying single base molecules of DNAs. On the other hand, nanogap nanopores can identify single base molecules of DNAs, RNAs, and peptides by detecting tunneling currents generated between nanogap electrodes. The Human Genome Project provided a complete genetic road map composed of 3 billion chemical base pairs that constitute human DNA. Many expected that the end of the Human Genome Project meant the dawn of personalized medicine and therapeutics based on genomic information. However, the throughputs and costs associated with DNA sequencing are barriers for the realization of personalized medicine and therapeutics.1-5 First- and second-generation DNA sequencing technologies identify base molecules via light emission. They require polymerase chain reaction (PCR) techniques for amplification of sequencing templates such that sufficient material is available for generating detectable signals. In addition, firstand second-generation DNA sequencing technologies require fluorescent labels. In contrast, third-generation DNA sequencing technologies directly detect single base molecules based on changes in electric currents, such that neither PCR amplification nor fluorescent probes are necessary.4,5 Determining the complete human genome using first-generation technologies takes 3 months and costs approximately $10 million, while employing second-generation technologies takes 2 months and costs approximately $0.1 million. Such long analysis times and exorbitant costs are not acceptable for practical applications. However, the sequence of a complete human genome can be determined in 1 day for $1000 using third-generation technologies, which rely on nanopores. The use of nanopores is therefore expected to result in a technical leap in DNA sequencing technologies.2,6-10 Nanopores can be classified as three types: bionanopores, solid-state nanopores, and nanogap nanopores (Figure 1 and Table 1).2 Although the materials used to fabricate detection

probes based on these three types of nanopores differ, they all target common biopolymer analytes carrying genomic information within biological systems.

Figure 1. Schematic diagrams and typical current-time profiles of a (a) bionanopore, (b) solid-state nanopore, and (c) nanogap nanopore when an ssDNA molecule passes through each nanopore due to electrophoresis generated between a pair of electrodes. The electric currents are characterized by their maximum currents Ip and current durations td. In (a), a channel protein is assembled on a cell membrane. When a molecule enters the nanopore, the ion current decreases as the molecular volume increases. In (b), a typical nanopore is formed on an SiN membrane. Unlike bionanopores, no single-molecule resolution has been obtained because there is no molecular recognition capability. In (c), nanogap electrodes are formed on a solid-state nanopore or nanochannel. Single molecules can be identified via tunneling currents generated between the nanoelectrodes.

Bionanopore devices identify base molecules passing through a nanopore by detecting changes in the ionic current flowing parallel to the nanopore when a voltage is applied across the membrane (Figure 1a). Negatively charged singlestranded DNA (ssDNA) molecules flow downward through the nanopore due to electrophoresis. When no molecule passes through the nanopore, a large ionic current is able to flow through it. When a molecule enters the nanopore, the ionic current decreases as the molecular volume increases. Bionanopores can read DNA sequences because the devices can identify small differences in the molecular volume as a function of changes in the ionic current. However, the mechanical durability and strength of these bionanopores are very low, because channel proteins and cell membranes are very fragile. Solid-state nanopores have been developed as an alternative approach for overcoming these issues (Figure 1b).2,6,10 Solidstate nanopores have the same configuration as bionanopores,

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but with greater durability. However, unlike bionanopores, solid-state nanopores do not identify the base molecules of DNA by detecting changes in the ionic current. This critical difference between bionanopores and solid-state nanopores is due to the differences in their molecular recognition abilities. Bionanopores have the ability to recognize base molecules via the detection of small differences in the molecular volume. The fixed diameter of solid-state nanopores prevents the identification of small differences in the molecular volume using ionic currents. Thus, to leverage the mechanical durability and strength of solid-state nanopores, nanogap nanopore devices have been developed. A nanogap nanopore is formed from a pair of nanoelectrodes and a solid-state nanopore (Figure 1c).2,10 This device can identify a single base molecule of DNA by measuring the tunneling current generated between the nanoelectrodes, not the ionic current, because each of the four base molecules has a different conductance.11-15 Table 1. Characteristics of bionanopores, solid-state nanopores, and nanogap nanopores. Bionanopore

Solid-state nanopore

Nanogap nanopores

Detection probe

Ionic current

Ionic current

Tunneling currents

Analytes

dsDNA ssDNA RNA Protein

dsDNA ssDNA RNA Protein

ssDNA ssRNA Peptide

Spatial resolution

Single base molecule dsDNA ssDNA RNA Single protein

dsDNA ssDNA RNA Single protein

Single base molecule Single amino acid molecule

Speed control method

Chemical modified nanopore Electrophoresis Temperature gradient Salts gradient

Chemical modified nanopore Electrophoresis Temperature gradient Salts gradient

Electrophoresis Electroosmosis Temperature gradient Salts gradient

ds and ss indicate double- and single-stranded, respectively.

Nanopore devices are advantageous compared with biosensors using antigen-antibody reactions and DNA hybridization because they offer the possibility of performing multidetection with selectivity.16-21 As a result, nanopore devices have attracted significant attention. DNA chips can detect target DNA molecules via optical or electrochemical signals generated via hybridization of probe and target ssDNA molecules using glass chips modified with a complementary chain of the probe ssDNA molecule. This detection mechanism is based on molecular recognition, with double-stranded DNA (dsDNA) molecules formed via hydrogen bonds, and has very high selectivity for detection of the target DNA. However, using the same probe DNA molecules, it is difficult to detect target DNA molecules with non-complementary sequences of base molecules with high precision. This situation is also true for biosensors utilizing molecular recognition via molecular interactions. Such biosensors are designed to detect a single

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analyte with high precision based on molecular interactions between a probe and the target molecule. They digitally detect whether the analyte exists using optical or electrical signals generated by the probe molecule. Therefore, most biosensors using molecular recognition do not measure the physical properties of analytes. Bionanopore devices allow selective translocation of DNA, RNA22-28, and protein29-32 molecules based on the molecular recognition ability of channel proteins and can identify molecules by measuring the changes in ionic currents. In addition, bionanopore devices can be used to perform multidetection, because the changes in ionic currents indicate the different volumes of single molecules passing through the nanopores. When used as DNA sequencers, the four base molecules of DNA can be identified.33 Thus, bionanopore devices have the ability to detect both selective translocation and multiple analytes using the molecular recognition and ionic current blockade capabilities of the channel protein, respectively. In other words, bionanopores realize selective multi-detection. It should be noted that with respect to identifying biopolymers, this distinguished selectivity of channel proteins may be limited to the detection of DNA. However, recent reports have suggested that bionanopore devices based on channel proteins may also be useful for the identification of RNA22-28 and other protein29-32 molecules. Solid-state nanopore devices have the same device configuration as bionanopore devices, but have no molecular recognition ability. It is very difficult for nanodevices to identify different molecular types with similar molecular volumes because they operate by detecting changes in ionic currents, which only indicate the molecular volumes passing through the nanopores. Until recently, solid-state nanopores were thought to have higher selectivities for analytes because they allow substances with diameters less than those of the nanopores to pass through. However, recent studies revealed that changes in ionic currents are detected by very thin solid-state nanopores (small thickness to diameter ratio) when substances with diameters greater than that of the nanopores approach the pore entrance.34-38 This finding implies that changes in the ionic currents do not directly correspond to the molecular volumes passing through the pores. On the other hand, unlike bionanopores, solid-state nanopores are advantageous for the detection of substances, such as single molecules and aggregates,39-41 passing through them because they do not undergo selective translocation. Therefore, solid-state nanopores can perform multi-detection, but with low selectivity. However, recent studies have shown that modification of nanopore surfaces with recognition molecules imparts molecular recognition capabilities to solid-state nanopores.42-44 Nanogap nanopores are solid-state nanopores with the added capability of detecting specific molecular electronic structures. Because tunneling currents conducted via single molecules located between nanogap electrodes can be determined using highest occupied molecular orbital (HOMO) or lowest unoccupied molecular orbital (LUMO) energy levels and the electronic interactions between the single molecules and the electrodes, these nanodevices can perform multi-detection of molecules with different electronic structures.45-51 On the other hand, their selectivity for molecules with similar electronic structures is low. However, when the electrodes in these devices are modified with recognition molecules for specific analytes, the selectivity of nanogap nanopores for the detec-


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tion of specific analytes is very high, but limited to those analytes. One approach to realizing multi-detection with high precision without the need for such modification is the introduction of multiple parameters, such as the simultaneous measurement of ionic and tunneling currents. 52 53 54 In this manner, researchers are attempting to develop nanopore devices capable of detecting many types of analytes using the same device platform that go beyond the operating principle of biosensors based on molecular recognition. The ideal goal is the development of nanopore devices with selective multi-detection, and the detection of many types of analytes with high precision is a key issue for realizing this goal. In fact, the US National Institutes of Health (NIH) launched a $1,000 genome sequencing project with the goal of achieving a label-free, low cost, and high-throughput DNA sequencer for use in personalized medicine and therapeutics based on genomic information. The target device was a nanogap nanopore device. Herein, considering this goal of selective multi-detection, developments in nanopore devices over the past two years are reviewed. Advances in nanotechnology have enabled the development of nanogap nanopores as improved solid-state nanopores, which were initially created to address mechanical durability issues associated with bionanopores. These nanopore devices have been developed using different materials,10 e.g., channel proteins for bionanopores32,55-57, and graphene53,58-79 and silicon semiconductor materials80 for solidstate and nanogap nanopores. Emerging devices based on electric current changes, and not on tunneling currents, have also been proposed.53,54 In addition, from the viewpoint of the practical application of nanopore devices, the possible mechanisms for controlling the speed of single molecules passing through nanopores,8,81 which is a key issue for obtaining high accuracy and throughput, are briefly reviewed. BIONANOPORES Bionanopores are composed of channel proteins and cell membranes (Figure 1a and Table 2). The most famous channel protein used in bionanopores is α-hemolysin, which has a nanopore diameter of 1.4 nm and a molecular height of approximately 10 nm (Figure 2). It took 20 years to achieve the identification of single base molecules in DNA after the first report of a DNA sequencer based on bionanopores.82 The channel protein α-hemolysin was first used to identify homopolymers, such as poly(deoxyadenylic acid) (polydA) and poly(deoxycytidylic acid) (polydC), using ionic current blockades,83,84 but a breakthrough was required for realization of single-molecule spatial resolution. The biggest challenge was the development of a mechanism for sufficiently reducing the translocation speed of ssDNA molecules in order to obtain single-molecule resolution using ionic current measurements.8 This barrier was conquered by modifying the inner nanopore surface with a cyclodextrin that had a smaller diameter than that of the nanopore.33 Using this strategy, sequencing of DNA has been realized with α-hemolysin and a complex of Mycobacterium smegmatis porin A (MspA) with Phi29 DNA polymerase,85,86 which denatures dsDNA to form ssDNA and makes the ssDNA flow slowly into the nanopore. Research based on bionanopores is divided into two streams: a search for channel proteins with the same selective translocation capacity as α-hemolysin and the development of

multi-detection using α-hemolysin. In both cases, the detection target is other biopolymers in addition to DNA, specifically proteins. Cytolysin A (ClyA), which has a minimum pore diameter of 3.3 nm and thus is larger than that of B-form dsDNA, has been shown to pass both ssDNA and dsDNA selectively, but sequencing of DNA has not yet been realized55. In a nanopore device based on aerolysin, which has a minimum diameter of 1 nm, the selective translocation of oligosaccharides (glycosaminoglycans) has been demonstrated for identification of saccharides with the different degrees of polymerization using an ionic current blockade and the dwell time.87 In addition, using this channel protein, selective translocation and detection of the thermal unfolding of proteins with an ionic current blockade have been explored, but the identification of proteins has not yet been achieved.32 Selective translocation of peptides has also been explored using the Nocardia farcinica channel, which has a minimum pore diameter of 2 nm or less, and it was revealed that a peptide molecule (oligoarginine) adheres to the nanopore entrance when the electrophoresis voltage is small, while it can be translocated upon the application of a large electrophoresis voltage.57 Therefore, the effort to identify biopolymers using new channel proteins is underway, and the development of new detection devices based on channel proteins with selective translocation abilities is in the initial stages.

Figure 2. Schematic figures of α-hemolysin. The smallest diameter of the nanopore and the molecular height are 1.4 nm and 10 nm, respectively. The channel proteins α-hemolysin and MspA can identify single base molecules in DNA molecules, while other channel proteins can only identify single biopolymers due to changes in ionic currents flowing through the nanopores.

Meanwhile, α-hemolysin and MspA are currently being used to develop multi-detection devices. In nanopore devices based on α-hemolysin, the detection of an unfolded protein has been performed using an enzyme,30,31 and this research has progressed, with the identification of other biopolymers in addition to DNA molecules. Specifically, for the direct sequencing of RNA using α-hemolysin, a study was performed in a manner similar to that used for the direct sequencing of DNA.33 When using homopolymers of ssRNA, previous research revealed that the four base molecules could be identified using an ionic current blockade, but that single-molecule resolution could not be achieved because the translocation speed was too high.83 As with the development of the direct DNA sequencer, the four base molecules could be identified due to changes in the ionic currents by fixing the base mole-



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cules of RNA in the interior of the nanopore using an enzyme.26 To achieve the identification of single base molecules of RNA via ionic currents, it was necessary to combine an enzyme (PNPase cuts RNA one base molecule at a time) with the head of ssRNA and modify the α-hemolysin with aminocyclodextrin adapters in order to enable translocation of the single base molecules of the one-molecule units.25 Therefore, in the future, direct RNA sequencers are expected to function like DNA sequencers. Separately, the detection of miRNA by hybridizing target miRNA with programmable oligonucleotide probes or polycationic peptide-PNA probes, and not by engineering α-hemolysin, is being investigated.27 In this device, the probe is first selectively translocated into a nanopore, and then miRNA is pulled by the probe and dragged into the nanopore. This method is still in the initial stages, and researchers are studying the selective translocation of miRNA. Table 2. Characteristics of bionanopores Material

Smallest diameter

Analytes

Spatial resolution

Ref .

αHemolysin

1.5 nm

ssDNA ssRNA microRNA Protein

Single base molecules Single RNA Single microRNA Single protein

33 2426 22, 27, 28 30, 31

MspA

1.2 nm

ssDNA 5 mC 5hmC

Single base molecule Single base molecule Single base molecule

85 86 86

Aerolysin

1.0 nm

Protein Olygosaccharide

Single protein Single olygosaccharide

32 87

N. farcinica channel

~ 2.0 nm

Peptide

Single peptide

57

ClyA

3.3 nm

ssDNA dsDNA

Single DNA Single DNA

55 55


Beyond the development of direct DNA and RNA sequencers, the detection of proteins has been attempted using αhemolysin. A significant challenge for detecting proteins is the need for them to unfold and then be dragged into the nanopores. First, in an effort to confirm selective translocation of a protein, an enzyme (ClpX) was used to unfold proteins (S1, S2-35, and S2-148).31 In this case, detection of an ionic current blockade confirmed that the unfolded protein was selectively translocated. In another study, a protein (V5-C109 thioredoxin) was unfolded by placing an oligo-(dC)30 tag on it, with the force of the tag dragging the protein into the nanopore.30 When using this method, cerin and phosphocerin groups in the protein were identified via two-dimensional mapping of the ionic current and the electric current noise, but the resolution re-

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quired for one amino acid molecule has not yet been obtained.29 Thus, in order to detect single molecules with small differences in their molecular volumes, parameters in addition to the ionic current are necessary, and multidimensionalization of the detection parameters may be required for selective multi-detection. Based on reports regarding single-base molecule resolution for DNA and RNA, the development of a method for slowly transporting unfolding proteins into nanopores is the key for realizing selective recognition of single molecules. SOLID-STATE NANOPORES Unlike bionanopores, solid-state nanopores do not have selective translocation capacity and are therefore used for the multi-detection of other biopolymers in addition to DNA and the real-time monitoring of vital functions88,89 (Figure 3 and Table 3). For biomolecule detection using SiN-based solidstate nanopores, the detection of histone and nucleosome,40 a combination of RNA and a drug molecule (benzimidazole derivatives), a conformation of an antiviral RNA drug target,90 and a combination of a γ-modified synthetic peptide nucleic acid probe and dsDNA89 have been achieved using ionic current blockades. In addition to detecting full biomolecules, significant progress has been made in tracing individual transcription events in real time and the conformational changes of RNA polymerase (RNAP) during the transcription process, during which the ionic current blockade is measured when a combination of DNA and RNAP is trapped at the nanopore entrance.88 In the same manner, the process of transfection via electroporation can be observed using an ionic current blockade when a single cell is trapped near the nanopore entrance.91 Solid-state nanopores are therefore used as a platform for tracing the single-molecule dynamics of vital functions.

Figure 3. Schematic diagrams of typical solid-state nanopores formed using (a) SiN and (b) graphene membranes. The graphene nanopore is supported on a SiN membrane. The translocation events of biopolymers can be detected using ionic currents flowing through the nanopores, but the single molecules in the biopolymers cannot be identified.

The development of solid-state nanopores that can both detect many types of molecules and identify single molecules has also been investigated. Achieving sufficient spatial resolution for the identification of a single base molecule is not possible with SiN-based solid-state nanopores, because the SiN membranes are at least 30-nm-thick.6 Given that the ionic current blockade is determined by the access resistance and a quantity that is proportional to the volume of the molecule present in the nanopore, thin-film materials are required in order to establish the limit of spatial resolution for solid-state nanopores. Accordingly, solid-state nanopores were fabricated using HfO2, which is stronger than SiN and has a better chemical resistance.92 The film thickness of the HfO2 in these solid-


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state nanopores was 2–7 nm, and the translocation of ssDNA and dsDNA was observed using ionic current blockades, but the spatial resolution needed for a single base molecule was not achieved. The spatial resolution obtained from the ionic current blockades for single and several-layer graphene-based solid-state nanopores has also been explored, but only translational events for single dsDNA molecules were observed, and single-molecule resolution was not demonstrated again.67,76 To overcome hydrophobic interactions between DNA and the surface of graphene, solid-state nanopores fabricated using chemically modified graphene,93 MoS2 (6.5 Å-thick layers),94 and DNA Origami95-98 were studied; they too were found to lack single-base molecular resolution. The difficulty is associated with the diameter and thickness of the nanopores. When either value is greater than 1 nm, the access resistance mainly contributes to the ionic current blockade. 34-38 Therefore, for the measurement of biopolymers such as DNA and RNA, the contribution of the access resistance of the base molecule that is present outside of the nanopores increases, and it is difficult to detect the changes in the volume of a single base molecule passing through the nanopores using the ionic current blockade. However, considering the development history of bionanopores, single base molecule resolution may be achieved for solid-state nanopores if the translocation speed of the single molecules can be sufficiently decreased such that small changes in the ionic currents can be measured with high precision. Though, it should be noted that if single base molecule resolution is not achieved for solid-state nanopores using controlled translocation speeds, the possibility that the single base molecule resolution observed for bionanopores may originate from their single molecule recognition ability. The most direct method for imparting molecular recognition capacity, which makes selective translocation possible, to solid-state nanopores is modification of the nanopore surfaces with recognition molecules. SiN-based solid-state nanopores covalently tethered on their surfaces to nucleoporin 98 kDA (Nup98) or nucleoporin 153 kDa (Nup153) exhibited selective transportation of bovine serum albumin (BSA) and importin beta (Impβ).42 After modification of 40-nm-diameter solidstate nanopores with Nup98 and Nup153, the diameters were reduced to 10 nm and 25 nm, respectively. Interestingly, the ion current blockade was not affected by the surface modification, but differences were observed in the electric current duration. Both proteins passed through the unmodified nanopores at the same frequency, while only Impβ passed through the nanopores modified by Nup98, and only a few BSA molecules passed through the nanopores modified with Nup153. In a second example, the surfaces of SiN-based solid-state nanopores were covered with gold and a self-assembled monolayer was formed on the gold surface.43 The nitrilotriacetic acid (NTA) receptor was then embedded in the self-assembled monolayer via gold-thiol bonds. This modified solid-state nanopore was shown to detect histidine-tag (His-tag), which participates in intermolecular interactions with the Ni atoms in the NTA. Moreover, when the gold surface was covered with Ni-NTA bonded to His-tag, the nanopore detected immunoglobulin G (IgG) antibody. When selective detection is performed using solid-state nanopores modified with recognition molecules, the target analytes are identified not by the ionic current blockade, but by the electric current duration due to the small changes in the ionic current blockade. Unfortunately, this approach requires greater detection time. Specifically, because single-molecule detection in nanopores must be per-

formed in a certain time sequence, the throughput of this detection method is low. To increase the throughput, the number of nanopores should be increased via integration, which is a well-known technology in the semiconductor industry. Table 3. Characteristics of solid-state nanopores Smallest diameter

Material

Analytes

Ref.

3 nm 3.7 nm 3–20 nm

RNA + drug

90 89 88

SiN/SiO2/SiN

20 nm

Histone Nucleosome

40

HfO2/SiNx

< 2 nm (HfO2)

ssDNA dsDNA

91

Graphene/SiO2

22 nm (Monolayer graphene)

λ-DNA

73

Graphene/SiNx

5 nm (1–2 Layer graphene)

10kb-DNA

79

Al2O3/graphene/Al2O3

9 nm (graphene)

dsDNA dsDNA protein

76

TiO2/graphene/SiN/SiO2

8nm (3–15 Layer graphene)

15kb-DNA

67

Chemical modified graphene

5–15 nm

ssDNA

92

MoS2/SiNx

5 nm (1 or fewlayer MoS2)

λ-DNA

93

DNA Origami/SiN

15 nm (DNA gami)

dsDNA

94

SiN

dsDNA + γPNA RNAP + DNA (Tracing gene expression transcription)

+

ori-

Chemical modified SiN

40 nm (SiN) 25 nm (Modified SiN)

Protein

42

Chemical Au/SiN

20–25 nm (Modified Au)

His-tagged protein IgG antibody

43

modified


The development of solid-state nanopores has also revealed a very important concept. When modifying a device that can detect many types of molecules with molecules having strong selectivity for a specific analyte due to strong intermolecular interactions, high selectivity is achieved, but the multidetection capacity is lost. As a result, both the accuracy of the information regarding the physical quantities of the analytes and the throughput per device decrease. However, it is possi-



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ble that the decrease in information about the physical quantity of a sample may be due to the limited ability of solid-state nanopores to detect single-molecule analytes. In fact, the diameters of reported modified solid-state nanopores were larger than the sizes of the analytes. Therefore, attempts have been made to detect single analyte molecules using recognition molecules placed in nanogap nanopores. NANOGAP NANOPORES The ideal structure of nanogap nanopores shown in Figure 1c has not yet been developed, but the operating principle of the device, which identifies single molecules via tunneling currents flowing between nanoelectrodes, has been proven using scanning tunneling microscopy (STM)14,15,74,99,100 and mechanically controllable break junctions (MCBJ) 11-13,101 (Figure 4 and Table 4). Nanogaps fabricated using an STM chip and a gold substrate modified with a type of recognition molecule (4-mercaptobenzaimde) have been shown to identify single base molecules of DNA.14 The nanogap used in this study was the same size as of the single base molecules, and only one type of recognition molecule was used to recognize the single base molecules via hydrogen bond formation. The number and strength of the hydrogen bonds between each of the adenosine (A), cytosine (C), guanine (G), and tyrosine (T) residues and the recognition molecule were different. In addition, for A, C, and G, an methylated cytosine (mC) was also identified along with the single base molecules. Moreover, single base molecules on ssDNA were identified, but singlemolecule sequencing of DNA has not been achieved. Furthermore, when the STM chip and the palladium substrate were modified with a different recognition molecule (1H-imidazole2-carboxamide), four types of amino acids, one type of methylated amino acids, and one type of enantiomer were identified using the different numbers and strengths of the hydrogen bonds formed between the amino acids and the recognition molecule.100 The amino acid molecules were identified by analyzing their tunneling currents using the theory of machinelearning. A series of these research results have shown that many types of molecules can be detected with high selectivity if various interactions between the analytes and the recognition molecule are possible. Unlike solid-state nanopores modified with recognition molecules, the physical quantity of a single molecule can be measured only when the single molecule can be detected. However, due to the variation in the hydrogen bonding (number and strength) between the analytes and the recognition molecule, selective multi-detection is possible. It should be noted that selective multi-detection becomes difficult when the variation in hydrogen bonding is less than the types of analytes to be detected. For example, for amino acids, 20 types of hydrogen bonding schemes are required for the detection of all 20 amino acids.

Figure 4. Schematic diagrams of nanogap nanopores. (a) Single molecules can be identified via tunneling currents using an STM chip on a metal substrate modified with recognition molecules that interact with analytes via hydrogen bonds. (b) MCBJ for

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identification of single molecules via tunneling currents. (c) While fixed nanogaps formed on a SiN membrane cannot identify single base molecules in DNA, they can detect translocation events of single DNA molecules using tunneling currents.

Single-molecule identification experiments have also been performed using nanogap electrodes without the ability for molecular recognition. Nanogaps with spacings of approximately 1 nm were formed using mechanically controllable break junctions in which a gold wire fabricated on an insulating substrate was fractured using the three-point bending configuration. Using this method, all of the base molecules of DNA13 and RNA11, methylated cytosine12, and oxo-guanine12 were identified via tunneling currents. In addition, singlemolecule sequencing of three base molecules of DNA and seven base molecules of RNA was achieved. Furthermore,11 12 types of amino acids and phosphotyrosine were identified, and partial sequencing of peptides was demonstrated.101 Among nanopore devices, only MCBJ-based nanogap nanopore devices can sequence DNA, RNA, and peptides at the single-molecule resolution and clearly demonstrate multidetection. While a gold electrode does not have the ability to recognize molecules, tunneling currents can specify the type of single base molecules because they measure the electronic states of single analytes. If molecular recognition is considered to be the ability to determine the presence of a specific molecule, tunneling currents have molecular recognition ability, i.e., they exhibit selectivity, and MCBJ-based nanogap nanopore devices realize selective multi-detection. Table 4. Characteristics of nanogap nanopores Device

Detection probe

Analytes

Spatial resolution

Ref.

Functionalized STM probe

Recognitiontunneling current (Usage of molecular recognition)

DNA Peptide

Single base molecules Single amino acid molecules

14 100

MCBJ

Tunneling current

DNA (3 bases) RNA (7 bases) Methylated cytosine Oxo-guanine Peptide Phosphotyrosine

Single base molecules

11,1 2 11 12

Single amino acid molecules

12 101 101

Si-based nanogap nanopore

Tunneling current

DNA

Single λdsDNA(48.5 kbp)

102

C-based nanogap nanopore

Tunneling current

DNA

Single λdsDNA(48.5 kbp)

103

C indicates carbon electrode.


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Demonstration of proof of concept for nanogap nanopores using STM and MCBJ is accelerating the development of nanopore devices with fixed nanogap electrodes that have spacings of several nm. The development of ideal nanogap nanopores is performed using platinum52,102,103 and carbon104 as electrode materials. For both types of nanogap nanopores, an increase in the electric current has been observed when λDNA passes through the nanogaps, but single base molecular identification due to tunneling currents has not been realized. It is possible that, as with bionanopores, the fast translocation speed of single DNA molecules passing through the nanogaps does not allow measurement of the electric current at the pA level. EMERGING NANOPORESE Emerging nanopore devices different from both solid-state and nanogap nanopores have been developed (Figure 5 and Table 5). The new devices formed from semiconducting materials have a structure similar to field-effect transistors (FETs), and the nanopores are fabricated on a channel between the source and the drain. FET-based nanopores can potentially create large electric currents by amplifying small changes in the potentials caused by DNA translocation. Thus these devices present one possible approach to overcoming the difficulty of rapidly measuring pA-level electric currents generated when single DNA molecules quickly pass through a nanopore. Nanopores with a diameter of 10 nm formed on Si nanowires with diameters of 30–50 nm can detect both changes in the ionic currents flowing through the nanopores and the channel conductances of the nanowires when dsDNA passes through the nanopores, but single base molecule resolution has not yet been achieved.54 Nanopores with a diameter of 10 nm fabricated on graphene nanoribbons also exhibit both ionic current blockade and changes in graphene conductance, but single base molecule resolution has again not been achieved to date.53

Figure 5. FET-based solid-state nanopores. Nanopores are formed on (a) a Si nanowire and (b) a graphene ribbon. The two devices can detect translocation events of single DNA molecules due to changes in the ionic currents and sourcedrain conductances, however, they cannot achieve singlemolecule resolution. Table 5. Characteristics of FET-based nanopores Device

Detection probe

Analytes

Spatial resolution

Ref.

Graphene ribbon device

Graphene current (Local electric potential)

DNA

Single (2,713 bp) dsDNA

53

Sinanowirebased FET

FET conductance (Local potential

DNA

Single (2,6 kbp) dsDNA

54

caused by changes in the solution resistance)

Notably, these FET-based devices show large electric current changes on the order of 100 nA, which is greater than approximately 1000 times as large as those obtained with existing nanopore devices, and allow high-speed electric current measurements. To date, single base molecule resolution has been achieved at a measurement speed of hundreds of kHz. With these emerging nanopore technologies, however, it may be possible to realize single base molecule resolution at electric current measurement speeds of more than 1 MHz.

SELECTIVITY AND MULTI-DETECTION Ideal sensors are expected to detect many types of analytes with high selectivity. The purpose is to quickly examine how much of a specific material exists in a complex mixture. For example, it is desirable to be able to determine whether or not a specific influenza virus is present in a blood sample. To evaluate various methods, consider the detection of GCAT in an aqueous solution containing GCAT and CGTA. If the CGTA is modified with a fluorescent probe, or electrochemically active probes are prepared, it is possible to selectively detect GCAT optically or electrochemically using molecular recognition due to intermolecular interactions (hydrogen bonds), while CGTA will not be detected. Selectivity involves the identification of a specific existing material, and intermolecular interactions play an important role. However, when using molecular recognition, because only the target molecule is selectively detected without measuring the physical properties of the analyte, the high selectivity clearly conflicts with multi-detection. Next consider using nanopore devices in this same scenario. Bionanopores can detect both GCAT and CGTA by selectively pushing them through the nanopores and measuring the corresponding changes in the ionic currents. Thus, these devices make selective multi-detection possible. Because solidstate nanopores have no ability to recognize these types of molecules, they cannot detect the small differences in the molecular volumes, and thus the two DNA oligomers would not be identified. On the other hand, nanogap nanopores can identify these two types of DNA oligomers because they have the capacity for single molecule identification and the sequencing of single DNA molecules. In other words, nanogap nanopores realize multi-detection by identifying single base molecules based on the measurement of the physical quantity specific to each single molecule. 45-51 Nanogap nanopores are therefore not just sensors, but measurement systems for the identification of molecular fingerprints, as is the case with nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. This ability is the critical difference between nanogap nanopores and sensors that rely on optical and electrochemical probes. Most importantly, nanogap nanopores perform multidetection without the selectivity provided by recognition molecules. However, when defining selectivity as the ability to identify an existing material, nanogap nanopores have selec-



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tivity because they can identify existing materials using a physical quantity (the tunneling current) specific to a single molecule. Therefore, in the current scenario with a mixture of GCAT and CGTA in order to determine whether GCAT is present, all of the molecules contained in the mixture must be evaluated. With a sensor that uses molecular recognition, however, the rapid evaluation of only the target analyte is an important feature of its selectivity. It is possible to analyze 1 M GCAT and CGTA in a realistic amount of time, but in the case of a 1aM solution, it is not impossible to perform the analysis in a realistic amount of time without preconcentration of the sample. This situation is expected to be true for all sensors based on molecular recognition. The advantages of nanogap nanopores with respect to detection time have not yet been established. The limits of detection with respect to solution concentrations must first be clarified. Bionanopores and nanogap nanopores have both selectivity and multi-detection capacity, but this discussion presupposes that high precision and throughput can be obtained for single molecule analyses based on these nanopores. One factor that determines the precision and throughput is the translocation speed of a single molecule flowing through the nanopores, and the development of methods for controlling single molecule fluid dynamics is therefore an important research target.8,81 CONTROLLING SINGLE MOLECULE FLUID DYNAMICS Detecting in nanopore devices involves the detection of picoampere-level ionic and tunneling currents, and their high precision is directly related to their ability to measure an electric current at a high signal-to-noise ratio. In order to improve the signal-to-noise ratio, it is necessary to reduce the translocation speed of the analytes. A key breakthrough in the development of bionanopores has therefore been the development of a method for obtaining sufficiently slow translocation speeds that allow single molecule resolution. This method involves the chemical modification of the nanopores. In all nanopore devices, an electrophoresis voltage is applied between the electrodes on both the cis and trans chambers in order to drag single molecules into the nanopores. The typical thickness of a nanopore membrane is less than 50 nm, and the diameters of nanopores are less than 10 nm. When an electrophoresis voltage of 0.1 V is applied, a voltage of approximately 0.1 V is applied to the nanopore with a thickness of 50 nm, because nanopores have a high electrical resistance. In other words, a very strong electric field of 0.1 V/50 nm = 20 KV/cm is applied to the nanopore. 6,73,79,105-108 Because the electrophoretic speed of negatively charged biomolecules is proportional to the strength of the electric field, single DNA molecules pass through nanopores at very fast speeds of 10−5 s/base.8,81 Because the strength of the electric field decreases as the applied voltage decreases, decreasing the electrophoretic voltage is expected to be one method for decreasing the translocation speed. However, because there are no technologies currently available for the generation of small voltages of 1 µV with the low voltage noises and ionic currents requires to decrease the detection limit, decreasing the electrophoretic voltage is not a practical method for reducing the translocation speed. An alternative approach involves measurement of the ionic and tunneling currents at much higher speeds than the translocation speed. However, the physical limit for measuring

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pA-level electric currents is controlled by the Johnson noise,109,110 which has a maximum speed of approximately 1 MHz. At present, the maximum measuring speed of all-round measuring instruments available on the market is 250 KHz. Therefore, to be able to measure electric currents with high precision and single-molecule resolution, it is necessary to develop a method for increasing the speed of current measurements and decreasing the translocation speed of single molecules. Studies focused on increasing the speed of electric current measurements have been performed using solid-state nanopores.107 A valid method for reducing the noise of a slight electric current is installing an electric current amplifier as close to the electrodes as possible. Solid-state nanopores integrated with CMOS (complementary metal-oxide semiconductor) preamplifiers exhibit signal-to-noise ratios above 5 at the 1 MHz bandwidth. This electric current measurement technology allows the detection of electric signals at 1 MHz. The development of a technology for controlling the translocation speed of single molecules has mainly been approached by considering the fluid dynamics in solid-state and nanogap nanopores (Figure 6). In bionanopores, a speedcontrol method has been developed based on the chemical modification of the nanopores and the use of enzymes. 30,31,33,85 The conditions under which a channel protein can perform its functions are, however, determined by the environment, including the temperature, ion concentration, and so on, and the margin for these environmental conditions is narrow. In addition, as mentioned above, because it takes significant time to detect analytes using molecular recognition based on intermolecular interactions, the throughput per bionanopore device is low.

Figure 6. Single-molecule speed control methods. A negatively charged ssDNA flows downward through nanopores due to electrophoresis. (a) In bionanopores, the translocation speed of a single DNA molecule can be changed using the electrophoresis voltages. (b) In solid-state and nanogap nanopores, the translocation speed of a single DNA molecule can be controlled using both the electrophoresis and gate voltages. When a negative gate voltage is applied to the gate electrode, cations accumulate on the nanopore surface, resulting in upward electroosmotic flow. (c) On the other hand, when a positive gate voltage is applied, anions are accumulated on the nanopore surface, resulting in downward electroosmotic flow. Thus, a negative gate voltage decreases the translocation speed, while a positive voltage increases it.

For solid-state and nanogap nanopores, there are passive and active methods for controlling the translocation speed. The passive methods use the viscosity,111 the temperature gradient,112-114 or the ion concentration gradient of the analyte solution.115-119 The viscosity of the solution is adjusted by adding glycerol, which has a higher viscosity than water.111 When


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using this method, a five-fold reduction in the translocation speed has been obtained. Temperature and ion concentration gradients exist on both sides of the membrane; when the ion concentration gradient was adjusted, a three-fold reduction in the translocation speed was obtained.114,117 It should also be noted that chemical modification of solid-state nanopores using molecules that can interact with DNA has resulted in translocations speeds 13–25 times slower than that observed without modification.120 Single-molecule speed control technologies for solid-state nanopores have also been developed from the viewpoint of fluid mechanics. In this case, the translocation speed is controlled using electrophoresis and electroosmotic flow.121-124 When negatively charged ssDNA molecules pass through an SiO2 nanopore with a gate electrode, and when negative and positive voltages are applied to the upper and the under sides of the membrane, respectively, the ssDNA molecules flow downward through the nanopore due to electrophoresis. It is well known that SiO2 has a surface charge of approximately −50 mC/m2.125,126 Thus, cations accumulate on the surface and then flow upward through the nanopore, resulting in an upward electroosmotic flow. In addition, when a negative voltage is applied to the gate electrode, more cations accumulate on the SiO2 surface, resulting in a stronger upward electroosmotic flow, which decreases the translocation speed of the DNA molecules. In contrast, when a positive voltage is applied to the gate electrode, anions accumulate on the SiO2 surface, thus resulting in a downward electroosmotic flow and an increase in the translocation speed of the DNA molecules. This speed control technology based on adjustment of the electroosmotic flow using the gate voltage offers a high level of control and has high compatibility with solid-state and nanogap nanopores, because the technology can be achieved by incorporating a gate electrode into the devices. Another speed control method of interest uses the difference in the pressures applied to both sides of the membrane. Using this method, an eight-fold reduction in the translocation speed was obtained.127 The fluid dynamics of single molecules within nanopores has not yet been fully clarified, and research studies designed to provide greater understanding of the influence of the ion concentration, temperature, electrophoresis, electroosmotic flow, and water pressure will be important in the future. Specifically, understanding the dynamics of single molecules passing through nanopores will provide important insights for improving the precision of single molecule identification using nanopores. OUTLOOK From the viewpoints of selectivity and multi-detection, we have reviewed the recent progress in nanopore technology with a focus on the detection of biomolecules. Some nanopore devices offer both selectivity (defined as the ability to detect a certain analyte) and multi-detection because they can identify a certain analyte or single molecules comprising a certain analyte. This ability stems from the capacity to measure molecular volumes and electronic structures specific to single molecules. Although selectivity conflicts with multi-detection in bulk measurements using recognition molecules, the coexistence of these two abilities is a remarkable feature of single-molecule science.

At present, only the measurement and analysis of electric currents and the duration of electric current are performed using nanopore devices, but further selective multi-detection is expected to become possible using other parameters, such as inductance (L), capacitance (C), and resistance (R) data obtained from single-molecule impedance measurements. The current operating principles of nanopores are mainly based on quantum chemistry and quantum mechanics, which explain the origins of electric currents reflecting physical properties specific to single molecules, while single-molecule analysis methods using multiple physical characteristics (for example, fluid dynamics, electromagnetism, and ion transport) have not yet been developed. Specifically, the combination of an electric current change and multiple physical parameters could make possible the development of a time-of-flight (TOF)-type single molecule mass spectrometer (Figure 7).128 This mass spectrometer may allow us to detect biologically active single biomolecules flowing in solution. In addition, considering that changes in ionic currents reflect the volumes of analytes passing through nanopores, even though nanopore devices can perform selective multi-detection, both nanopore and pretreatment devices for separation and extraction should be integrated on a single device chip for practical use, because analyzing all of the molecules in sample solutions at a singlemolecule resolution will decrease the accuracy and throughput.

Figure 7. Schematic diagram of a single-molecule mass spectrometer based on nanopore devices. Electric currents generated between the counter electrodes detect the translocation events of single molecules in solution. The time of flight can be calculated using the electric signals, while the translocation speed can be controlled using the gate voltage.

As clearly demonstrated in DNA sequencers, nanopores are one part of the analytical system trinity: device, measurement, analysis. All parts of the trinity must be developed simultaneously in order to obtain useful results. To date, however, device and measuring technique developments have been achieved. Therefore, in the near future the development of analytical methods will be very important, because a significant amount of data is produced by nanopore devices. However, it should be noted that the data produced by nanopore devices are the result of single molecule measurements, and it is necessary to determine whether the conventional statistical methods established for infinitely existing molecules can be applied to the analysis of single molecule data. For example, histograms constructed from approximately 1,000 single-molecule conductance values have been analyzed using a Gaussian function, but the application of the function has not been validated. In addition, the hidden Markov theo-



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ry,129 which is used to assign base sequences from the raw data generated by DNA sequencers, presupposes that each phenomenon is independent. However, in single-molecule measurements, each base molecule comprising DNA molecules is covalently bonded, and a change in the configuration and conformation of a single base molecule propagates through the neighborhood base molecules. Thus it is important to determine whether the hidden Markov model can be applied for the analysis of single-molecule data, because each phenomenon is not independent. In the initial studies of bionanopores, the dynamics of single DNA molecules passing through nanopores were studied from the viewpoint of the thermodynamics of the system formed by a nanopore and a DNA molecule.130 Reconsidering nanopores from the viewpoint of thermodynamics, it has been found that unstable biomolecules exist in nanopores, while the system formed by a nanopore and a biomolecule is stable. For example, unfolded proteins within nanopores are unstable entropically compared with folded proteins.131 In other words, while instability existing within the system, stability of the overall system is realized. As another example, when attempting to see the crystal structure of an enzyme from a chemical perspective, it is found that metal complexes exist in the enzymes and become active centers, but these metal complexes are not expected to exist in simple substances. Proteins exist in the neighborhood of metal complexes, and enzymes formed by proteins and metal complexes exist stably, resulting in the realization of instability within a stable system. Nanopore devices therefore potentially allow the study of single molecules that cannot exist as simple substances, but are stable as part of the nanopore system, as well as the observation of the nature of biologically active single molecules. A similar approach may be possible using metal organic frameworks (MOFs),132 but a critical difference between nanopore devices and MOFs is the ability of nanopore devices to evaluate single molecules and to measure the nature of only single molecules. Nanopore devices are therefore expected to become a platform for exploring biological functions and dynamics and the chemical reactions of biomolecules.

AUTHOR INFORMATION Biography Masateru Taniguchi obtained his Ph.D. in chemistry from Kyoto University in 2001. He then became a postdoc in the Institute of Scientific and Industrial Research in Osaka University. In 2002, he became an assistant professor in Osaka University. In 2007, he became a researcher of PRESTO (Precursory Research for Embryonic Science and Technology), Japan Science and Technology Agency. From 2008 to 2011, he was an associate professor at the Osaka University. Since 2011, he is a professor of Bionanotechnology, the Institute of Scientific and Industrial Research, Osaka University. His current activities involve the development of single-molecule analytical technologies for DNA, RNA, and protein sequences for genomic and proteomic applications toward disease diagnosis and genomic and personalized medicine.

ACKNOWLEDGMENT This research is partially supported by KAKENHI Grant No. 26220603.

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