Hadi
Arjmandi-Tash
,
Liubov A.
Belyaeva
and
Grégory F.
Schneider
*
Faculty of Science, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333CC Leiden, The Netherlands. E-mail: g.f.schneider@chem.leidenuniv.nl
First published on 27th November 2015
Graphene and other two dimensional (2D) materials are currently integrated into nanoscaled devices that may – one day – sequence genomes. The challenge to solve is conceptually straightforward: cut a sheet out of a 2D material and use the edge of the sheet to scan an unfolded biomolecule from head to tail. As the scan proceeds – and because 2D materials are atomically thin – the information provided by the edge might be used to identify different segments – ideally single nucleotides – in the biomolecular strand. So far, the most efficient approach was to drill a nano-sized pore in the sheet and use this pore as a channel to guide and detect individual molecules by measuring the electrochemical ionic current. Nanoscaled gaps between two electrodes in 2D materials recently emerged as powerful alternatives to nanopores. This article reviews the current status and prospects of integrating 2D materials in nanopores, nanogaps and similar devices for single molecule biosensing applications. We discuss the pros and cons, the challenges, and the latest achievements in the field. To achieve high-throughput sequencing with 2D materials, interdisciplinary research is essential.
Key learning points(1) [Mechanism] To learn the working principle of nanopores for biomolecule detection and sequencing.(2) [General] To learn the advantages and the challenges of the implementation of 2D materials in nanopore schemes. (3) [Mechanism] To learn about nanogap and the other proposals as the next generations of biomolecular sensors. |
Ionic channels, naturally occurring in biological nanopores e.g. α-hemolysin and MspA, embedded in a lipid bilayer membrane were the first type of the nanopores experimentally probed, with now the proof that sequence information can be obtained.1 Solid-state nanopores, however, are more stable alternatives.2 The tunability of their physical and chemical properties, and their compatibility for mass-production are significant advantages.2
The channel of a nanopore in a freestanding silicon-based membrane typically contains hundreds of bases simultaneously. Consequently such long channel nanopores are not capable of addressing and detecting single nucleotides composing a DNA strand. The integration of two-dimensional (2D) materials, especially graphene in nanopore systems, has drawn a lot of interest in recent years.3–6 As the thinnest possible materials with thicknesses comparable to the spacing between the nucleotides, monolayers of 2D materials promise very high spatial resolution, potentially eligible to DNA sequencing.7Fig. 1a compares the resolution achievable by nanopores in silicon-based materials (long channel nanopores) and nanopores in 2D materials (2D nanopores).
Fig. 1 Nanopores for DNA detection. (a) Schematic representation of DNA translocation through long channel nanopores (membrane thickness ≫ spacing between nucleotides) and 2D nanopores (membrane thickness ≈ spacing between nucleotides). (b) Schematic representation of a typical nanopore device: an electrostatic field applied between two Ag/AgCl electrodes immersed in respectively the cis and trans reservoirs drives electrophoretically the ions and DNA molecules through the nanopore. The top inset figure shows the distribution of the field lines close to the nanopore. The dashed lines, composing a cylinder and two hemispheres, estimate the effective nanopore volume. The bottom inset figure illustrates the architecture of a 2D nanopore.6 (c) Measured current in a graphene nanopore system in the absence and presence of dsDNA in the buffer solution. Adapted with permission from ref. 3. Copyright 2010 American Chemical Society. (d and e) Simulated ionic current (d) and the conformations of poly(A–T)20 and poly(G–C)20 duplexes (e) passing through a 2D nanopore of d = 2.4 nm in graphene; adapted with permission from ref. 15. Copyright 2011 American Chemical Society. |
Large, continuous and stable films of 2D materials can now be grown chemically to form free-standing membranes.8–11 Some of the 2D materials, particularly graphene, are electronically conductive with highly mobile and gate tunable charge carriers. Such properties open up new modalities for single molecule detection, e.g. by probing the doping effect of a translocating DNA molecule on the conductivity of graphene.12 Hexagonal boron nitride (h-BN) and molybdenum disulfide (MoS2) are the other 2D materials with demonstrated capabilities in nanopore applications.13,14
The far-too-rapid translocation of DNA molecules through nanopores, however, makes the detection of the individual nucleotides very challenging, both for 2D and long channel nanopores. Additionally, the low mechanical stability of atomically thin membranes is the Achilles' heel of 2D nanopores, adding considerable noise to the measured signal and reducing the achievable resolution. Due to such limitations, attempts to sequence DNA with 2D nanopores have not yet met the initial hopes. Instead, the platform has been extensively used to successfully detect the translocation of individual double-stranded3–5 and single-stranded6 DNA molecules.
Considering the rapidly growing interest in the application of 2D nanopores and nanogaps, this article serves as a comprehensive tutorial, reviewing the latest progress in the field. Background literature, fabrication methods and important achievements are presented in each section. We explain the remaining challenges and, importantly, offer appropriate solutions. This review bridges chemistry and physics and suggests new development routes in this still young and emerging research field.
Identifying the building blocks of biomolecules (i.e. sequencing) is the driving force and the ultimate goal of nanopore analysis: theoretical calculations confirmed the possibility of distinguishing base pairs composing a double-stranded DNA (dsDNA) in graphene nanopore systems,15 as the ionic current largely depends on the conformation (stretching) of the translocating molecule. While the externally applied electrostatic field tends to stretch and deform base pairs occupying the nanopore, intermolecular hydrogen bonding in A–T and G–C base pairs (A: adenine, T: thymine, G: guanine, C: cytosine) resists against the deformation to some extent. Additionally, harsh electrostatic force can even break the hydrogen bonds and locally deform the molecules.15 The G–C base pair, however, is more robust against stretching than the A–T pair, as the former possesses an extra hydrogen bond. It was shown by computer simulation that an optimal driving force (∼1 V in this simulation) can sufficiently deform the A–T base pair so that the resulting ionic current can be distinguished from the G–C pair signal.
Exfoliated or chemically grown, the flakes have to be transferred onto a substrate with a previously drilled opening as shown in the bottom inset of Fig. 1b. The substrate provides a mechanical support for graphene while the opening in the supporting membrane allows biomolecules to translocate. Square or circular openings with sizes of hundreds of nanometers to few micrometers have been used3–5 (Table 2). The supporting membrane should be thin enough to effortlessly drill the opening, but with enough mechanical stability to support graphene. In practice, by partially etching a micro-scale window in the center of doped silicon wafers, free standing silicon nitride (SiNx) membranes with few tens of nanometers thicknesses are fabricated and used. Focused electron or ion beams are standard techniques to sculpt microscale openings in such membranes. In a next step, a 2D material is transferred onto the substrate covering the opening. The interface between the substrate and the 2D material should be well sealed against any ionic leakage to maximize sensitivity, for example using an elaborate transfer process.16 As the final step, a nanopore is drilled in the 2D material. Few techniques have been successfully used for drilling such nanopores.
Fig. 2 Methods for sculpting 2D nanopores. (a) TEM image of a nanopore sculpted with a high energy (300 kV) electron beam in a monolayer graphene at room temperature; adapted with permission from ref. 3. Copyright 2010 American Chemical Society. The inset schematically illustrates the process of breaking carbon–carbon bonds with such an energetic electron beam. (b) High resolution TEM image of a nanopore sculpted by high temperature STEM at 600 °C; adapted by permission from Macmillan Publishers Ltd: Nat. Commun.,10 copyright 2013. (c) TEM image showing an array of similar and small (d ≈ 2 nm) nanopores sculpted by STEM at 600 °C; adapted with permission from ref. 10. Copyright 2013 American Chemical Society. (d) TEM image of nanopores formed by argon ion bombardment followed by electron exposure: red arrows indicate the position of the randomly distributed nanopores. The inset shows a very small nanopore (d ≈ 5.8 Å) obtained with this technique. Images are adapted from ref. 21. (e) SEM image of nanopores photothermally formed in a graphene membrane: examples of gold nanoparticles and nanopores are visible in the image. Adapted with permission from ref. 22. Copyright 2014 American Chemical Society. (f) High resolution TEM image of an edge of a nanopore formed in graphene using the photothermal method: The inset schematically describes the processes leading to the pore formation. Adapted with permission from ref. 22. Copyright 2014 American Chemical Society. |
Technique | Smallest nanopore | Crystallinity | Parallela | Drilling SiNxb | Reproducibility | Ref. |
---|---|---|---|---|---|---|
a The possibility for sculpting several nanopores at the same time. b The possibility for drilling SiNx membrane simultaneously with the 2D material. | ||||||
TEM | d ≈ 2 nm | Destroyed | No | Yes | Poor | 3–5, 9 and 12 |
High-T STEM | d ≈ 2 nm | Preserved | No | Yes | Yes | 10 and 19 |
Atom by atom growth | d ≈ 6 Å | Not clear | Yes | No | No | 21 |
Photothermal | 2 × 60 nm2 | Partially preserved | Yes | No | No | 22 |
Molecule | Membrane | Geometry | Translocation speeda | Ref. | ||
---|---|---|---|---|---|---|
Material | # Layers | Opening | Nanopore (nm) | |||
a The translocation speed is calculated based on the reported translocation time for an unfolded DNA. The reported data in this column are respectively: the translocation speed, the diameter of nanopore and the applied transmembrane voltage. b The membrane is covered with 5 nm of TiO2. c Covering the surface of graphene and the perimeter of the nanopore with hydrophilic layer makes the effective nanopore diameter smaller. | ||||||
dsDNA | Exfoliated graphene | 1–8 | d = 5 μm | 5–25 | 56 ns per bp, 22 nm, 200 mV | 3 |
dsDNA | CVD graphene with/without 5 nm of TiO2 | 3–15 | d = 1.5 μm | 5–10 |
30 ns per bp, 8 nm, 100 mV
14 ns per bp, 6 nm, 100 mVb |
4 |
dsDNA | CVD graphene | 1–2 | 200 × 200 nm2 | 5–23 | Not reported | 5 |
ssDNA & dsDNA | CVD graphene | 1 | 200 × 200 nm2 | 3–7 | 10 ns per bp, 3.3 nm, 160 mV | 6 |
dsDNA | CVD h-BN | 2–3 | 300 × 300 nm2 | 5–14 |
20 ns per bp, ∼6 nm, 100 mV
16 ns per bp, ∼6 nm, 160 mV |
13 |
dsDNA | CVD graphene with 5 nm Al2O3 | 1 | n/a | 10 | n/a | 12 |
ssDNA | Exfoliated graphene | 1 | d = 1.5 μm | 3–20 |
∼19 ns per nt, 15 nmc, 200 mV
∼26 ns per nt, 10 nmc, 200 mV ∼36 ns per nt, 5 nmc, 200 mV |
19 |
dsDNA | Exfoliated MoS2 | 1–4 | 200 × 200 nm2 up to 500 × 500 nm2 | 2–20 | ∼20 ns per bp, 20 nm, 200 mV | 14 |
Fig. 3 Implementation of 2D nanopores for detecting biomolecules. (a) Examples of typical conductance signals for DNA translocating through nanopores: the color coding depicts the different folding configurations. Adapted with permission from ref. 3. Copyright 2010 American Chemical Society. (b) Ionic conduction through nanopores of similar diameters in graphene (d ≈ 8 nm) and SiNx (d ≈ 6 nm, L = 40 nm) membranes. The experiments were both performed in 1 M KCl, 10 mM Tris (pH = 8.1). Numerous translocation events are visible as sharp current dips in both time traces. Adapted with permission from ref. 4. Copyright 2010 American Chemical Society. (c and d) Graphical representation of simulated ionic current density through a (c) 2D nanopore (d ≈ 2.5 nm, L = 0.6 nm) and (d) a long channel nanopore (d ≈ 2.5 nm, L = 5 nm); insets show the same simulation in the presence of a translocating dsDNA strand (modeled as insulating cylinder with d = 2.1 nm). The color coding represents the current density and ranges from j = 0 to 2 GA m−2 and j = 0 to 4 GA m−2 for the main and inset figures respectively. The figure is adapted from ref. 6. (e) Influence of the diameter of nanopores in single-layer graphene on the average current blockade (δI); the dashed line shows results originating from simulation. The figure is adapted from ref. 6. (f) STEM image of a 2D nanopore in graphene (d = 5 nm) before (inset) and after (main panel) the incubation with single stranded circular M13 DNA; the white blob at the center of the nanopore is presumably a DNA molecule clogging the nanopore. Adapted by permission from Macmillan Publishers Ltd: Nat. Commun.,19 copyright 2013. (g) AFM images of the surface of HOPG before (inset) and after (main panel) incubation with M13 ssDNA; adapted by permission from Macmillan Publishers Ltd: Nat. Commun.,19 copyright 2013. |
A general model for the conductance of a nanopore (G) is proposed as:23,24
(1) |
Fig. 3b compares the ionic current measured from a 2D nanopore in graphene (d = 8 nm) and from a nanopore with a similar diameter (d = 6 nm) in a SiNx membrane of L = 40 nm.4 The baseline current in the 2D nanopore is several times larger than the one in the SiNx membrane. This observation is consistent with eqn (1) since the reduction of the membrane thickness yields a higher conductance. Indeed, ions feel much less resistivity passing through nanopores in 2D materials, compared to long channel nanopores.
Although achieving higher resolution is an important motivation for using 2D nanopores, some reports argue about the efficiency of using monolayer membranes.25 Molecular dynamic simulations showed that once a negatively charged ssDNA enters the nanopore, the change of the ion distribution results in an electric double layer near the surface of the DNA strand, reducing the sensitivity of the ionic current to the size of the bases. The effect is stronger in thin (monolayer) graphene membrane; hence the relatively thick membranes (few layer membranes) are claimed to be more efficient for sequencing ssDNA.
Several approaches can reduce the mechanical vibration of 2D nanopores in buffer solutions. The process for the fabrication of free standing membranes may induce some mechanical stress in the final membrane which limits the mechanical fluctuations. Additionally, the concentration of salt in the buffer solution also affect the mechanical vibrations: the more concentrated the salts, the more ions colliding the membrane and the less mechanical stability.19 Increasing the working temperature also has similar consequences.26
Additionally, the spacing between the border of the nanopore and the DNA (i.e. the relative size of the nanopore versus the size of the DNA molecule) is another parameter affecting the sensitivity of nanopores. Simulations demonstrated that in long channel nanopores, the ionic current density is uniformly distributed throughout the channel width as the edges of 2D nanopores strongly localize the ionic current6 (Fig. 3c and d). Therefore, when the diameter of the nanopore approaches the diameter of the translocating DNA (insets of Fig. 3c and d), the localized current is more sensitive to the spatial spreading of the DNA building blocks. Indeed, sub-nanometer resolution and ultra-high sensitivity to extremely small changes in the local diameter of the translocating molecule can be achieved with a tightly dimensioned nanopore. The results were confirmed experimentally (Fig. 3e).6
A rapid UV/ozone treatment can turn graphene into graphene oxide with hydrophilic properties. The oxidation by itself may not be enough as 30 percent of the devices measured after the treatment4 did not show enough wettability to translocate DNA. Instead, depositing a thin layer of hydrophilic TiO2 films on both sides of the graphene membrane further improved the wettability of the devices.
The composition of the buffer solution is another parameter affecting the interaction between the molecule and the graphene surface. Salty solutions (3 M KCl) and high pH of the buffer solution (up to 10) minimize the interaction of DNA with the surface, leading to faster DNA translocation.6 Note that a further increase in the pH (up to ∼12) may denature dsDNA into ssDNA. In a separate study, 3M of KCl did not prevent DNA from interacting with graphitic surfaces:19 AFM mappings showed that ssDNA still sticks rather strongly to the surface of HOPG (Fig. 3g). Instead, a more general approach based on non-covalently functionalizing graphene with hydrophilic ethylene glycol groups reduced DNA–graphene interactions.19 A reduced hydrophobic interaction between the aromatic graphene and nucleotides, at the cost of just a 1–2 nm increase in the channel length, was demonstrated upon coating graphene with such an ultrathin layer.
Fig. 4 Important challenges of 2D nanopores for sequencing biomolecules. (a) Conductance of 2D nanopores in graphene as a function of the diameter of the nanopore: the data in the main panel is measured from nanopores sculpted using high temperature STEM. Dashed green line represents the best fitting with eqn (1) revealing L = 1.2 ± 0.1 nm. Adapted by permission from Macmillan Publishers Ltd: Nat. Commun.,19 copyright 2013. The inset shows the resistance of 2D nanopores in graphene membranes (number of layers ranging between 1 to 8) sculpted by room temperature TEM as a function of the diameter. The best fitting with eqn (1) reveals a membrane thickness of 9 nm. Adapted with permission from ref. 3. Copyright 2010 American Chemical Society. (b) Translocation time as a function of the diameter of 2D nanopores in graphene membranes: different experiments (blue and red) compared in the main6 and inset19 panels show opposite trends. The vertical and horizontal axes in the inset have the same unit as the main panel. The inset panel is adapted by permission from Macmillan Publishers Ltd: Nat. Commun.,19 copyright 2013. (c) Translocation speed of dsDNA through a graphene nanopore with a diameter of 8 nm plotted as a function of the transmembrane potential: the membrane is composed of a graphene coated with 5 nm of TiO2. Dashed line shows the linear fit of the data. Adapted with permission from ref. 4. Copyright 2010 American Chemical Society. (d) Current blockade amplitudes plotted as a function of the translocation time for double stranded (blue circles, measured at pH = 10) and single stranded (black circles, measured at pH = 12.5) DNA molecules. The data were measured in a graphene nanopore of d = 3.3 nm. The dsDNA molecules translocate through the nanopore in a fully unfolded configuration. The data are adapted from ref. 6. (e) Simulation of the translocation of ssDNA through 2D nanopores in one-, two-, and three-layer graphene membranes: (top) Snapshots showing translocating ssDNA (poly(dT)20) through membranes with different number of layers, (bottom) plot represents the number of translocating nucleotides as a function of time (t). For single layer graphene (green) the whole ssDNA translocates in a single step, while for two- and three-layer membranes multiple steps are observed (red and black). The data are simulated for a 500 mV bias voltage. Adapted with permission from ref. 27. Copyright 2012 American Chemical Society. |
The bandwidth of the measurement electronics sets an upper limit to the achievable temporal resolution. The combination of the membrane with the ions at its both sides can be regarded as a parallel plate capacitor which adds high frequency noise to the measured signal. To attenuate this noise, it is inevitable to use a low-pass filter of ∼10 kHz as a part of the electronic detection scheme.28 Using such a filter sets a maximum temporal resolution of ∼50 μs. The achieved resolution is therefore much inferior to the required resolution to resolve single nucleotides.
Slowing down the translocation speed of the DNA is an important objective followed by many researchers, particularly when using biological nanopores. In fact, a DNA translocating through a nanopore experiences three different forces, namely the electrophoretic driving force, the nanopore-molecule interaction force, and the drag (viscosity) force from the buffer solution. While the electrophoretic driving force accelerates the translocation of DNA, the two others impede against the translocation. Several parameters modulate those forces and therefore the translocation speed of DNA; they will be discussed in the following sections.
The shape of the nanopore may also affect the translocation speed: in circular nanopores, increasing the bias voltage generally accelerates the translocation. However simulations show that broken rotational symmetry in elliptical nanopores affects the DNA atomic conformation inside the nanopore: DNA molecules may reshape in conformations which are unfavorable for translocation, slowing down the molecules.27
Manipulating the net charge on the DNA is the more efficient method to modulate the translocation speed. Ions in the buffer, such as magnesium chloride, can coordinate on the DNA and strongly screen the net charges.29 Additionally, protons in the solutions with low pH values can partially neutralize the negative charge on the DNA molecules and reduce the translocation speed.30
Single stranded DNA shows stronger hydrophobic interaction with hydrophobic graphene as the coupling of the two strands in a dsDNA minimizes the overall hydrophobicity of the molecule.27Fig. 4d compares the translocation events of double stranded and single stranded DNA through a 2D nanopore in graphene of d = 3.3 nm.6 As a result of the ultra-small area of the nanopore, only unfolded dsDNA can translocate. Double stranded DNA shows uniform translocation characteristic since the events possess very similar durations. In contrast, the translocation time for ssDNA ranges from ∼50 μs up to more than 400 μs. Strong hydrophobic interaction between graphene and ssDNA can explain the divergent translocation times.
A more detailed study about the DNA–membrane hydrophobic interaction was performed by probing the translocation of single and double stranded DNA through nanopores in graphene–Al2O3 stacked membranes.31 Different configurations of stacking layers, including graphene/Al2O3/graphene, Al2O3/graphene and standalone Al2O3, have been examined. The results are summarized in Table 3. Translocation of the ssDNA is slower through membranes with graphene layers compared to standalone Al2O3, pointing out the strong hydrophobic interaction between graphene and ssDNA. The fact that a double stranded DNA molecule exhibits much faster translocation compared to single stranded DNA in similar membranes (graphene/Al2O3/graphene) is another confirmation that in ssDNA purines and pyrimidines are more accessible for pi-stacking interaction with graphene.
Membrane configuration | Translocation ratea | |
---|---|---|
a The translocation is measured at 300 mV bias and for nanopres with similar diameters. | ||
Graphene–Al2O3–graphene (hydrophobic–hydrophilic–hydrophobic) | 5.5 μs per nt (for ssDNA) | |
Al2O3–graphene (hydrophilic–hydrophobic) | 4.7 μs per nt (for ssDNA) | |
Al2O3 (hydrophilic) | 1.8 μs per nt (for ssDNA) | |
Graphene–Al2O3–graphene (hydrophobic–hydrophilic–hydrophobic) | 0.4 μs per bp (for dsDNA) |
In addition to the naturally occurring hydrophobic interaction between the DNA and 2D materials, electrostatic interaction also can be induced. Chemically functionalizing the nanopore with positively and negatively charged groups is a way to achieve nanopores with different charge states: as DNA is also charged (naturally), the DNA–membrane electrostatic interaction can affect the translocation speed. Simulations illustrated that the translocation through a nanopore (r = 1.2 nm) in a positively charged membrane (+3.6e) can happen almost twice faster than a similar nanopore in a negatively charged membrane (−3.6e).15 Indeed the repulsive interaction between the negatively charged DNA molecule and nanopore rim reduces the effective nanopore diameter.
Electrically biasing the conducting 2D membrane is another approach to induce (modulate) an electrostatic interaction. Stacked membranes in which an electrically connected graphene membrane is sandwiched between two dielectric materials is a model systems for this purpose.20 Even though the reduced translocation speed is a favorable consequence of strong DNA–membrane interaction, such interactions should still be avoided: the interaction can reduce the stability of the device by clogging the nanopore.19 Increasing the noise level is another drawback.4
Note that even if the translocation speed remains the same in long and short channels, the time that a DNA nucleotide occupies the channel in longer channels is larger; hence enhanced temporal resolution can be achieved. Indeed, simulations show that single-nucleotide steps could be identified in bi- and three-layer graphene nanopores27 (Fig. 4e). The co-presence of multiple bases in the nanopore is the drawback limiting the spatial resolution.
Experimentally, nanopores drilled in few layer graphene yielded slower translocations (more than twice as slow) compared to similar nanopores drilled in 20 nm-thick SiNx membranes.3 Similar comparison with hexagonal boron nitride (h-BN) membranes13 reveals an opposite trend i.e. faster translocation through h-BN nanopores in comparison than in SiNx ones. Such contradictionary observations may also point out the influence of chemical composition of the membrane material on the strength of the DNA–membrane interactions.
The relatively uncontrollable and arbitrary conformations of the nucleotides affect the ionic current blockade. Nevertheless simulations showed that the dependency of the signal to the nucleotide type is stronger than to its conformation.27 Hence even in presence of conformational instabilities, single nucleotide differentiation is possible.
The magnitude of the low frequency noise – known as 1/f noise – in nanopores in graphene and other 2D materials is normally much larger (up to 100 times) than in long channel nanopores.13,14,32 As a source of a considerable reduction in the signal-to-noise ratio, the high amplitude of 1/f noise is an important challenge in 2D nanopores. It was demonstrated that for nanopores in SiNx membranes the amplitude of the low frequency noise is inversely dependent on the number of ionic charge carriers ∝ 1/N:33 a principle known as Hooge's relation. However experimental results do not show such a dependency in monolayer graphene nanopores.32 The reasons behind this discrepancy are not yet understood.
The origin of the high-amplitude low-frequency noise in 2D nanopores is under debate. Charge fluctuations in the membrane are potentially capable of inducing noise in the ionic current. Protonation and de-protonation of the carboxyl groups formed at the nanopore rim may cause such fluctuations and can be triggered by varying the pH of the environment. However experimental results did not confirm any strong dependency of the noise to the pH of the buffer solution.32 We note that those experiments were performed at relatively high KCl concentration (1 M); more decisive conclusions could be drawn by lowering the ionic strength of the buffer solution.
The incomplete wetting of the hydrophobic graphene has been hypothetized as the source of the measured noise.4 To cope with this effect, the hydrophobicity of the graphene membrane was modified by a rapid UV/ozone exposure, yielding graphene oxide. The improved wetting of the membrane on the cost of inducing defects, however, did not lead to a remarkable reduction of the noise level. Importantly, the evaporation of a hydrophilic TiO2 layer on graphene considerably reduced the noise. The idea of covering the membrane with hydrophilic materials was followed later by20 in which relatively thick membranes of multi-stacked graphene and Al2O3 layers were made. The top hydrophilic Al2O3 layer was supposed to provide adequate wetting during the experiments. Indeed very low 1/f noise, comparable with long channel nanopores and considerably less than in pure 2D nanopores was measured.
Mechanical instability of the membranes is another parameter which is suspected to cause the noise: the fluctuation of the membrane may lead to the fluctuation of the ion flux. Increasing the thickness of the membrane is an approach to improve its mechanical stability. Indeed prominent trends in lowering the 1/f noise level while increasing the thickness (i.e. the number of layers) of graphene and h-BN membranes were measured.17,32 Such results point out the effect of the mechanical fluctuations as an important source of low frequency noise in 2D nanopore devices. In fact conventional solid state membranes (normally thicker than 20 nm) are mechanically more rigid and stable compared to few layer 2D materials. We note that unlike the interpretation of early reports,4 the reduced noise level measured by covering graphene with other materials4 or in stacked systems20 can be explained in terms of the increased mechanical stability, rather than the modification of the hydrophobicity of the graphene membrane. Minimizing the area of the free-stranding membrane is an approach to improve the stability of the membrane without affecting the surface properties. In fact reduced noise levels have been reported in two samples with very small free standing graphene areas.6
Comparing the approaches to improve the mechanical stability of the membrane we note that minimizing the SiNx opening blocks a large amount of the pin-holes in graphene which would result in an enhanced signal. The important disadvantage of using thick graphene layers or of depositing other materials is that the increased thickness of the membrane eludes the mono-atomic nature of graphene which potentially provides single base resolution.
An optical method was successfully adjusted to measure translocating molecules in a 2D nanopore.22 Fluorescently-tagged DNA molecules were electrostatically driven through a nanopore in a graphene membrane made by photothermal sculpting (see the fabrication section, Fig. 2e). The water immersed objective lens of a fluorescent microscope, focused on the nanopore, can be used to monitor and record the dynamics of the DNA translocation. Gold nanoparticles serve as antennas to enhance the fluorescence signal, as they possess a surface plasmon resonance peak matching the absorption peak of the fluorescence dye. Subsequent snapshots in Fig. 5a illustrate the dynamics of the DNA translocation through the nanopore from the moment it enters the nanopore till the moment it is nearly fully stretched (∼18 μm in length). Illustrated by a brighter spot, the fluorescence intensity close to the antenna (marked by the red arrow in the last snapshot) is four to five times higher than at the center (blue arrow), showing the plasmonic resonance effect.
Fig. 5 Alternative detection methods based on 2D nanopores. (a) Subsequent snapshots obtained using a confocal scanning fluorescence microscope showing the translocation of a λ-DNA. Adapted with permission from ref. 22. Copyright 2014 American Chemical Society. (b) Proposed electrical scheme for detecting single nucleotides of a ssDNA passing through a graphene nanopore by probing electronic current across the graphene; Adapted with permission from ref. 34. Copyright 2010 American Chemical Society. (c–e) Graphene nanoribbon used for the electrical detection of the translocation of a DNA strand through a nanopore sculpted in the nanoribbon, (c): the schematic representation of the fabricated device, (d): time series of the synchronized measurement of DNA translocations (ionic current through the nanopore and electrical current through the nanoribbon) (e): zoomed-in view of a single translocation event marked inside the boxes in (d). Adapted with permission from ref. 12. Copyright 2013 American Chemical Society. (f) Schematic representation of the proposed nanochannel device promoting DNA–graphene interaction for single nucleotide sequencing; Adapted by permission from Macmillan Publishers Ltd: Nat. Nanotechnol.,36 copyright 2011. |
Another novel technique for DNA sequencing was theoretically investigated.34,35 The device contains a graphene nanoribbon containing a nanopore supported by a hard material e.g. SiO2 (Fig. 5b). Simulations showed that passing through the nanopore, each nucleotide would generate currents in the graphene nanoribbon due to the doping effect. Indeed, the electrostatic interaction between the graphene nanoribbon and translocating nucleotides modulates the conductance of the nanoribbon. The conductance spectrum of each individual nucleotide is unique and sufficiently different for each of the four nucleotides. The calculations predict that the detected current is independent of the orientation of the bases, which is an important requirement for the generalization of the approach. The sensitivity of the device inversely depends on the density of the available electronic states in the graphene nanoribbon: the wider the ribbon, the higher the density of states and lower the sensitivity. The efficiency of the scheme for detecting single DNA molecules was probed experimentally by correlating ionic current (typical nanopore application) and electrical current through the nanoribbon (Fig. 5c to e).12 Unlike the ionic current in which blocking the nanopore ends up with drops in the ionic current, both drops and peaks can be measured in electrical current due to the ambipolar nature of graphene. Strong correlation between ionic and electric current measurements was achieved.
As a result of the monoatomic channel length of 2D nanopores, the molecule–nanopore rim interaction is very small in 2D nanopore devices which can result in stochastic motion of the molecules through the nanopore. Indeed conformational instability as a result of the stochastic motion overlaps the distribution of the signals from different nucleotides. To cope with this problem, a large modification in the nanopore architecture was proposed.36 The proposed device, schematically shown in Fig. 5f composed of a nanoscale fluidic channel locally covered by a graphene nanoribbon bridge. The combination can be regarded as a long nanopore through which DNA molecules – driven by an external field – translocates. The bridge grips the translocating nucleotides firmly by temporarily forming π–π interaction. Hereby, the conformational fluctuation of the nucleotides is limited. The simulations showed that the distinct doping effects of the stacked nucleotides can be measured as electrical signals across the graphene nanoribbon and be used to discriminate between the nucleotides. The possibility of multi-stacking nucleotides to graphene nanoribbon is an important limitation of the technique.
The considerably large noise in graphene nanopores has been attributed to the low mechanical instability of the membrane as a result of its atomic thickness. Indeed simulations predict that the noise level reduces by using trilayer graphene membrane of ∼1 nm thickness.37 A monolayer MoS2 is ∼1 nm thick and is relatively (three times) thicker than a monolayer graphene. Hence on a cost of the slight reduction of the resolution of the device, higher signal to noise ratio is predicted in the 2D nanopores in MoS2 membranes. In an early work, a signal to noise ratio of 10 has been measured for 2D nanopores in MoS2 which is almost three times higher than what can be achieved in graphene 2D nanopores.14
DNA–membrane interaction is another important parameter to select the right material. Molecular dynamics simulations show that in contrary to the graphene, DNA does not stick significantly to the surface of MoS2, even without any additional surface treatments.26 This result is in agreement with experiments.14 Indeed, the presence of the hydrophilic Mo sites in the structure of the MoS2 membranes reduces the DNA–membrane interaction. In contrary to MoS2, experimentations with h-BN membranes demonstrate remarkably higher interaction with DNA molecules (in comparison to SiNx and graphene6) even at strong alkaline and salty condition (pH = 10, KCl concentration of 3 M).13
The electrically conducting nature of MoS2, its low interaction with DNA and the high enough mechanical stability which a free standing MoS2 membrane promise a good replacement for graphene in nanopore applications.
DNA sequencing with 2D nanopores met important challenges. The very rapid translocation of the nucleotides through the nanopore is far beyond the sensitivity of the ionic current. Several approaches can be taken to slow down DNA. Separately, the large 1/f ionic current noise which severely affects the signal to noise ratio, is another major challenge, very particular to 2D nanopores. The origin of this noise is not entirely clear yet, although the existing reports point the mechanical fluctuations of the membrane as the most probable source. Improving the mechanical stability of the membrane by reducing the area of freestanding part seems to be the most efficient technique to overcome the limitation. Utilizing thicker 2D materials (e.g. MoS2) is another potential approach.
Alternative detection schemes – not necessarily based on the ionic conduction – have been proposed to overcome the existing difficulties. The conducting nature of 2D materials with externally tunable conductivity is the important supplement of graphene and some other 2D materials to develop such methods. In principle, schemes based on the electronic conduction could be faster than the existing measurement approach based on the ionic current. Theoretical propositions and experimental results of such schemes will be discussed in details in the next section.
Fig. 6 Tunneling current for single-molecule detection. (a) Individual electron tunneling through a rectangular potential barrier: ψ is the wave function of the tunneling electron. (b) Schematic illustration of a nanogap between metallic electrodes (e.g. break junction) for single-molecule detection: two electrodes (yellow) are separated by a subnanometer-sized gap in which a peptide molecule is placed. Electronic properties of the peptide affect the tunneling current between the electrodes. The scale bar shows 5.5 Å. Adapted by permission from Macmillan Publishers Ltd: Nat. Nanotechnol.,44 copyright 2014. (c) Tunneling current for the visualization of dsDNA molecules using STM: DNA molecules are visible as white strands. Reprinted from ref. 41, copyright 2003, with permission from Elsevier. (d) Current distributions for thymidine 50-monophosphate (TMP), cytidine 50-monophosphate (CMP) and guanosine 50-monophosphate (GMP) measured by using a break junction with a gap of ∼1 nm. The distribution of the measured current for each nucleotide is unique. Adapted by permission from Macmillan Publishers Ltd: Nat. Nanotechnol.,43 copyright 2010. |
The detection and sequencing of single molecules could be implemented in a simple device composed of two electrodes separated by a small gap through which a biomolecule would translocate (Fig. 6b). Upon applying an electrical potential, a current would be established between the electrodes, tunneling through the translocating biomolecules. For DNA, the different nucleotides composing the translocating DNA strand – as they possess distinct local density of electronic states and different molecular sizes – were predicted to yield tunneling currents with intensities significantly different for each nucleotide.39,40 Separately, the method potentially provides temporal resolution that would allow recording several data points per nucleotide. As the nucleotides pass one-by-one across the gap, recording the tunneling current over time should, in principle, resolve the particular sequence of the nucleotides in the strand.
Molecular break junction is another platform employing the principle of tunneling current for detection. Smoothly pulling the two ends of a metallic rod (e.g. a thin wire) yields, in time, a thinner and thinner wire, a few atoms wide junction, a single atom junction, and finally a gap (Fig. 6b). The shape and the width of the gap are controlled by adjusting the applied force on the rod: metallic electrodes with nanogaps as narrow as ∼5 Å have been achieved with this technique.42 In a remarkable experiment, a gold break junction was immersed in aqueous solutions containing nucleotides. Three nucleotides, namely thymidine 5′-monophosphate (TMP), guanosine 5′-monophosphate (GMP) and cytidine 5′-monophosphate (CMP), were probed.43 In each case, unique tunneling current signatures were detected (Fig. 6d). Similarly, a recent work confirmed the efficiency of the break junction method for the identification of twelve (out of 20) different amino acids, including the detection of post-translational modifications of single peptide molecules.44
STM and break junction methods have been studied extensively for various purposes in the past and offer a rather simple and fast solution to investigate biomolecules with tunneling current. However remarkable limitations exist, preventing their direct use for sequencing applications: STM performs best at low temperature (few Kelvins) and ultrahigh vacuum which is not adequate for biological specimens; break junctions do not provide per se a fluidic channel to guide long stretches of unfolded biomolecules through the gap. Additionally, the formation of parallel conducting channels reduces the resolution of the techniques in water. Such limitations can be overcome by employing so-called graphene in-plane electrodes.
The concept of using in-plane graphene electrodes for DNA sequencing (Fig. 7a) was investigated theoretically by Henk Postma in 2010:39 while a nanogap width of less than 1.5 nm (a distance narrow enough for detecting a transversal tunneling current and large enough for a DNA molecule to be able to translocate) would yield specific tunneling current signatures for each nucleotide, small metric variations in the width of the nanogap drastically impact the measured current. Important parameters that influence the detected signal and are crucial for achieving the single nucleotide resolution will be discussed in details in the next sections.
Fig. 7 (a) Tunneling current for DNA sequencing with graphene electrodes. (b) Simulations of the tunneling current expected for ssDNA with the sequence CGG CGA GTA GCA TAA GCG AGT CAT GTT GT between two graphene electrodes in a nanogap configuration of two different widths (w = 1.27 nm and w = 1.72 nm). The different bases are represented with different colors (G in red, C in blue, T in purple, and A in green). Adapted with permission from ref. 39. Copyright 2010 American Chemical Society. (c) Theoretical calculations of the effect of the angle of rotation of deoxyadenosine monophosphate (dAMP) on the zero-bias transmission spectra of dAMP translocating in a graphene nanogap: the different curves correspond to different angles of rotations of dAMP with respect to the graphene edge. Adapted with permission from ref. 40. Copyright 2011 American Chemical Society. (d) Possible ranges of tunneling current for dAMP, dTMP, dGMP, dCMP nucleotides translocating through a nanogap in graphene (with diverse orientation and proximity to the electrodes): calculations were done under an applied bias of 1 V and for a gap width of 1.47 nm. Adapted with permission from ref. 40. Copyright 2011 American Chemical Society. (e) Illustration of the concept of DNA sequencing with tunneling current with a nanopore in bilayer graphene: a DNA molecule translocates through the nanopore while recording the current tunneling from the rim of the nanopore in the top graphene layer to the rim of the nanopore in the bottom graphene layer. (f) Tunneling conductance calculated for ssDNA molecule passing through a nanopore in bilayer graphene (see panel (e) for an illustration of the working device). The amplitude of the rim-to-rim tunneling current signs for individual nucleotides. The figure is adapted from ref. 47. |
(2) |
Purine based nucleotides | Pyrimidine based nucleotides | |||
---|---|---|---|---|
dGMP | dAMP | dTMP | dCMP | |
a Measured for the energy range of −1 eV < E < 1 eV. | ||||
Transmissiona | 10−20–10−6G0 | 10−20–10−6G0 | 0–10−8G0 | 0–10−8G0 |
Width of the peak | Broad ∼0.1–0.2 eV | Broad ∼0.1–0.2 eV | Narrow ∼0.05–0.10 eV | Narrow ∼0.05–0.10 eV |
Position of the peak | Close to Fermi level E − Ef ≤ 0.5 eV | Far from Fermi level E − Ef ≥ 0.5 eV | Far from Fermi level E − Ef ≥ 0.5 eV | Far from Fermi level E − Ef ≥ 0.5 eV |
Functionalization of the electrodes is an effective approach to minimize the conformational uncertainty. The effect of the hydrogen bonds formed in between single nucleotide from a single-stranded DNA and the individual gold atoms at the electrodes (functionalized by purine and pyrimidine molecules) were investigated theoretically.45 The results showed a giant improvement in the sensitivity of the device: more than one order of magnitude difference in tunneling current between the nucleotides. Furthermore, the stabilization of the DNA bases against any possible thermal fluctuations dramatically reduced the electrical noise. Slowing down the translocation of DNA is another benefit of generating hydrogen-bonding interactions between the translocating nucleotides and the nanogap electrodes. Applying a large bias voltage can lead to similar results. Indeed, the transverse electric field established between the biased electrodes can be very large and stronger than the driving transmembrane field.46 The resultant stabilization of the translocating DNA preserves the sensitivity of the readout for different bases (distinguishability).
In the most simple design of a nanogap (Fig. 7a), the surface of the graphene electrodes is in contact with the buffer solution. A parallel ionic current may flow in between the electrodes and reduce the sensitivity of the signal. Covering the graphene with a self-assembled monolayer or with deposited materials can reduce this parallel current. The small contribution of the parallel current between unpassivated carbon atoms at the edges can be minimized by a calibration measurement before and after each DNA translocation.
In a real device the current in between the large layers of graphene can be several orders of magnitude larger than the conductance through the edges of the nanopore. In theory, the interlayer conduction depends on the relative orientation angle in between the layers. Therefore, the two layers can be electrically decoupled at some certain twist angles.47,48 The twisted bilayer graphene can be grown chemically, however the twist angle is not controllable. Yet the implementation of a sequencing device based on bilayer graphene would still require a solid (experimental) protocol for the fabrication of well-defined and tunable twisted bilayer graphene. Deposition of the electrodes on one layer without electrically touching the other one is another fabrication challenge.
Using the insulating few- (mono-) layer hexagonal boron nitride (h-BN) in-between graphene layers reduces the interlayer conductance.49 It is postulated that in the presence of the molecule in the nanopore, the transmitted current through the two possible paths (i) via the molecules, and (ii) via the h-BN layer, may show quantum interference which could further improve the sensitivity of the device.49 The relative orientation (stacking) of the graphene/h-BN/graphene crystals affects the electrical isolation of the graphene layers. We note that the separation of the two graphene electrodes by the h-BN layer increases the tunneling length, decreasing the tunneling current. Separately, increasing the tunneling channel length also implies that more nucleotides may contribute to the tunneling current (at least if more than one layer of h-BN are used to separate the two graphene electrodes) which decreases the resolution of the measurement.
The complex fabrication of the electrodes with a narrow gap (<1.5 nm) is certainly the most important challenge which has prevented the realization of graphene based in-plane electrodes sensors so far. Additionally, theoretical works confirmed the efficiency of the detection scheme for single stranded DNA. As such, ssDNA must first translocate through the nanogap in an unfolded conformation, for example in the presence of unfolding agents such as urea. Separately, a nanopore in a bilayer graphene intrinsically has a physical gap in between the two layers, which could be used also to distinguish nucleotides in a DNA strand.
Nanogap in 2D materials is a proposition which potentially can overcome some of the sequencing challenges. Their fabrication is, however, even more complicated than nanopores. While the tunneling current has been predicted to be very sensitive to the available electronic states in nucleotides and therefore may provide more accurate readings, all predictions are based on theoretical work (except for some of peculiar experimental studies with break junction). Certainly, the transmembrane potential used to drive the molecules could be isolated further from the measurement circuit to limit the high frequency noise. The electrodes could also be supported on relatively thick membranes providing higher mechanical stability.
Very importantly, small nanogaps with a width of less than ∼1.5 nm are also required to establish a measureable tunneling current. While high temperature STEM techniques showed the feasibility for the fabrication, tunneling has not been measured so far with graphene for example. Additionally, tunneling currents are very sensitive to the gap width and only a few percent increase in the gap width dramatically reduce the signal by orders of magnitude. Additionally, conventional nanofabrication methods are hardly capable to produce such short nanogaps, explaining why the idea has not been realized yet experimentally.
An alternative to longitudinal nanogaps was proposed theoretically: the distance between the layers in a bilayer graphene can be regarded as a natural gap between the two basal plane graphene electrodes. This proposition may ease the fabrication in the sense that the gap can be achieved naturally and reproducibly. However the isolation of the two conducting layers is hard to achieve, even in twisted graphene bilayers.
To conclude, the approaches to sequence DNA with 2D materials are still at a very early stage. The existing nanofabrication methods failed in realizing sequencing. But the principle is still viable: an atomic layer of a 2D material – particularly a graphene edge – can scan a genome and record its sequence as DNA is scanned. It is now clear that developing unconventional fabrication methods are vital (particularly the one that does not need a transmission electron microscope, nor a cleanroom).
It is hard to predict now whether 2D crystals will ever meet the goal of DNA sequencing one day. Certainly, however, in the quest of this goal, new science will be discovered setting out even more horizons for the research.
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