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Amit Meller - One of the best experts on this subject based on the ideXlab platform.
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optical recognition of converted dna nucleotides for single molecule dna sequencing using nanopore arrays
Nano Letters, 2010Co-Authors: Ben Mcnally, Alon Singer, Zhiliang Yu, Zhiping Weng, Amit MellerAbstract:We demonstrate the feasibility of a nanopore based single-molecule DNA sequencing method, which employs multicolor readout. Target DNA is converted according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state Nanopores are then used to sequentially strip off the beacons, leading to a series of detectable photon bursts, at high speed. We show that signals from multiple Nanopores can be detected simultaneously, allowing straightforward parallelization to large nanopore arrays.
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Chemically modified solid-state Nanopores.
Nano letters, 2007Co-Authors: Meni Wanunu, Amit MellerAbstract:Nanopores are extremely sensitive single-molecule sensors. Recently, electron beams have been used to fabricate synthetic Nanopores in thin solid-state membranes with subnanometer resolution. Here we report a new class of chemically modified nanopore sensors. We describe two approaches for monolayer coating of Nanopores: (1) self-assembly from solution, in which Nanopores ∼10 nm diameter can be reproducibly coated, and (2) self-assembly under voltage-driven electrolyte flow, in which we are able to coat 5 nm Nanopores. We present an extensive characterization of coated Nanopores, their stability, reactivity, and pH response.
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characteristics of solid state nanometre pores fabricated using a transmission electron microscope
Nanotechnology, 2007Co-Authors: Ben Mcnally, Min Jun Kim, Kazuyoshi Murata, Amit MellerAbstract:Solid-state Nanopores can be used to detect nucleic acid structures at the single molecule level. An e-beam has been used to fabricate Nanopores in silicon nitride and silicon dioxide membranes, but the pore formation kinetics, and hence its final structure, remain poorly understood. With the aid of high-resolution TEM imaging as well as TEM tomography we examine the effect of Si3N4 material properties on the nanopore structure. In particular, we study the dependence of membrane thickness on the nanopore contraction rate for different initial pore sizes. We explain nanopore formation kinetics as a balance of two opposite processes: (a) material sputtering and (b) surface-tension-induced shrinking.
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rapid fabrication of uniformly sized Nanopores and nanopore arrays for parallel dna analysis
Advanced Materials, 2006Co-Authors: Min Jun Kim, Meni Wanunu, David C. Bell, Amit MellerAbstract:Nanometer-sized pores can be used to detect and characterize biopolymers, such as DNA, RNA, and polypeptides, with single-molecule resolution. Experiments performed with the 1.5 nm pore a-hemolysin (a-HL) demonstrated that singlestranded DNA and RNA molecules can be electrophoretically threaded through a pore, and that the ion current flowing through the pore contains information about the biopolymer sequence: its type, length, and secondary structure. The a-HL nanopore has been used to study the unzipping kinetics of DNA hairpin molecules under stationary or time-varying forces, to detect DNA hybridization kinetics, and to study the interaction of DNA with bound proteins using nanopore force spectroscopy. In addition, a-HL can be biochemically modified for various sensing tasks, such as analyte detection and ligand–receptor interactions. Solid-state Nanopores can be fabricated in thin Si3N4 and SiO2 membranes, using either an Ar beam or an electron beam (e-beam) in a transmission electron microscope (TEM), as well as in a variety of materials using other techniques. Solid-state Nanopores offer several advantages over phospholipid-embedded protein channels, namely, their size can be tuned with nanometer precision and they exhibit an increased mechanical, chemical, and electrical stability. Recent studies using solid-state pores have begun to emerge, demonstrating the detection of single-stranded and double-stranded DNA molecules. A major advantage of solid-state Nanopores is that they can, in principle, be integrated into devices compatible with other detection schemes in addition to ion current measurements. In particular, optical-based methods offer straightforward parallelism through the simultaneous probing of many Nanopores. Optical methods for sensing single molecules can be implemented by labeling the biomolecules and/or the Nanopores. Although protein pores embedded in a phospholipid bilayer can be interrogated optically to detect single molecules, a stable, long-timescale probing is very complicated since the pores readily diffuse in the bilayer, leading to aggregation and destabilization of the membrane. In contrast, Nanopores fabricated in solid-state materials are static, and are therefore more compatible with optical probing. In this paper, we extend state-of-the-art techniques by demonstrating the rapid fabrication of finely tuned Nanopores and nanopore arrays. The Nanopores were fabricated in thin Si3N4 films using the intense e-beam of a field-emission TEM. By maximizing the e-beam density at the specimen we achieved a nearly fivefold decrease in the fabrication time of a single nanopore (ca. 30 s). Investigation of pore contraction/expansion dynamics under different irradiation conditions enabled nanopore fabrication in the range of 2–20 nm with exceptional size control (<0.5 nm variability). Since the Nanopores were fabricated sequentially (i.e., using one e-beam), both the reduction in fabrication time and size control were crucial for the manufacturing of nanopore arrays. The 3D nanopore shape was extensively characterized by performing TEM tomography, as well as by ion-current measurements through the pores. Finally, the detection of double-stranded DNA molecules through 4 nm diameter Nanopores was demonstrated by monitoring their translocation under an applied bias. The starting materials for TEM processing were either fabricated in house or by Protochips Inc. (Raleigh, NC), using the following procedure: low-pressure chemical vapor deposition (LPCVD) was used to form a Si3N4 film (20 or 50 nm thick) on one side of a 500 lm thick Si wafer. A 100 lm× 100 lm window was then fabricated in the wafer using photolithography and standard wet-etching. Nanopore fabrication was then carried out in the thin Si3N4 membrane using a JEOL 2010F field-emission TEM. Alignment of the e-beam involved the adjustment of the condenser stigmatism to the familiar triangle-shaped beam, using a large condenser aperture. The resulting electron-energy-density distribution displayed a threefold aberration. The condenser lens was then used to fully converge the beam to an intense point with a triangular halo of low intensity. The column was then aligned using standard high-resolution transmission electron microscopy alignment procedures. After the alignment procedure, Nanopores with diameters (d) in a range from 3 to 6 nm were directly drilled using an e-beam intensity of ca. 2.5× 10 e nm and a CO M M U N CA IO N
Giovanni Maglia - One of the best experts on this subject based on the ideXlab platform.
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Preparation of Fragaceatoxin C (FraC) Nanopores.
Methods in molecular biology (Clifton N.J.), 2020Co-Authors: Natalie Lisa Mutter, Giovanni Maglia, Gang Huang, Nieck Jordy Van Der Heide, Florian Leonardus Rudolfus Lucas, Nicole Stéphanie Galenkamp, Carsten WlokaAbstract:Biological Nanopores are an emerging class of biosensors with high-end precision owing to their reproducible fabrication at the nanometer scale. Most notably, nanopore-based DNA sequencing applications are currently being commercialized, while nanopore-based proteomics may become a reality in the near future.Although membrane proteins often prove to be difficult to purify, we describe a straightforward protocol for the preparation of Fragaceatoxin C (FraC) Nanopores, which may have applications for DNA analysis and nanopore-based proteomics. Recombinantly expressed FraC Nanopores are purified via two rounds of Ni-NTA affinity chromatography before and after oligomerization on sphingomyelin-containing liposomes. Starting from a plasmid vector containing the FraC gene, our method allows the production of purified Nanopores within a week. Afterward, the FraC Nanopores can be stored at +4 °C for several months, or frozen.
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An Engineered ClyA Nanopore Detects Folded Target Proteins by Selective External Association and Pore Entry
2016Co-Authors: Misha Soskine, Annemie Biesemans, Hagan Bayley, Benjamien Moeyaert, Stephen Cheley, Giovanni MagliaAbstract:Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological Nanopores generally perform better than artificial Nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at −50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA Nanopores decorated with covalently attached aptamers. These Nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores
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single molecule analyte recognition with clya Nanopores equipped with internal protein adaptors
Journal of the American Chemical Society, 2015Co-Authors: Misha Soskine, Annemie Biesemans, Giovanni MagliaAbstract:Nanopores have been used to detect molecules, to sequence DNA, or to investigate chemical reactions at the single-molecule level. Because they approach the absolute limit of sensor miniaturization, Nanopores are amenable to parallelization and could be used in single-cell measurements. Here we show that single enzymes can be functionally and reversibly trapped inside the confined space of a ClyA nanopore. Remarkably, the binding of ligands to the internalized proteins is mirrored by specific changes to the nanopore conductance. Conveniently, the manipulation of the charge of the protein allowed increasing of the residence time of the protein inside the nanopore. Nanopores with internalized protein adaptors can be used to study proteins in real time or can be incorporated into inexpensive portable devices for the detection of analytes with high selectivity.
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an engineered clya nanopore detects folded target proteins by selective external association and pore entry
Nano Letters, 2012Co-Authors: Misha Soskine, Annemie Biesemans, Hagan Bayley, Benjamien Moeyaert, Stephen Cheley, Giovanni MagliaAbstract:Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis and applications in sensing. Biological Nanopores generally perform better than artificial Nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analytes proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at −50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA Nanopores decorated with covalently attached aptamers. These Nanopores selectively captured and internalized cognate protein analytes, but excluded non-cognate analytes, in a process that resembles transport by nuclear pores.
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controlled translocation of individual dna molecules through protein Nanopores with engineered molecular brakes
Nano Letters, 2011Co-Authors: Marcela Rinconrestrepo, Hagan Bayley, Ellina Mikhailova, Giovanni MagliaAbstract:Protein Nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein Nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the α-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, for example, in combination with solid-state Nanopores.
Ulrich F. Keyser - One of the best experts on this subject based on the ideXlab platform.
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Single protein molecule detection by glass Nanopores
ACS Nano, 2013Co-Authors: Wenhong Li, Alana M. Thackray, Nicholas A.w. Bell, Silvia Hernández-ainsa, Vivek V. Thacker, R. Bujdoso, Ulrich F. KeyserAbstract:Nanopores can be used to detect and analyze single molecules in solution. We have used glass Nanopores made by laser-assisted capillary-pulling, as a high-throughput and low cost method, to detect a range of label-free proteins: lysozyme, avidin, IgG, β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and β-galactosidase in solution. Furthermore, we show for the first time solid state nanopore measurements of mammalian prion protein, which in its abnormal form is associated with transmissible spongiform encephalopathies. Our approach provides a basis for protein characterization and the study of protein conformational diseases by nanopore detection.
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DNA origami Nanopores
Nano Letters, 2012Co-Authors: Christian R. Engst, Nicholas A.w. Bell, Marc Ablay, Giorgio Divitini, Tim Liedl, Caterina Ducati, Ulrich F. KeyserAbstract:We demonstrate the assembly of functional hybrid Nanopores for single molecule sensing by inserting DNA origami structures into solid-state Nanopores. In our experiments, single artificial Nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state Nanopores with diameters around 15 nm. We show that these hybrid Nanopores can be employed for the detection of λ-DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state Nanopores and DNA self-assembly.
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optical tweezers for force measurements on dna in Nanopores
Review of Scientific Instruments, 2006Co-Authors: Ulrich F. Keyser, Cees Dekker, J Van Der Does, Nynke H DekkerAbstract:We demonstrate the means to integrate two powerful and widely used single-molecule techniques, viz., optical tweezers and solid-state Nanopores. This setup permits simultaneous spatial sampling and high-resolution force measurements of nucleic acids and proteins. First, we demonstrate the rapid spatial localization of Nanopores using our custom-built inverted microscope and ionic current measurements. This is made possible by including a specialized flow cell for silicon-based Nanopores with an optical window for a high-numerical aperture microscope. Subsequently, we can insert individual DNA molecules into a single nanopore and arrest the DNA during voltage-driven translocation. To detect the position of the trapped particle in the optical trap with high accuracy in the presence of the nanopore, the optical tweezers uses reflected light from the bead for detection. Consequently, we can use our setup to directly determine the force on a DNA molecule in a solid-state nanopore. Finally, we suggest a number ...
Michael S Strano - One of the best experts on this subject based on the ideXlab platform.
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predicting gas separation through graphene nanopore ensembles with realistic pore size distributions
ACS Nano, 2021Co-Authors: Zhe Yuan, Ananth Govind Rajan, Michael S Strano, Rahul Prasanna Misra, Daniel BlankschteinAbstract:The development of nanoporous single-layer graphene membranes for gas separation has prompted increasing theoretical investigations of gas transport through graphene Nanopores. However, computer simulations and theories that predict gas permeances through individual graphene Nanopores are not suitable to describe experimental results, because a realistic graphene membrane contains a large number of Nanopores of diverse sizes and shapes. With this need in mind, here, we generate nanopore ensembles in silico by etching carbon atoms away from pristine graphene with different etching times, using a kinetic Monte Carlo algorithm developed by our group for the isomer cataloging problem of graphene Nanopores. The permeances of H2, CO2, and CH4 through each nanopore in the ensembles are predicted using transition state theory based on classical all-atomistic force fields. Our findings show that the total gas permeance through a nanopore ensemble is dominated by a small fraction of large Nanopores with low energy barriers of pore crossing. We also quantitatively predict the increase of the gas permeances and the decrease of the selectivities between the gases as functions of the etching time of graphene. Furthermore, by fitting the theoretically predicted selectivities to the experimental ones reported in the literature, we show that Nanopores in graphene effectively expand as the temperature of permeation measurement increases. We propose that this nanopore "expansion" is due to the desorption of contaminants that partially clog the graphene Nanopores. In general, our study highlights the effects of the pore size and shape distributions of a graphene nanopore ensemble on its gas separation properties and calls into attention the potential effect of pore-clogging contamination in experiments.
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addressing the isomer cataloguing problem for Nanopores in two dimensional materials
Nature Materials, 2019Co-Authors: Ananth Govind Rajan, Kevin S Silmore, Jacob Swett, Alex W Robertson, Jamie H Warner, Daniel Blankschtein, Michael S StranoAbstract:The presence of extended defects or Nanopores in two-dimensional (2D) materials can change the electronic, magnetic and barrier membrane properties of the materials. However, the large number of possible lattice isomers of Nanopores makes their quantitative study a seemingly intractable problem, confounding the interpretation of experimental and simulated data. Here we formulate a solution to this isomer cataloguing problem (ICP), combining electronic-structure calculations, kinetic Monte Carlo simulations, and chemical graph theory, to generate a catalogue of unique, most-probable isomers of 2D lattice Nanopores. The results demonstrate remarkable agreement with precise nanopore shapes observed experimentally in graphene and show that the thermodynamic stability of a nanopore is distinct from its kinetic stability. Triangular Nanopores prevalent in hexagonal boron nitride are also predicted, extending this approach to other 2D lattices. The proposed method should accelerate the application of nanoporous 2D materials by establishing specific links between experiment and theory/simulations, and by providing a much-needed connection between molecular design and fabrication. Nanopores in 2D materials have various possible lattice isomers, making relevant quantitative analysis difficult. An isomer-cataloguing framework is developed to address this problem, demonstrating remarkable agreement between simulated and experimental data.
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addressing the isomer cataloguing problem for Nanopores in two dimensional materials
Nature Materials, 2019Co-Authors: Ananth Govind Rajan, Kevin S Silmore, Jacob Swett, Alex W Robertson, Jamie H Warner, Daniel Blankschtein, Michael S StranoAbstract:The presence of extended defects or Nanopores in two-dimensional (2D) materials can change the electronic, magnetic and barrier membrane properties of the materials. However, the large number of possible lattice isomers of Nanopores makes their quantitative study a seemingly intractable problem, confounding the interpretation of experimental and simulated data. Here we formulate a solution to this isomer cataloguing problem (ICP), combining electronic-structure calculations, kinetic Monte Carlo simulations, and chemical graph theory, to generate a catalogue of unique, most-probable isomers of 2D lattice Nanopores. The results demonstrate remarkable agreement with precise nanopore shapes observed experimentally in graphene and show that the thermodynamic stability of a nanopore is distinct from its kinetic stability. Triangular Nanopores prevalent in hexagonal boron nitride are also predicted, extending this approach to other 2D lattices. The proposed method should accelerate the application of nanoporous 2D materials by establishing specific links between experiment and theory/simulations, and by providing a much-needed connection between molecular design and fabrication.
Cees Dekker - One of the best experts on this subject based on the ideXlab platform.
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sds assisted protein transport through solid state Nanopores
Nanoscale, 2017Co-Authors: Laura Restrepoperez, Aleksei Aksimentiev, Shalini John, Chirlmin Joo, Cees DekkerAbstract:Using Nanopores for single-molecule sequencing of proteins – similar to nanopore-based sequencing of DNA – faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through Nanopores. Here, we combine molecular dynamics (MD) simulations with single-molecule experiments to investigate the utility of SDS (Sodium Dodecyl Sulfate) to unfold proteins for solid-state nanopore translocation, while simultaneously endowing them with a stronger electrical charge. Our simulations and experiments prove that SDS-treated proteins show a considerable loss of the protein structure during the nanopore translocation. Moreover, SDS-treated proteins translocate through the nanopore in the direction prescribed by the electrophoretic force due to the negative charge impaired by SDS. In summary, our results suggest that SDS causes protein unfolding while facilitating protein translocation in the direction of the electrophoretic force; both characteristics being advantageous for future protein sequencing applications using solid-state Nanopores.
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dna translocations through solid state plasmonic Nanopores
Nano Letters, 2014Co-Authors: Francesca Nicoli, Cees Dekker, Daniel Verschueren, Misha Klein, Magnus P JonssonAbstract:Nanopores enable label-free detection and analysis of single biomolecules. Here, we investigate DNA translocations through a novel type of plasmonic nanopore based on a gold bowtie nanoantenna with a solid-state nanopore at the plasmonic hot spot. Plasmonic excitation of the nanopore is found to influence both the sensor signal (nanopore ionic conductance blockade during DNA translocation) and the process that captures DNA into the nanopore, without affecting the duration time of the translocations. Most striking is a strong plasmon-induced enhancement of the rate of DNA translocation events in lithium chloride (LiCl, already 10-fold enhancement at a few mW of laser power). This provides a means to utilize the excellent spatiotemporal resolution of DNA interrogations with Nanopores in LiCl buffers, which is known to suffer from low event rates. We propose a mechanism based on plasmon-induced local heating and thermophoresis as explanation of our observations.
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hybrid pore formation by directed insertion of α haemolysin into solid state Nanopores
Nature Nanotechnology, 2010Co-Authors: Adam R Hall, Andrew Scott, Dvir Rotem, Kunal Mehta, Hagan Bayley, Cees DekkerAbstract:Most experiments on Nanopores have concentrated on the pore-forming protein α-haemolysin (αHL) and on artificial pores in solid-state membranes. While biological pores offer an atomically precise structure and the potential for genetic engineering, solid-state Nanopores offer durability, size and shape control, and are also better suited for integration into wafer-scale devices. However, each system has significant limitations: αHL is difficult to integrate because it relies on delicate lipid bilayers for mechanical support, and the fabrication of solid-state Nanopores with precise dimensions remains challenging. Here we show that these limitations may be overcome by inserting a single αHL pore into a solid-state nanopore. A double-stranded DNA attached to the protein pore is threaded into a solid-state nanopore by electrophoretic translocation. Protein insertion is observed in 30-40% of our attempts, and translocation of single-stranded DNA demonstrates that the hybrid nanopore remains functional. The hybrid structure offers a platform to create wafer-scale device arrays for genomic analysis, including sequencing.
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solid state Nanopores
Nature Nanotechnology, 2007Co-Authors: Cees DekkerAbstract:The passage of individual molecules through nanosized pores in membranes is central to many processes in biology. Previously, experiments have been restricted to naturally occurring Nanopores, but advances in technology now allow artificial solid-state Nanopores to be fabricated in insulating membranes. By monitoring ion currents and forces as molecules pass through a solid-state nanopore, it is possible to investigate a wide range of phenomena involving DNA, RNA and proteins. The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.
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optical tweezers for force measurements on dna in Nanopores
Review of Scientific Instruments, 2006Co-Authors: Ulrich F. Keyser, Cees Dekker, J Van Der Does, Nynke H DekkerAbstract:We demonstrate the means to integrate two powerful and widely used single-molecule techniques, viz., optical tweezers and solid-state Nanopores. This setup permits simultaneous spatial sampling and high-resolution force measurements of nucleic acids and proteins. First, we demonstrate the rapid spatial localization of Nanopores using our custom-built inverted microscope and ionic current measurements. This is made possible by including a specialized flow cell for silicon-based Nanopores with an optical window for a high-numerical aperture microscope. Subsequently, we can insert individual DNA molecules into a single nanopore and arrest the DNA during voltage-driven translocation. To detect the position of the trapped particle in the optical trap with high accuracy in the presence of the nanopore, the optical tweezers uses reflected light from the bead for detection. Consequently, we can use our setup to directly determine the force on a DNA molecule in a solid-state nanopore. Finally, we suggest a number ...
