Nanopore sensors for biomolecular characterization
11698359 · 2023-07-11
Assignee
Inventors
Cpc classification
C12Q2522/101
CHEMISTRY; METALLURGY
C12Q2522/101
CHEMISTRY; METALLURGY
C12Q2537/164
CHEMISTRY; METALLURGY
G01N33/48721
PHYSICS
C12Q2537/164
CHEMISTRY; METALLURGY
International classification
Abstract
Provided herein are methods and devices for characterizing a biomolecule parameter by a nanopore-containing membrane, and also methods for making devices that can be used in the methods and devices provided herein. The nanopore membrane is a multilayer stack of conducting layers and dielectric layers, wherein an embedded conducting layer or conducting layer gates provides well-controlled and measurable electric fields in and around the nanopore through which the biomolecule translocates. In an aspect, the conducting layer is graphene.
Claims
1. A method for characterizing a biomolecule parameter, said method comprising the steps of: providing a hybrid biological solid-state nanopore in a membrane comprising a conductor-dielectric stack, wherein said membrane separates a first fluid compartment from a second fluid compartment and said nanopore fluidly connects said first and said second fluid compartments and said conductor comprises a conductor nanoribbons formed from an atomically thin electrically conducting layer of graphene and said solid-state nanopore has a constant diameter; providing the biomolecule to said first fluid compartment; applying an electric field across said membrane; driving said biomolecule through said hybrid biological solid-state nanopore to said second fluid compartment under said applied electric field; and monitoring an electrical parameter across the membrane or along a plane formed by the membrane as the biomolecule transits the hybrid biological solid-state nanopore by field effect sensing with the conductor nanoribbon, thereby characterizing said biomolecule parameter wherein said conductor-dielectric stack comprises: a plurality of graphene layers that form an outermost graphene layers and a one or more central graphene layer positioned between the outermost graphene layers, wherein adjacent graphene layers are separated by a dielectric layer; and said method further comprises: independently energizing the outermost graphene layers to slow down passage of the biomolecule and the central graphene layer for measuring a time-course of electric potential or transverse current along said conductor nanoribbon during said biomolecule transit through said hybrid biological solid-state nanopore, thereby characterizing a sequence or length of said biomolecule; and said hybrid biological solid-state nanopore comprises a protein.
2. The method of claim 1, wherein said biomolecule parameter is selected from the group consisting of: polynucleotide sequence; polynucleotide methylation state from a methylation-dependent protein bound to a polynucleotide sequence; presence of a protein-polynucleotide binding event; polypeptide sequence; and biomolecule secondary structure.
3. The method of claim 1, further comprising field effect gating by independently electrically biasing one or more of said graphene layers to provide electrical gating of said hybrid biological solid-state nanopore and wherein said biasing is by electrically connecting an electrode to an individual conductor layer embedded in the conductor-dielectric stack, and said biasing modifies an electric field in the hybrid biological solid-state nanopore generated by the applied electric field across the membrane.
4. The method of claim 1, wherein said dielectric layer comprises Aluminum Oxide, Tantalum Oxide, Silicon Dioxide, or Silicon Nitride.
5. The method of claim 1, wherein said electrical parameter is selected from one or more of the group consisting of: current or current blockade through the hybrid biological solid-state nanopore; conductance; resistance; impedance; electric potential; and translocation time of said biomolecule through said hybrid biological solid-state nanopore.
6. The method of claim 1, wherein said dielectric is deposited by atomic layer deposition.
7. A device for characterizing a biomolecule parameter, said device comprising: a membrane comprising: a first surface and a second surface opposite said first surface, wherein said membrane separates a first fluid compartment comprising said first surface from a second fluid compartment comprising said second surface, and at least one solid-state nanopore traversing said membrane between said first surface and said second surface; a conductor/dielectric stack positioned between said first surface and said second surface, wherein said conductor/dielectric stack comprises at least three conductor layers that form a top, a bottom and one or more middle conductor layers, with adjacent conductor layers separated by a dielectric layer; and a hybrid biological solid-state nanopore comprising a protein through said membrane solid-state nanopore that fluidically connects said first compartment and said second compartment, wherein said solid-state hybrid biological solid-state nanopore has a constant diameter; a power supply in electrical contact with said top and bottom conductor layers of the membrane to provide an electric potential difference between said first fluid compartment and said second fluid compartment; and a detector configured to detect an electrical current through said hybrid biological solid-state nanopore as a biomolecule transits said hybrid biological solid-state nanopore under an applied electric potential difference between said first and second fluid compartments, wherein said detector comprises a field effect sensor formed from the power supply in electrical contact with at least one of said middle conductor layer that is an atomically thin electrically conducting a graphene nanoribbon layer, wherein said electric potential difference of said top and bottom conductor layers provides independently controlled gating, and said at least one middle conductor layer is electrically isolated from said top and bottom conductor layers for control and characterization of the hybrid biological solid-state nanopore electric field.
8. The device of claim 7, wherein said top and/or bottom conductor layers form one or more gate electrodes, and each of said one or more gate electrodes is a graphene conductor layer in said conductor/dielectric stack, wherein the gate electrode is electrically connected to a source electrode powered by said power supply.
9. The device of claim 7, wherein said conductor layer has a thickness that is less than or equal to 3 nm at the hybrid biological solid-state nanopore, and said electrical contact comprises a Ti/Au pad in electrical contact with said conductor layer and an electrically conductive wire in electrical contact with said Ti/Au pad and said power supply, wherein said Ti/Au pad is electrically isolated from any of said first and second fluid compartment.
10. The method of claim 1, wherein said nanoribbon further comprises electrical contacts for measuring a transverse current along said nanoribbon during transit of a biomolecule through said hybrid biological solid-state nanopore.
11. The method of claim 1, wherein said hybrid biological solid-state nanopore has a diameter that is selected from a range between 5% and 95% of a width of the nanoribbon.
12. The device of claim 8, wherein each of said one or more gate electrodes is in electrical isolation to provide independent control of the electric field in and/or adjacent to the hybrid biological solid-state nanopore.
13. The device of claim 7, comprising two or more independently biased gate electrodes.
14. The device of claim 7, wherein vertically adjacent conductor nanoribbons have an overlap region of about 10000 nm.sup.2 in area.
15. The method of claim 1, wherein there are at least two vertically adjacent graphene layers that are nanoribbons, through which said hybrid biological solid-state nanopore traverses in a direction that is transverse to a longitudinal direction of said graphene nanoribbon, and wherein vertically adjacent graphene nanoribbons have an offset longitudinal direction with respect to each other.
16. The device of claim 7, wherein there are at least two vertically adjacent graphene layers that are nanoribbons, through which said hybrid biological solid-state nanopore transits in a transverse direction to a longitudinal direction of said graphene nanoribbon, and wherein vertically adjacent nanoribbons have an offset longitudinal direction with respect to each other that is between 10° and 130°.
17. The device of claim 7, wherein the hybrid biological solid-state nanopore comprises a specific recognition sequences or receptors attached to a surface of the membrane nanopore.
18. The device of claim 7, wherein said protein contains an aperture that is the nanopore.
19. The method of claim 1, wherein said protein is a polymerase, nuclease, histone, helicase, transcription factor, alpha hemolysin or Mycobacterium smegmatis porin A.
20. The device of claim 7, wherein said protein is a polymerase, nuclease, histone, helicase, transcription factor, alpha hemolysin or Mycobacterium smegmatis porin A.
21. The device of claim 7, wherein said protein is coaxially aligned with said solid-state nanopore.
Description
DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
DETAILED DESCRIPTION
(23) “Biomolecule” is used broadly herein to refer to a molecule that is relevant in biological systems. The term includes, for example, polynucleotides, DNA, RNA, polypeptides, proteins, and combinations thereof. The biomolecule may be naturally occurring or may be engineered or synthetic. A “biomolecule parameter” refers to a measurable or quantifiable property of the biomolecule. The parameter may be a constant for the biomolecule, such as the sequence or a sequence portion. The parameter may vary for a particular biomolecule depending on the state or conditions of the biomolecule, such as for a biomolecule parameter that is a methylation state, binding event and/or secondary structure. An “electrical parameter” refers to a parameter that can be electrically measured or determined and that relates to the biomolecule parameter. Accordingly, electrical parameter may be electrical in nature, or may itself by a non-electrical parameter that is determined based on an underlying parameter that is electrical in nature, such as transit or translocation time, flux, or translocation frequency.
(24) “Methylation” refers to DNA having one or more residues that are methylated. For example, in all vertebrate genomes some of the cytosine residues are methylated. DNA methylation can affect gene expression and, for some genes, is an epigenetic marker for cancer. Two different aspects of DNA methylation can be important: methylation level or content as well as the pattern of methylation. “Methylation state” is used broadly herein to refer to any aspect of methylation that is of interest from the standpoint of epigenetics, disease state, or DNA status and includes methylation content, distribution, pattern, density, and spatial variations thereof along the DNA sequence. Methylation detection via nanopores is further discussed in U.S. Pub. No. 2012/0040343 (168-08).
(25) In addition, biomolecule parameter refers to a quantitative variable that is measurable and is affected by the biomolecule transit through a nanopore, such as for example, translocation speed through a nanopore, variations in an electrical parameter (e.g., changes in the electric field, ionic current, resistance, impedance, capacitance, voltage) in the nanopore as the biomolecule enters and transits the pore, changes arising from biochemical reaction between the biomolecule and a nanopore surface region functionalized with a chemical moiety such as the release of pyrophosphotes, changes in pH including via a chemical moiety having exonuclease or endonuclease function.
(26) “Dielectric” refers to a non-conducting or insulating material. In an embodiment, an inorganic dielectric comprises a dielectric material substantially free of carbon. Specific examples of inorganic dielectric materials include, but are not limited to, silicon nitride, silicon dioxide, boron nitride, and oxides of aluminum, titanium, tantalum or hafnium. A “high-k dielectric” refers to a specific class of dielectric materials, for example in one embodiment those dielectric materials having a dielectric constant larger than silicon dioxide. In some embodiments, a high-k dielectric has a dielectric constant at least 2 times that of silicon dioxide. Useful high-k dielectrics include, but are not limited to Al.sub.2O.sub.3, HfO.sub.2, ZrO.sub.2, HfSiO.sub.4, ZrSiO.sub.4 and any combination of these. In an aspect, any of the methods and devices provided herein have a dielectric that is Al.sub.2O.sub.3.
(27) “Conductor-dielectric stack” refers to a plurality of layers, with at least one layer comprising an electrical conductor and another layer a dielectric. In an embodiment, a layer may be geometrically patterned or deposited, such as in a nanoribbon configuration including a conductor layer that is a conducting nanoribbon having a longitudinal direction that is transverse to the passage formed by the nanopore. In an aspect, the stack comprises 2 or more layers, 3 or more layers, or a range that is greater than or equal to 5 layers and less than or equal to 20 layers. In an aspect, adjacent conductor layers are separated from each other by a dielectric layer. In an aspect the outermost layers are conducting layers, dielectric layers, or one outermost layer that is dielectric and the other outermost layer at the other end of the stack is a conductor. In an aspect, local electric field may be applied and controlled near the membrane surface by selectively patterning a dielectric layer that covers an underlying conductor layer that is electrically energized. Any of the methods and devices provided herein have a conducting layer that is grapheme. As exemplified herein, the term graphene can be replaced, as desired, with other atomically thin electrically conducting layers, such as MoS.sub.2, doped silicon, silicene, or ultra-thin metal.
(28) “Fluid communication” or “fluidly connects” refers to a nanopassage that permits flow of electrolyte, and specifically ions in the electrolyte from one side of the membrane (e.g., first fluid compartment) to the other side of the membrane (e.g., second fluid compartment), or vice versa. In an aspect, the fluid communication connection is insufficient to permit biomolecule transit between sides without an applied electric field to facilitate transit through the nanopore. This can be controlled by combination of nanopore geometry (e.g., diameter), nanopore surface functionalization, applied electric field through the nanopore and biomolecule and fluid selection.
(29) “Specific binding” refers to an interaction between two components wherein one component has a targeted characteristic. Binding only occurs if the one component has the targeted characteristic and substantially no binding occurs in the absence of the targeted characteristic. In an embodiment, the targeted characteristic is a nucleotide type (e.g., A, T, G, C), an amino acid, or a specific sequence of nucleotides.
(30) The invention may be further understood by the following non-limiting examples. All references cited herein are hereby incorporated by reference to the extent not inconsistent with the disclosure herewith. Although the description herein contains many specificities, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of the invention. For example, thus the scope of the invention should be determined by the appended claims and their equivalents, rather than by the examples given. PCT Pub. No. WO 2010/080617, U.S. Pat. Pub. No. 2012/0040343 and U.S. Pat. Pub. No. 2011/0226623, filed Dec. 17, 2010) are specifically incorporated by reference to the extent not inconsistent herewith for the systems, devices and methods provided therein as related to biomolecule characterization by transit of the biomolecule through a nanopore under an applied electric field.
EXAMPLE 1: Graphene-Al.SUB.2.O.SUB.3 .Nanopores
(31) Graphene, an atomically thin sheet of carbon atoms densely packed into a two-dimensional honeycomb lattice possesses remarkable mechanical, electrical and thermal properties. The comparable thickness of a graphene monolayer to the 0.32-0.52 nm spacing between nucleotides in ssDNA, makes this material particularly attractive for electronic DNA sequencing. This example describes the development and characterization of novel graphene based Al.sub.2O.sub.3 nanopore sensors for the analysis of DNA and DNA-protein complexes. The nanopore is fabricated in a graphene-dielectric-graphene-dielectric stack, facilitating the independent biasing of each graphene layer. This structure is mechanically robust, exhibits stable conductance in ionic solution, is pH sensitive and is compatible with the integration of graphene nanoribbons and tunneling electrodes for graphene based nanopore DNA sequencing. In addition, the remarkable response of this platform to solution pH enables a sequencing by synthesis approach using ionic current alone. This platform is also well suited for use in diagnostics due to the single protein sensitivity demonstrated, particularly in methylation detection as shown here, applicable to cancer diagnostics.
(32) Fabrication of Graphene-Al.sub.2O.sub.3 Nanopores. A 300 nm diameter pore is first formed using a focused ion beam (FIB) tool in a free standing Al.sub.2O.sub.3 membrane (
(33) Electrical Characterization of Graphene-Al.sub.2O.sub.3 Nanopores. The current-voltage characteristics of graphene-Al.sub.2O.sub.3 nanopores are shown in
(34) Detection of dsDNA using graphene-Al.sub.2O.sub.3 Nanopores. To study the transport properties of graphene-Al.sub.2O.sub.3 nanopores, experiments are performed involving the translocation of λ-DNA, a 48.5 kbp long, dsDNA fragment extracted and purified from a plasmid. Given the relatively small persistence length of dsDNA (54±2 nm), λ-DNA is expected to assume the shape of a highly coiled ball in high salt solution with a radius of gyration, ∫R.sub.g=√{square root over (2l.sub.pL)}≈1.33 μm as shown in
(35) Two distinct blockade levels are observed in λ-DNA translocation experiments, a shallow blockade corresponding to linear dsDNA transport, and a deeper blockade level corresponding to folded DNA transport as seen in
(36) The λ-DNA translocation experiments described in this example show that the graphene-Al.sub.2O.sub.3 nanopore is highly sensitive at detecting not only the presence of a single molecule, but also discriminating its subtle secondary structure (folded or unfolded). Indeed, this system may read the topographic structure of protein bound DNA fragments and or secondary structures that form in ssRNA. Below, protein-DNA binding experiments involving estrogen receptor α to its cognate binding sequence are described.
EXAMPLE 2: Detection of Protein-DNA Complexes with Single Protein Resolution
(37) The translocation of protein-DNA complexes through a graphene/Al.sub.2O.sub.3 nanopore with the resolution of a single protein is shown in
(38) Another system examines recombination protein A, known to form stable nucleoprotein filaments on double-stranded DNA in the presence of magnesium and ATPγS. This model protein plays a central role in homologous recombination and DNA repair in prokaryotes. RecA-coated DNA molecules were prepared and provided by NABsys (Providence, R.I., USA) using a documented process (Smeets et al. Nano Lett. 2008, 9:3089-3095). The transport of this protein-DNA complex through a graphene-Al.sub.2O.sub.3 nanopore should induce significantly deeper current blockades relative to native dsDNA, as the effective diameter of this nucleoprotein filament is 7.5±0.5 nm.
(39) This example confirms that a multilayered graphene/Al.sub.2O.sub.3 nanopore can measure a biological parameter related to a single protein bound to dsDNA, and can be used in applications for detecting and spatially mapping single bound proteins on a DNA molecule.
EXAMPLE 3: Methylation Analysis
(40) Current methods for gene based methylation analysis are highly labor intensive, require large sample volumes, suffer from high per run cost and in most cases lack the sensitivity needed to derive useful clinical outcomes. In contrast, a nanopore based approach to methylation analysis for early cancer detection, though a radical departure from current clinical paradigms, may deliver the sensitivity and speed needed in extracting useful clinical information, relevant to patient outcome. Nanopore based techniques are well suited for gene based methylation analysis due to their ability to (1) detect target molecules at extremely low concentrations from minute sample volumes, (2) detect a combination of methylation aberrations across a variety of genes (important in monitoring disease progression and prognosis), (3) detect subtle variations in methylation patterns across alleles that would not be detected using bulk ensemble averaging methods such as PCR and gel-electrophoresis, (4) perform rapid methylation analysis (hundreds of copies of the same gene analyzed in minutes), (5) reduce cost (small reagent volumes needed), (6) simplify experimental and analysis steps by eliminating cumbersome PCR, DNA sequencing and bisulfite conversion steps.
(41) Analysis Protein bound Methylated DNA using Electrical Current Spectroscopy. The nanopore based methylation analysis process is illustrated in
(42) MeCP, MBD1 and MBD2 are selected as they bind specifically and exclusively to a single methylated CpG dinucleotides in vitro, and have been identified as critical components in transcriptional repression. The specificity of these proteins are used to label methylation sites along a methylated DNA molecule. The MBD-DNA complex is introduced into the cis chamber of the nanopore fluidic setup as shown in
(43) Methylation Determination: A single methylated DNA molecule from an unmethylated DNA fragment of equal length using nanopore based current spectroscopy methods (
(44) Methylation Quantification and Mapping: Current spectroscopy allows for the mapping of methylation sites along a specific DNA fragment and to quantify overall level of methylation. The process is illustrated in
(45) This raises the question as to the spatial resolution of the technique. DNase I footprinting confirm that the MBD of MeCP2 protects a total of 12-14 nucleotides surrounding a single methylated CpG pair. As the MBD of MeCP2 and MBD2 are homologous, we expect that MBD2 will cover approximately 12-14 bp of DNA upon binding also. Additional methyl CpG dinucleotides within this 12-14 bp domain are not available to bind to other MBD2 molecules, thereby limiting the spatial resolution of this technique. It is therefore expected that the nanopore platform can resolve individual MBD molecules positioned along a single DNA strand with good resolution given its high signal-to-noise ratio. The length-wise topographic reading process described in this example allows for quantification of methylation levels and to map methylation distributions along a single DNA fragment, and can be extended to the analysis of specific genes. This highly sensitive nanopore based methylation analysis technique is useful in medical diagnostics.
EXAMPLE 4: pH Dependent Response of Graphene-Al.SUB.2.O.SUB.3 .Nanopores
(46) Because of the high surface-to-volume ratio in nanopores, surfaces potentially have a very large effect on pore conductance at low salt concentrations. The surface charge characteristics and pH response of graphene-Al.sub.2O.sub.3 nanopores in particular can help facilitate a sequencing by synthesis approach by monitoring local changes in pH through the release of H.sup.+ ions during the incorporation of nucleotides using a DNA polymerase. At high salt concentrations, charge carriers in the solution dominate the ionic current through the pore. The conductance scales linearly with the number of charge carriers, as observed experimentally, and surface charge has negligible effect. At low KCl concentrations, however, the total current through the nanopore is a combination of the contributions of the bulk concentration of ions in solution and the counterions shielding the surface charge (electroosmotic flow). Above the isoelectric point of Al.sub.2O.sub.3 (˜pH 8-9), the surface charge in the pore is negative resulting in a double layer of condensed K ions, and below the isoelectric point, the surface charge is positive resulting in a double layer of condensed Cl counterions as shown in
(47) Conductance saturation is clearly observed at pH 10.9 as salt concentration is reduced, suggesting the presence of a highly charged, negative pore surface under these high pH conditions. In contrast, conductance saturation is not observed at pH 4 even at very low KCl concentrations (
(48) The pH response of graphene-Al.sub.2O.sub.3 nanopores is significantly more pronounced than the pH response of SiN and TiO.sub.2 nanopores as well as SiO.sub.2 nanochannels. This may in part be due to the presence of graphene in conjunction with the high surface charge density of Al.sub.2O.sub.3. Modulating the surface potential of the nanopore using solution pH can indeed modulate the conductance of the pore. This platform is suited to monitoring local pH during the incorporation of single nucleotides using DNA Polymerase, facilitating a sequencing by synthesis approach.
EXAMPLE 5: Graphene Gated Nanopores and Shaped Graphene Layers
(49) The concept of an electrically gated solid-state nanopore has been discussed, but the use of graphene as the gate material and the implementation of such a system was not previously demonstrated. A third electrode embedded in the nanopore is particularly attractive as it can be used to modify the electric fields in the pore and could be used to slow down or capture a translocating DNA molecule, a key step for implementation of nanopore sequencing. The effects of an insulated third electrode (30 nm thick TiN layer) on the conductances of both nanochannels and nanopores have been described. This example, however, discusses using graphene, of thickness only a few monolayers, as a nanopore electrode or a gate electrode. The realization of such a structure involves modifications to the architecture shown in
(50) Nanopore measurements with the graphene gate are conducted by tying the gate node to the source electrode, as shown in the schematic of
(51)
(52) A higher conductance level is seen at pH 10.9 and pH 7.6 with the gate connected relative to the floating case. In contrast, lower conductance is observed at pH 4 with the gate connected relative to the floating gate case. Though this current enhancement and reduction is more pronounced as the salt concentration is reduced suggesting an electrostatic effect, this result cannot be attributed solely to an electrostatic modulation of the field in the pore. It is likely that there are also electrochemical currents flowing through the contacted g2 layer, which are more pronounced at higher pH. This potentially explains the significant current amplification observed at 1M KCl, pH 10.9 conditions even though the Debye screening length at this concentration is only ˜0.3 nm. This is consistent with the notion that at high pH, OH— can disrupt the sp.sup.2 bonding of graphene resulting in charge transfer at the graphene fluid interface. This effect does not occur at low pH values, consistent with the lack of current enhancement observed in our experiments. The current modulation through the pore with the gate connected also cannot be attributed solely to leakage currents. Little variation in leakage current as a function of pH in the voltage range (−100 mV to 100 mV), identical to what is probed in gated nanopore measurements is observed. The results described in this application also suggest that the g2 layer may in fact be used as a trans electrode in the pore given the significant current transfer that is observed at this interface. This layer can serve as a sensitive electrode in future DNA translocation experiments. The application of local potentials in the pore via this third electrode is also useful in slowing or trapping DNA molecules in the pore.
(53) A Graphene Nanoribbon-Nanopore for DNA Detection and Sequencing: Theoretical-only feasibility of nucleotide discrimination using a graphene nanoribbon (GNR) with a nanopore in it was recently demonstrated. Nucleotide specific transverse currents through the ribbon are reported in those theoretical studies. This example uses a similar architecture for single molecule DNA sequencing.
(54) Nanoelectrodes in a Nanochannel as Voltage Sensors. The following electrode architecture (Wheatstone Bridge) in a nanochannel can facilitate the sensing of individual DNA molecules and DNA/protein complexes with very high spatial resolution, facilitating long range haplotype mapping of DNAs and sequencing using a voltage sensing approach. The architecture described here is shown in
(55) Graphene Nanoelectrodes in a Nanopore as Voltage Sensors. This example extends the two layer graphene/dielectric architecture to three layers for applications such as shown in
REFERENCES
(56) Wanunu, M. & Meller, A. Chemically Modified Solid-State Nanopores. Nano Letters 7, 1580-1585 (2007).
(57) Nam, S.-W., Rooks, M. J., Kim, K.-B. & Rossnagel, S. M. Ionic Field Effect Transistors with Sub-10 nm Multiple Nanopores. Nano Letters 9, 2044-2048 (2009).
(58) Stein, D., Kruithof, M. & Dekker, C. Surface-Charge-Governed Ion Transport in Nanofluidic Channels. Physical Review Letters 93, 035901 (2004).
(59) Karnik, R. et al. Electrostatic Control of Ions and Molecules in Nanofluidic Transistors. Nano Letters 5, 943-948 (2005).
(60) Nelson, T., Zhang, B. & Prezhdo, O. V. Detection of Nucleic Acids with Graphene Nanopores: Ab Initio Characterization of a Novel Sequencing Device. Nano Letters 10, 3237-3242 (2010).
EXAMPLE 6: Nano-Fabricated Graphene-Al.SUB.2.O.SUB.3 .Nanopores And Nanopore Arrays for the Sensitive Detection of DNA And DNA-Protein Complexes
(61) This example describes the fabrication of nanopores and nanopore arrays for the sensitive detection of single DNA molecules and DNA-protein complexes. High density arrays of ˜15 nm diameter nanopores are fabricated using electron beam lithography and reactive ion etch steps in SiN/Al.sub.2O.sub.3 membranes, facilitating high throughput analysis of single DNA molecules. The fabrication of single nanopores in ultra-thin graphene/Al.sub.2O.sub.3 membranes is also reported for detection of DNA-protein complexes. Single protein resolution at low salt concentrations is demonstrated.
(62) Nanopore DNA analysis is an emerging technique that involves electrophoretically driving DNA molecules through a nano-scale pore in solution and monitoring the corresponding change in ionic pore current. This versatile approach permits the label-free, amplification-free analysis of charged polymers (single stranded DNA, double stranded DNA and RNA) ranging in length from single nucleotides to kilobase long genomic DNA fragments with sub-nm resolution. Recent advances in nanopores suggest that this low-cost, highly scalable technology could lend itself to the development of third generation DNA sequencing technologies, promising rapid and reliable sequencing of the human diploid genome for under a $1000. To enable high throughput multiplexed sequencing using solid-state nanopores however, the fabrication of high density nanopore arrays is required. This example demonstrates an optimized process for the fabrication of ˜15 nm diameter nanopore arrays in suspended Al.sub.2O.sub.3/SiN membranes using electron beam lithography and dry etch processes, a platform technology well suited for parallel DNA analysis. The incorporation of graphene into solid-state nanopores also holds much promise. The comparable thickness of a graphene monolayer to the 0.32-0.52 nm spacing between nucleotides in single stranded DNA (ssDNA) makes this material particularly attractive for single nucleotide detection with application to electronic DNA sequencing. This example describes the fabrication of single nanopores in robust ultra-thin graphene/Al.sub.2O.sub.3 membranes and uses this architecture for the highly sensitive detection of single DNA-protein complexes. The model protein-DNA system used in these studies is Estrogen Receptor α (ERα) bound to a 50 basepair (bp) long probe containing its cognate binding sequence (Estrogen Response Element). These studies demonstrate the single protein sensitivity of this architecture and may be extended to the detection of various other DNA binding proteins, including transcription factors, nucleases and histones.
(63) The principle of nanopore sensing is analogous to that of a Coulter counter. A nano-scale aperture or nanopore is formed in an insulating membrane separating two chambers filled with conductive electrolyte. In the case of solid-state membranes, nanopores are formed via decompositional sputtering using a focused convergent electron beam to form a pore of cross-sectional diameter comparable to the 2.2 nm cross-sectional diameter of double stranded (ds) DNA. Charged molecules (e.g. DNA) are inserted into one of the fluidic chambers, and are electrophoretically driven through the pore under an applied electric potential thereby modulating the ionic current through the pore. The corresponding electronic signature reveals useful information about the structure and dynamic motion of the translocating molecule. This concept can be extended to sequencing in that if each passing nucleotide in ssDNA yields a characteristic residual ionic current, this current trace can then be used to extract sequence information.
(64) Experimental. Nanopore Array Fabrication. Free-standing Al.sub.2O.sub.3/SiN membranes are formed using a fabrication process documented previously. The membrane comprises a 350 Å thick Al.sub.2O.sub.3 layer deposited via atomic layer deposition (ALD) followed by a capping 430 Å thick SiN layer deposited via plasma enhanced chemical vapor deposition (PECVD). First, ZEP 520 e-beam resist dissolved in Anisole in a ratio of ZEP520:Anisole (2:3) is spun onto the free standing membrane (2000 rpm for 60 s), optimized to a final thickness of 750 Å for ˜10 nm feature definition. The ZEP520 coated chips are next baked at 200° C. for 2 minutes, followed by electron beam exposure using a JEOL JBX-6000FS Electron Beam Lithography (dose=10,000 μC/cm.sup.2). The array patterns are developed in Xylenes for 30 s followed by IPA for 30 s. A reactive ion etching (RIE) step is next used to transfer the array pattern in ZEP520 into the SiN. Etching is done in 60 sccm CF.sub.4: 6 sccm CHF.sub.3 at a power of 60 W and pressure of 80 mTorr. Etch rates of ˜600 Å/min versus ˜200 Å/min for ZEP520 are measured under these conditions. The ZEP520 and SiN etch windows serve as the mask for dry etching Al.sub.2O.sub.3, done in a PlasmaTherm SLR-770 Inductively Coupled Plasma (ICP) Reactive Ion Etcher. Etching is done in 10 sccm BCl.sub.3: 40 sccm Ar at an ICP power of 200 W, platen power of 20 W at a DC Bias ˜65V. An Al.sub.2O.sub.3 etch rate of ˜-220 Å/min versus 90 Å/min for SiN and 200 Å/min for ZEP520 is observed under these conditions.
(65) Fabrication of single nanopores in graphene/Al.sub.2O.sub.3 membranes. Free-standing Al.sub.2O.sub.3/SiN membranes are again formed using the membrane fabrication process documented previously. Large ˜300 nm diameter pores are milled in these membranes using a FEI DB235 focused ion beam (FIB) tool. Graphene films are grown using an Etamota chemical vapor deposition (CVD) system, on 1.4 mil copper foils purchased from Basic Copper. The foils are annealed under Ar/H.sub.2 flow for 45 minutes and graphene is grown under a CH.sub.4/H.sub.2/Ar flow at 1000° C., ˜500 mTorr for 20 min. The resulting Cu/graphene substrates are cooled to room temperature under Ar flow at a rate of ˜20° C./min. Graphene transfer to the receiving substrate proceeds as follows: graphene is coated with a bilayer of PMMA (495K and 950K), backside graphene on the copper foil is removed in an O.sub.2 plasma, and then the backside copper is etched in a 1M FeCl.sub.3 solution. The resultant PMMA/graphene film is wicked onto a glass slide, rinsed in DI water, rinsed in 10% HCl in DI to remove residual metal particles, followed by a final DI rinse, and wicked onto the receiving substrate. After the graphene dries on the receiving substrate, PMMA is removed in a 1:1 Methylene Chloride:Methanol solution. The transferred film is annealed in a CVD furnace at 400° C. under Ar/H.sub.2 flow to remove any residual PMMA. Next, a seed layer, such as a metal seed layer comprising 15 Å of metallic Al is evaporated on the graphene coated chip using a CHA SEC-600 E-Beam Evaporator. This layer completely oxidizes in air and serves as a seed layer for ALD Al.sub.2O.sub.3. 60 Å of Al.sub.2O.sub.3 is next deposited using ALD. A nanopore is drilled in the graphene/Al.sub.2O.sub.3 membrane using a focused convergent electron beam from a JEOL 2010F FEG-TEM with beam conditions similar to that used to drill pores in pure Al.sub.2O.sub.3 membranes.
(66) Results and Discussion. The nanopore array fabrication process is shown in
(67) The fabrication of individual nanopores in ultra-thin graphene/Al.sub.2O.sub.3 membranes is shown in
(68) The growth of primarily monolayer graphene using the CVD process employed here has been reported. Note the D peak seen in
(69) The translocation of protein-DNA complexes through a graphene/Al.sub.2O.sub.3 nanopore is shown in
(70) The fabrication of nanopores and nanopore arrays for the sensitive electrical detection of single DNA-protein complexes is demonstrated. The manufacture process allows for the formation of high density arrays of ˜15 nm diameter nanopores and greater, fabricated using electron beam lithography and reactive ion etch steps in suspended SiN/Al.sub.2O.sub.3 membranes. The process may further comprise individually addressing these pores with nano-scale electrodes to facilitate high throughput DNA analysis with application to DNA sequencing. The fabrication of single nanopores in ultra-thin graphene/Al.sub.2O.sub.3 membranes and the detection of DNA-protein complexes, specifically ERα/ERE, is also demonstrated. Importantly, a spatial resolution of a single protein is achieved using this platform at low salt concentrations.
REFERENCES
(71) Venkatesan, B. M. & Bashir, R. Nanopore Sensors for Nucleic Acid Analysis. Nat. Nanotechnol., 6:615-624 (Available online Sep. 18, 2011).
(72) Branton, D. et al. The potential and challenges of nanopore sequencing. Nat. Biotechnol. 26, 1146 (2008).
(73) Venkatesan, B. M. et al. Highly Sensitive, Mechanically Stable Nanopore Sensors for DNA Analysis. Adv. Mater. 21, 2771-2776 (2009).
(74) Venkatesan, B. M. et al. Lipid bilayer coated Al.sub.2O.sub.3 nanopore sensors: towards a hybrid biological solid-state nanopore. Biomed. Microdevices, 1-12 (2011).
(75) Venkatesan, B. M., Shah, A. B., Zuo, J. M. & Bashir, R. DNA Sensing Using Nanocrystalline Surface-Enhanced Al.sub.2O.sub.3 Nanopore Sensors. Adv. Funct. Mater. 20, 1266-1275 (2010).
(76) Li, X. et al. Large-Area Synthesis of High-Quality and Uniform Graphene Films on Copper Foils. Science 324, 1312-1314 (2009).
EXAMPLE 7: Nanopore Sensors for Nucleic Acid Analysis
(77) Nanopore DNA analysis is an emerging technique that involves electrophoretically driving DNA molecules through a nano-scale pore in solution and monitoring the corresponding change in ionic pore current. This versatile approach permits the label-free, amplification-free analysis of charged polymers (single stranded DNA, double stranded DNA and RNA) ranging in length from single nucleotides to kilobase long genomic DNA fragments with sub-nm resolution. Recent advances in nanopores suggest that this low-cost, highly scalable technology could lend itself to the development of third generation DNA sequencing technologies, promising rapid and reliable sequencing of the human diploid genome for under a $1000. The emerging role of nanopores in sequencing, genomic profiling, epigenetic analysis and medical diagnostics is described in this example.
(78) Sequencing the human genome has helped further understanding of disease, inheritance, and individuality. Genome sequencing has been critical in the identification of Mendelian disorders, genetic risk factors associated with complex human diseases, and continues to play an emerging role in therapeutics and personalized medicine. The growing need for cheaper and faster genome sequencing has prompted the development of new technologies that surpass conventional Sanger chain termination methods in terms of speed and cost. These novel second and third generation sequencing technologies, inspired by the $1000 genome challenge proposed by the National Institute of Health in 2004, are expected to revolutionize genomic medicine. Nanopore DNA sequencing is one such technology that is currently poised to meet this grand challenge.
(79) Nanopore DNA sequencing is attractive as it is a label-free, amplification-free single-molecule approach that can be scaled for high throughput DNA analysis. This technique typically requires low reagent volumes, benefits from relatively low cost and supports long read lengths, potentially enabling de novo sequencing and long-range haplotype mapping. The principle of nanopore sensing is analogous to that of a Coulter counter. A nano-scale aperture or nanopore is formed in an insulating membrane separating two chambers filled with conductive electrolyte. Charged molecules are electrophoretically driven through the pore under an applied electric potential thereby modulating the ionic current through the pore. The corresponding electronic signature reveals useful information about the structure and dynamic motion of the translocating molecule. This concept can be extended to sequencing as first proposed by Deamer, Branton, and Church in the 90's, in that if each passing nucleotide in single stranded DNA (ssDNA) yields a characteristic residual ionic current, this current trace can then be used to extract sequence information.
(80) Recent developments in biological nanopores suggest that nanopore sequencing is indeed feasible. Proof-of-principle experiments using biological α-hemolysin and MspA nanopores have shown significant progress in this direction. This example describes recent advances in this area along with new developments in solid-state and hybrid nanopore technology, in particular the incorporation of graphene that could enable single nucleotide discrimination and ultrafast sequencing. Efforts to slow down DNA translocation (
(81) Ionic current approaches have shown significant success in proof-of-principle sequencing experiments, particularly sequencing by exonuclease digestion and DI sequencing. Nanopore based optical approaches also show promise but require extensive conversion of DNA. Computational studies suggest that transverse electron tunneling and capacitive nanopore approaches may also facilitate ultrafast sequencing, though the experimental realization of these techniques is still pending.
(82) Biological Nanopores: Biological nanopores reconstituted into lipid bilayers present an attractive option for single molecule DNA analysis. Their versatility can be attributed to several factors: firstly, nature provides the cellular machinery to mass manufacture biological nanopores with an atomic level of precision that still cannot be replicated by the semiconductor industry; X-ray crystallographic information is available revealing pore structure with angstrom level resolution; techniques such as site directed mutagenesis can be used to tailor a pore's physical and chemical properties; and remarkable heterogeneity is observed amongst pores in terms of size and composition. In-vitro studies of DNA transport through biological nanopores have traditionally involved α-hemolysin, the structure of this heptameric protein pore shown in
(83) Most nanopore sequencing strategies to date have sought to actively or passively slow down the transport of ssDNA prior to electronic read-out. Active approaches typically incorporate enzymes to regulate DNA transport through the pore. An enzyme motor coupled to a nanopore is attractive for two reasons: (1) the enzyme-DNA complex forms in bulk solution enabling its electrophoretic capture in the pore and, (2) relatively slow and controlled motion is observed as the enzyme processively steps the DNA substrate through the pore. An elegant demonstration of this is the base-by-base ratcheting of ssDNA through α-hemolysin catalyzed by DNA Polymerase. Single nucleotide primer extension events were electronically observed only in the presence of a complementary nucleotide set, enabling sequencing. More recently, Lieberman et al. demonstrated the processive replication of ssDNA on α-hemolysin using phi29 DNA Polymerase. In addition to being able to resolve individual catalytic cycles, polymerase dynamics could also be discerned (dNTP binding, polymerase fingers opening-closing) using ionic current. A review on the controlled transport of DNA through α-hemolysin using DNA processing enzymes is provided by Deamer. Simpler, passive approaches to slowing down DNA also exist, for example, using nucleotide labeling, end termination of ssDNA with DNA hairpins, incorporating molecular brakes into the pore by lining the transmembrane domain with positively charged residues and so on, but no one approach has emerged in addressing the grand challenge of highly controlled, orientated molecule transport. Nucleotide labeling is quite attractive as the chemistry, charge, and size of the label can be varied potentially enabling “on the fly” sequencing, however labeling contiguous nucleotides in large genomic fragments presents challenges. A more versatile, label-free sequencing method was recently demonstrated by the Bayley group. In this study, Clarke et al. demonstrated the ability to continuously resolve indigenous mononucleotides (dAMP, dCMP, dGMP, dTMP) through α-hemolysin using resistive current measurements. Base selectivity was achieved by modifying a mutant α-hemolysin pore with an aminocyclodextrin adapter covalently bound within the β barrel of the transmembrane domain, thereby constricting the channel while enhancing the chemical specificity of the sensor. Raw mononucleotides were read with over 99% confidence under optimal operating conditions. By integrating this base identification platform with a highly processive exonuclease through either chemical attachment or genetic fusion, a nanopore based single molecule sequencing by digestion approach may indeed be feasible. Such an approach forms the basis for commercial sequencing efforts by Oxford Nanopore (Oxford, UK).
(84) Although α-hemolysin has by far dominated the biological nanopore sequencing landscape in the past, more efficient biological nanopores for sequencing have already begun to emerge. A structural drawback with α-hemolysin pertains to its ˜5 nm long cylindrical β barrel that accommodates up to ˜10 nucleotides at a time. Nucleotides located in this β barrel significantly modulate the pore current and subsequently dilute the ionic signature specific to a single nucleotide in the narrowest 1.4 nm pore constriction, reducing the overall signal-to-noise ratio in sequencing applications. This inherent structural limitation is overcome by a relatively new candidate in the nanopore sequencing arena, the channel porin Mycobacterium smegmatis porin A (MspA). MspA is an octameric protein channel that contains a single constriction of diameter ˜1.2 nm with a channel length of ˜0.5 nm, forming a tapered funnel shape (structural cross section shown in
(85) The application of biological nanopores to areas outside DNA sequencing also holds tremendous potential. One particular biological pore that could find useful applications in molecular diagnostics and DNA fingerprinting is the connector protein from the bacteriophage phi29 DNA packaging motor. The versatility of this protein nanopore stems from its relative size, the protein hub being comprised of twelve GP10 subunits that self-assemble to form a channel of inner diameter ˜3.6 nm. Interestingly, the open channel conductance of this nanopore is ˜5 times higher than that of α-hemolysin under similar conditions, suggesting the possibility of screening larger analytes including dsDNA, DNA protein complexes and amino acid polymers for protein sequencing. The translocation of dsDNA through a genetically engineered connector channel embedded in a lipid bilayer was recently demonstrated by Wendell et al. Unidirectional transport of dsDNA through this channel (from N-terminal entrance to C-terminal exit) was observed, suggesting a natural valve mechanism in the channel that assists dsDNA packaging during bacteriophage phi29 virus maturation. The capabilities of this exciting protein nanopore will become more apparent in years to come.
(86) Solid-State Nanopores. Despite the heterogeneity and remarkable sensitivity of biological nanopores, these sensors do exhibit some inherent disadvantages. The delicate nature of the mechanically supporting lipid bilayer, the sensitivity of biological pores to experimental conditions (pH, temperature, salt concentration), and challenges associated with large scale array integration for high throughput DNA analysis/sequencing limit the versatility of these biological platforms. Even with ongoing improvements to bilayer stability through the development of supported bilayers on solid and nanoporous substrates, varying bilayer lipid compositions, and the development of tethered bilayer architectures, the robustness and durability of solid-state membranes still significantly supersedes that of their biological counterparts. Coupled with advances in microfabrication techniques, solid-state nanopores are therefore fast becoming an inexpensive and highly versatile alternative to biological nanopores. Other advantages afforded by solid-state technology include the ability to tune nanopore dimensions with sub-nm precision, the ability to fabricate high density nanopore arrays, superior mechanical, chemical, and thermal characteristics over lipid-based systems and the possible integration with electrical and optical probing techniques.
(87) The first reports of DNA sensing using solid-state nanopores emerged from the Golovchenko lab in early 2001. Nanopores were formed in thin SiN membranes using a custom built feedback controlled ion beam sculpting tool, a process that yields true nanometer control over pore size. Today, most groups prefer to use a focused convergent electron beam from a field emission gun (FEG) TEM to decompositionally sputter nanopores in thin insulating membranes, a technique that has evolved since the 1980s. The fabrication of solid-state nanopores in thin insulating membranes is reviewed by Healy et al. and the application of this technology to single molecule biophysics is reviewed by Dekker. SiN has traditionally been the nanopore membrane material of choice due to its high chemical resistance and low mechanical stress, deposited via an optimized low pressure chemical vapor deposition process. This process is typically done at elevated temperature (˜800° C.) and lacks thickness control in the sub-nm regime. To effectively probe the local structure of DNA with the resolution of an individual nucleotide, insulating membranes of sub-nm thickness are required. In working towards this goal, a method of forming nanopores in ultra-thin insulating Al.sub.2O.sub.3 membranes using atomic layer deposition (ALD) is proposed, a process that yields angstrom level control over membrane thickness. The fabrication of nanopores in Al.sub.2O.sub.3 membranes using a focused electron beam revealed two interesting phenomena, the dose-dependent conversion of Al.sub.2O.sub.3 to metallic Al, applicable to the direct ‘write’ of nanoscale electrodes in the pore, and the controlled formation of α and γ nanocrystalline domains, permitting nano-scale surface charge engineering at the pore/fluid interface. Controlling the stoichiometry of the material in the pore and surface charge density is important given the impact of these parameters on 1/f noise and DNA transport velocities. Interestingly, slower DNA transport was observed in Al.sub.2O.sub.3 nanopores relative to SiN pores of similar diameter, attributed to strong electrostatic interactions between the positively charged Al.sub.2O.sub.3 surface and negatively charged dsDNA. Enhancing these interactions, either electrostatically or chemically, could further help reduce DNA transport velocities, a prerequisite for nanopore sequencing. The versatility of this ALD based technique also allows for: 1) the formation of membranes and nanopores in a variety of other high-k dielectric materials including TiO.sub.2 and HfO.sub.2, each with unique material properties and 2) the integration of metallic contacts and graphene layers directly into the membrane due to the low temperature nature of the ALD process (typically <250° C.). Though this approach has shown much promise, the fabrication of robust, insulating ALD membranes of sub-nm thickness has proven challenging due to ionic current leakage through pinholes in ultrathin films. The formation of sub-nm thick insulating membranes will therefore likely require a novel approach.
(88) Graphene: Graphene, an atomically thin sheet of carbon atoms densely packed into a two-dimensional honeycomb lattice possesses remarkable mechanical, electrical and thermal properties. The comparable thickness of a graphene monolayer to the 0.32-0.52 nm spacing between nucleotides in ssDNA, makes this material particularly attractive for electronic DNA sequencing. The first reports of single nanopores and nanopore arrays fabricated in suspended graphene films emerged from the Drndic lab in 2008. Subsequent TEM based studies by the Zettl group elucidated both the kinetics of pore formation in graphene and graphene edge stability (zig-zag versus armchair) in-situ. The detection of individual dsDNA molecules using graphene nanopores however, has only been recently demonstrated. In separate studies, the Golovchenko (Harvard), Dekker (Delft), and Drndic (University of Pennsylvania) labs reported the electron-beam based fabrication of 5-25 nm diameter nanopores in suspended graphene films, prepared through either chemical vapor deposition (CVD) or exfoliation from graphite. Nanopores were formed in as few as 1-2 monolayers of graphene with membranes exhibiting remarkable durability and insulating properties in high ionic strength solution. The conductance of pores in monolayer thin membranes exhibited some unique trends. The Harvard study showed a linear scaling of pore conductance with pore diameter, d.sub.pore, in monolayer thin membranes as opposed to the d.sub.pore.sup.2 scaling typically observed with pores in thick SiN membranes. An effective membrane thickness, h.sub.eff, of ˜0.6 nm was extracted for nanopores formed in a graphene monolayer. This result agrees with the theory in the limit as h.sub.eff.fwdarw.0 where the dominant resistance is not the pore resistance itself (R.sub.p 1 but rather the access ore, resistance (R.sub.access attributed to the potential drop in the electrolyte from the electrode to the nanopore), where R.sub.access scales inversely with d.sub.pore. In contrast, the Delft studies showed that the conductance of nanopores in a graphene monolayer scales as a function of d.sub.pore.sup.2, an intriguing result that suggests a cylindrical nanopore geometry of non-negligible thickness (R.sub.pore>R.sub.access). The origin of this d.sub.pore.sup.2 scaling may be due to a polymer coating (6-mercaptohexanoic acid) introduced on the graphene to reduce DNA adsorption. Furthermore, the Delft group reported similar conductance values for equidiameter pores formed in a single monolayer versus pores formed in membranes of thickness up to 8 monolayers. The latter result is plausible as nanopore formation in multilayer graphene is known to induce a terrace effect where the number of graphene layers monotonically decreases radially in the direction of the pore center, with regions of only monolayer thickness lining the pore edge (
(89) The translocation of dsDNA through graphene pores induced subtle fluctuations in the ionic current marking the transport of both folded and unfolded DNA structures, analogous to DNA induced current blockades in SiN nanopores. Translocation velocities ranged anywhere from 10-100 nts/μs, too fast for the electronic measurement of individual nucleotides. As a result, Garaj probed the theoretical spatial and geometric resolution of a graphene monolayer nanopore using computational analysis. Pseudo-static simulations of dsDNA in a 2.4-nm diameter graphene pore of thickness ˜0.6 nm revealed a resolution of ˜0.35 nm, identical to the size of an individual DNA nucleotide. This exciting result suggests that if DNA translocation could be sufficiently slowed in a graphene pore to say ˜1 nt/ms, single nucleotide detection is theoretically possible potentially facilitating electronic sequencing. To enable such advancements however, the quantitative aspects of DNA transport need to be better understood. For example, it still remains to be seen why under normalized conditions (salt concentration, voltage), nanopores in multilayer graphene (3-15 monolayers) give deeper DNA induced current blockades relative to pores in single layer graphene. One possible explanation is the terrace effect previously mentioned, though more detailed studies on graphene nanopore structure, properties and quantitative DNA transport are needed. A number of fundamental questions pertaining to sequencing also remain. For example, it is not clear whether single nucleotide resolution is experimentally realizable in the presence of thermodynamic fluctuations and electrical noise. Furthermore, the chemical and structural similarity amongst purines and pyrimidines could inherently limit the identification of individual nucleotides using ionic current alone through a bare graphene pore. Surface functionalization of graphene pores may be necessary to enhance nucleotide specificity, though such an approach may compromise resolution due to membrane thickening.
(90) Nanopore applications outside DNA sequencing. The more immediate application for solid-state nanopores is likely in medical diagnostics. A nanopore based diagnostic tool could: (1) detect target molecules at very low concentrations from minute sample volumes (perhaps shed DNA from tumor cells in patient serum); (2) simultaneously screen panels of biomarkers/genes (important in diagnosis, monitoring progression and prognosis); (3) provide rapid analysis at relatively low cost; and (4) eliminate cumbersome amplification and conversion steps such as PCR, bisulfite conversion, and Sanger sequencing. MicroRNA (miRNA) expression profiling is one application where solid-state nanopore technology could excel. The detection and accurate quantification of these cancer biomarkers will likely have important clinical implications, facilitating disease diagnosis, staging, progression, prognosis, and treatment response. Wanunu et al. recently demonstrated a nanopore based approach for the detection of specific microRNA sequences enriched from cellular tissue with sensitivities surpassing conventional micro-array technologies (
(91) Genetic analysis involving the detection of single nucleotide polymorphisms (SNPs) is another important diagnostic application tailored for nanopores. SNPs and point mutations have been linked to a variety of Mendelian diseases such as cystic fibrosis and Huntington's disease as well as more complex disease phenotypes. In proof-of-principle experiments, Zhao and coworkers demonstrated the sensitive detection of SNPs using ˜2 nm diameter SiN nanopores. Using the nanopore as a local force actuator, the binding energies of a DNA binding protein and its cognate sequence relative to a SNP sequence could be discriminated (
(92) Hybrid Biological/Solid-State Nanopores. A major drawback with solid-state nanopore technology at present is the inability to chemically differentiate analytes of the same approximate size. This lack of chemical specificity can be overcome through surface modification of the pore via the attachment of specific recognition sequences and receptors, in essence forming a hybrid structure. A chemically sensitive nanopore may be necessary to uniquely identify nucleotides in sequencing applications or to differentiate and quantify target proteins in diagnostic applications. Chemical functionalization and its effect on the electrical properties of polymer nanopores was recently demonstrated by Siwy. Surface functionalization can also be used to introduce DNA sequence specificity. In studies involving DNA hairpin functionalized SiO.sub.2 nanopores, higher flux and smaller translocation times were observed for the passage of perfect complementary (PC) ssDNA versus single base mismatched probes (1 MM), a highly sensitive strategy for the detection of SNPs (
(93) The concept of a hybrid biological solid-state nanopore was recently advanced by Dekker and co-workers, through the direct insertion of genetically engineered α-hemolysin into 2.4-3.6 nm diameter SiN nanopores. A simple yet elegant strategy was devised to control the orientation of α-hemolysin in the solid-state pore. By chemically linking a long dsDNA tail to the protein pore as shown in
(94) The advances described here suggest that nanopores will likely play an increasing role in medical diagnostics and DNA sequencing in years to come. As new optical and electronic approaches for the detection and sequencing of DNA molecules emerge, including single molecule evanescent field detection of sequencing-by-synthesis in arrays of nano-chambers (Pacific Biosciences), sequencing by ligation on self-assembled DNA nanoarrays (Complete Genomics), and the detection of H.sup.+ ions released during sequencing-by-synthesis on silicon field effect transistors from multiple polymerase-template reactions (Ion Torrent), the goal of direct read ‘on the fly’ sequencing of a single molecule using a biological or solid-state nanopore still remains a highly attractive grand challenge. The exciting possibility of performing long base reads on unlabeled ssDNA molecules in a rapid and cost-effective manner could revolutionize genomics and personalized medicine. This fascinating prospect continues to drive innovation in both academic and commercial settings, including large scale investment from the NIH and private sector investment from companies including Roche/IBM, Oxford Nanopore, and NABsys. Current trends suggest that significant hurdles inhibiting the use of biological nanopores in sequencing (high translocation velocity, a lack of nucleotide specificity) have been resolved. Similarly, if DNA translocation through solid-state nanopores could be slowed down to ˜3 Å/ms (length of a single nucleotide moving in a millisecond through a sensor region with spatial resolution of ˜3 Å), and if nucleotides could be identified uniquely with an electronic signature, a 1 million base long molecule could be sequenced in less than 20 minutes. Scaling this technology to an array of 100,000 individually addressed nanopores operating in parallel could enable the sequencing of a 3 billion bp human genome with 50 fold coverage in less than 1 hour.
(95) To achieve this, novel architectures that add functionality at the nanopore interface are likely needed, such as the electronically gated nanopores and nanochannels provided herein, the integration of single-walled carbon nanotubes, and graphene nanoribbons and nanogaps embedded in a nanopore. IBM's approach to sequencing using a DNA nanopore transistor architecture is equally intriguing. Using molecular dynamics, the IBM group demonstrated the controlled base-by-base ratcheting of ssDNA through a nanopore formed in a multilayered metal-oxide membrane using alternating electric fields applied across the metal layers. An experimental demonstration of this result has not yet been shown however. Recent experimental advances using scanning tunneling microscopy are also exciting and suggest the possibility of identifying nucleotides using electron tunneling (nucleotide specific tunneling currents being associated with differences in the HOMO-LUMO gaps of A,C,G,T) and the partial sequencing of DNA oligomers. The use of nanofabricated metallic gap junctions to measure nucleotide specific electron tunneling currents is particularly fascinating in that if a tunneling detector of this nature could be embedded in a nanopore and DNA could be sufficiently slowed, the goal of solid-state nanopore sequencing may be attainable. Exemplary nanopore architectures for sequencing are shown in
(96) Efforts to fabricate nanogap-nanopore tunneling detectors are currently underway, though the path to sequencing is not trivial given thermal fluctuations of bases within the nanopore (whether individual nucleotides or contiguous nucleotides in ssDNA) and electrical noise. Hence a statistical approach involving many repeat sampling events of each nucleotides/molecule will likely be needed to obtain sequence information. Additionally, as tunneling currents are exponentially dependent on barrier widths and heights (based on the effective tunnel distance and molecule orientation), a two point measurement might inherently provide only limited information. Perhaps a measurement setup analogous to a 4 point probe is needed, however, reliably fabricating such a structure with sub-nm precision is still a formidable task. It should also be noted that for certain applications, all 4 bases might not need to be uniquely identified. Investigators have been using binary conversion of nucleotide sequences (A/T=0, and G/C=1), to successfully map short DNA and RNA fragments to the genome for marker discovery and identification of genomic alterations. Hence, even the direct sequencing with binary identification of nucleotide pairs in dsDNA using nanopores could be of significant prognostic and diagnostic value.
(97) In summary, significant advances have been made over the past few years in both biological and solid-state nanopores for label-free ‘on the fly’ sequencing of DNA molecules. There is no doubt that nanopores will stay as an important enabler of generation three sequencing technologies in the race towards affordable and personalized DNA sequencing.
(98) Exemplary embodiments of certain devices and methods are provided in
(99) Referring to
REFERENCES
(100) Thomas, P. D. & Kejariwal, A. Coding single-nucleotide polymorphisms associated with complex vs. Mendelian disease: Evolutionary evidence for differences in molecular effects. Proc. Natl Acad. Sci. USA 101, 15398-15403 (2004).
(101) The International HapMap, C. A haplotype map of the human genome. Nature 437, 1299-1320 (2005).
(102) Mardis, E. R. Next-Generation DNA Sequencing Methods. Annu. Rev. Genom. Human Genet. 9, 387-402 (2008).
(103) Metzker, M. L. Sequencing technologies—the next generation. Nat. Rev. Genet. 11, 31-46 (2010).
(104) http://www.genome.gov/12513210.
(105) Coulter, W. H. Means for counting particles suspended in a fluid. U.S. Pat. No. 2,656,508 (1953).
(106) Church, G., Deamer, D. W., Branton, D., Baldarelli, R. & Kasianowicz, J. Characterization of individual polymer molecules based on monomer-interface interactions. U.S. Pat. No. 5,795,782 (1995).
(107) Deamer, D. W. & Branton, D. Characterization of Nucleic Acids by Nanopore Analysis. Acc. Chem. Res. 35, 817-825 (2002).
(108) Rhee, M. & Burns, M. A. Nanopore sequencing technology: research trends and applications. Trends Biotechnol. 24, 580-586 (2006).
(109) Dekker, C. Solid-state nanopores. Nat. Nanotechnol. 2, 209-215 (2007).
(110) Healy, K. Nanopore-based single-molecule DNA analysis. Nanomedicine 2, 459-481 (2007).
(111) Branton, D. & et al. The potential and challenges of nanopore sequencing. Nat. Biotechnol. 26, 1146 (2008). This review article assesses the feasibility of various nanopore sequencing techniques that are currently under development (both optical and electrical).
(112) Deamer, D. W. Nanopore analysis of nucleic acids bound to exonucleases and polymerases. Annu Rev Biophys 39, 79-90 (2010). This review article provides a historical perspective on the field of nanopore DNA sequencing and elaborates on nanopore based enzyme mediated sequencing approaches.
(113) Iqbal, S. & Bashir, R. Nanopores: Sensing and Fundamental Biological Interactions, Edn. 1. (Springer, N.Y., 2011).
(114) Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. USA 93, 13770-13773 (1996).
(115) 16. Akeson, M., Branton, D., Kasianowicz, J. J., Brandin, E. & Deamer, D. W. Microsecond Time-Scale Discrimination Among Polycytidylic Acid, Polyadenylic Acid, and Polyuridylic Acid as Homopolymers or as Segments Within Single RNA Molecules. Biophys. J. 77, 3227-3233 (1999).
(116) Meller, A. & Branton, D. Single molecule measurements of DNA transport through a nanopore. Electrophoresis 23, 2583-2591 (2002).
(117) Benner, S. et al. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nat. Nanotechnol. 2, 718-724 (2007).
(118) Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. A Single-Molecule Nanopore Device Detects DNA Polymerase Activity with Single-Nucleotide Resolution. J. Am. Chem. Soc. 130, 818-820 (2008).
(119) Lieberman, K. R. et al. Processive Replication of Single DNA Molecules in a Nanopore Catalyzed by phi29 DNA Polymerase. J. Am. Chem. Soc. 132, 17961-17972 (2010). A polymerase coupled to a nanopore as shown here, could be used for both strand sequencing as well as mechanistic studies of enzyme function.
(120) Olasagasti, F. et al. Replication of individual DNA molecules under electronic control using a protein nanopore. Nat. Nanotechnol. 5, 798-806 (2010).
(121) Rincon-Restrepo, M., Mikhailova, E., Bayley, H. & Maglia, G. Controlled Translocation of Individual DNA Molecules through Protein Nanopores with Engineered Molecular Brakes. Nano Lett. 11, 746-750 (2011).
(122) Mitchell, N. & Howorka, S. Chemical Tags Facilitate the Sensing of Individual DNA Strands with Nanopores13. Angew. Chem. Int. Ed. 47, 5565-5568 (2008).
(123) Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265-270 (2009). This study forms the basis for Oxford Nanopore's sequencing by exonuclease digestion approach using biological α-hemolysin.
(124) Stoddart, D., Heron, A. J., Mikhailova, E., Maglia, G. & Bayley, H. Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc. Natl Acad. Sci. USA 106, 7702-7707 (2009).
(125) Faller, M., Niederweis, M. & Schulz, G. E. The Structure of a Mycobacterial Outer-Membrane Channel. Science 303, 1189-1192 (2004).
(126) Derrington, I. M. et al. Nanopore DNA sequencing with MspA. Proc. Natl Acad. Sci. USA 107, 16060-16065 (2010). Proof-of-principle experiments demonstrating DI sequencing using MspA are shown in this work.
(127) Wendell, D. et al. Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nat. Nanotechnol. 4, 765-772 (2009).
(128) Jing, P., Haque, F., Shu, D., Montemagno, C. & Guo, P. X. One-Way Traffic of a Viral Motor Channel for Double-Stranded DNA Translocation. Nano Lett. 10, 3620-3627 (2010).
(129) Groves, J. T., Ulman, N. & Boxer, S. G. Micropatterning fluid lipid bilayers on solid supports. Science 275, 651-653 (1997).
(130) Mager, M. D. & Melosh, N. A. Nanopore-Spanning Lipid Bilayers for Controlled Chemical Release. Adv. Mater. 20, 4423-4427 (2008).
(131) White, R. J. et al. Ionic conductivity of the aqueous layer separating a lipid bilayer membrane and a glass support. Langmuir 22, 10777-10783 (2006).
(132) Venkatesan, B. M. et al. Lipid bilayer coated Al.sub.2O.sub.3 nanopore sensors: towards a hybrid biological solid-state nanopore. Biomed. Microdevices, 1-12 (2011).
(133) Chung, M. & Boxer, S. G. Stability of DNA-Tethered Lipid Membranes with Mobile Tethers. Langmuir 27, 5492-5497 (2011).
(134) Langford, K. W., Penkov, B., Derrington, I. M. & Gundlach, J. H. Unsupported planar lipid membranes formed from mycolic acids of Mycobacterium tuberculosis. J. Lipid Res. 52, 272-277 (2011).
(135) Knoll, W., Köper, I., Naumann, R. & Sinner, E.-K. Tethered bimolecular lipid membranes—A novel model membrane platform. Electrochim. Acta 53, 6680-6689 (2008).
(136) Storm, A. J., Chen, J. H., Ling, X. S., Zandbergen, H. W. & Dekker, C. Fabrication of solid-state nanopores with single nanometer precision. Nat. Mater. 2, 537-540 (2003).
(137) Venkatesan, B. M. et al. Highly Sensitive, Mechanically Stable Nanopore Sensors for DNA Analysis. Adv. Mater. 21, 2771-2776 (2009).
(138) Kim, M. J., Wanunu, M., Bell, D. C. & A. Meller Rapid Fabrication of Uniformly Sized Nanopores and Nanopore Arrays for Parallel DNA Analysis. Adv. Mater. 18, 3149-3153 (2006).
(139) Nam, S.-W., Rooks, M. J., Kim, K.-B. & Rossnagel, S. M. Ionic Field Effect Transistors with Sub-10 nm Multiple Nanopores. Nano Lett. 9, 2044-2048 (2009).
(140) McNally, B. et al. Optical Recognition of Converted DNA Nucleotides for Single-Molecule DNA Sequencing Using Nanopore Arrays. Nano Lett. 10, 2237-2244 (2010). Each nucleotide is converted to a preset nucleotide specific sequence and hybridized with fluorescent probes. Hybridized probes are sequentially cleaved off during transport through <2 nm diameter pores allowing optical sequence read out.
(141) Li, J. et al. Ion-beam sculpting at nanometre length scales. Nature 412, 166-169 (2001).
(142) Salisbury, I. G., Timsit, R. S., Berger, S. D. & Humphreys, C. J. Nanometer scale electron beam lithography in inorganic materials. Appl. Phys. Lett. 45, 1289-1291 (1984).
(143) Healy, K., Schiedt, B. & Morrison, A. P. Solid-state nanopore technologies for nanopore-based DNA analysis. Nanomedicine 2, 875-897 (2007).
(144) Venkatesan, B. M., Shah, A. B., Zuo, J. M. & Bashir, R. DNA Sensing Using Nanocrystalline Surface-Enhanced Al.sub.2O.sub.3 Nanopore Sensors. Adv. Funct. Mater. 20, 1266-1275 (2010).
(145) Hoogerheide, D. P., Garaj, S. & Golovchenko, J. A. Probing Surface Charge Fluctuations with Solid-State Nanopores. Phys. Rev. Lett. 102, 256804 (2009).
(146) George, S. M. Atomic Layer Deposition: An Overview. Chem. Rev. 110, 111-131 (2009).
(147) Geim, A. K. Graphene: Status and Prospects. Science 324, 1530-1534 (2009).
(148) Fischbein, M. D. & Drndic, M. Electron beam nanosculpting of suspended graphene sheets. Appl. Phys. Lett. 93, 113107-113103 (2008). This is the first report of nanopore fabrication in a suspended graphene sheet using a focused convergent electron beam and has served as the inspiration for subsequent studies involving DNA transport through graphene nanopores.
(149) Girit, Ç. Ö. et al. Graphene at the Edge: Stability and Dynamics. Science 323, 1705-1708 (2009).
(150) Garaj, S. et al. Graphene as a subnanometre trans-electrode membrane. Nature 467, 190-193 (2010).
(151) Merchant, C. A. et al. DNA Translocation through Graphene Nanopores. Nano Lett. 10, 2915-2921 (2010). Along with experimental data showing DNA translocation through a graphene monolayer nanopore, this paper importantly calculates the theoretical spatial resolution of a monolayer thin graphene nanopore sensor.
(152) Schneider, G. g. F. et al. DNA Translocation through Graphene Nanopores. Nano Lett. 10, 3163-3167 (2010).
(153) Hall, J. E. Access resistance of a small circular pore. J. Gen. Physiol. 66, 531-532 (1975).
(154) Song, B. et al. Atomic-Scale Electron-Beam Sculpting of Near-Defect-Free Graphene Nanostructures. Nano Lett. 11, 2247-2250 (2011).
(155) Li, J., Gershow, M., Stein, D., Brandin, E. & Golovchenko, J. A. DNA molecules and configurations in a solid-state nanopore microscope. Nat. Mater. 2, 611-615 (2003).
(156) Storm, A. J. et al. Fast DNA Translocation through a Solid-State Nanopore. Nano Lett. 5, 1193-1197 (2005).
(157) Wanunu, M., Morrison, W., Rabin, Y., Grosberg, A. Y. & Meller, A. Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient. Nat. Nanotechnol. 5, 160-165 (2010).
(158) Calin, G. A. & Croce, C. M. MicroRNA signatures in human cancers. Nat. Rev. Cancer 6, 857-866 (2006).
(159) Volinia, S. et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl Acad. Sci. USA 103, 2257-2261 (2006).
(160) Wanunu, M. et al. Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors. Nat. Nanotechnol. 5, 807-814 (2010).
(161) Strathdee, G. & Brown, R. Aberrant DNA methylation in cancer: potential clinical interventions. Expert Rev. Mol. Med. 4, 1-17 (2002).
(162) Lee, W. H., Isaacs, W. B., Bova, G. S. & Nelson, W. G. CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: A new prostate cancer biomarker. Cancer Epidem. Biomar. 6, 443-450 (1997).
(163) Laird, P. W. The power and the promise of DNA methylation markers. Nat. Rev. Cancer 3, 253-266 (2003).
(164) Mirsaidov, U. et al. Nanoelectromechanics of Methylated DNA in a Synthetic Nanopore. Biophys. J. 96, L32-L34 (2009).
(165) Wanunu, M. et al. Discrimination of Methylcytosine from Hydroxymethylcytosine in DNA Molecules. J. Am. Chem. Soc. 133, 486-492 (2010).
(166) Botstein, D. & Risch, N. Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease. Nat. Genet. (2003).
(167) Zhao, Q. et al. Detecting SNPs Using a Synthetic Nanopore. Nano Lett. 7, 1680-1685 (2007).
(168) Singer, A. et al. Nanopore Based Sequence Specific Detection of Duplex DNA for Genomic Profiling. Nano Lett. 10, 738-742 (2010).
(169) Iqbal, S. M., Akin, D. & Bashir, R. Solid-state nanopore channels with DNA selectivity. Nat. Nanotechnol. 2, 243-248 (2007).
(170) Wanunu, M. & Meller, A. Chemically Modified Solid-State Nanopores. Nano Lett. 7, 1580-1585 (2007).
(171) Siwy, Z. S. & Howorka, S. Engineered voltage-responsive nanopores. Chem. Soc. Rev. 39, 1115-1132 (2009).
(172) Kowalczyk, S. W. et al. Single-molecule transport across an individual biomimetic nuclear pore complex. Nat. Nanotechnol. advance online publication (2011).
(173) Yusko, E. C. et al. Controlling protein translocation through nanopores with bio-inspired fluid walls. Nat. Nanotechnol. 6, 253-260 (2011).
(174) Hall, A. R. et al. Hybrid pore formation by directed insertion of alpha-haemolysin into solid-state nanopores. Nat. Nanotechnol. 5, 874-877 (2010).
(175) Vercoutere, W. et al. Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel. Nat. Biotech. 19, 248-252 (2001).
(176) Eid, J. et al. Real-Time DNA Sequencing from Single Polymerase Molecules. Science 323, 133-138 (2009).
(177) Drmanac, R. et al. Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanoarrays. Science 327, 78-81 (2010).
(178) http://www.iontorrent.com.
(179) http://www.genome.gov/27527584.
(180) Karnik, R., Duan, C., Castelino, K., Daiguji, H. & Majumdar, A. Rectification of Ionic Current in a Nanofluidic Diode. Nano Lett. 7, 547-551 (2007).
(181) Jin, X. & Aluru, N. Gated transport in nanofluidic devices. Microfluid. Nanofluid., 1-10 (2011).
(182) Liu, H. et al. Translocation of Single-Stranded DNA Through Single-Walled Carbon Nanotubes. Science 327, 64-67 (2010).
(183) Nelson, T., Zhang, B. & Prezhdo, O. V. Detection of Nucleic Acids with Graphene Nanopores: Ab Initio Characterization of a Novel Sequencing Device. Nano Lett. 10, 3237-3242 (2010).
(184) Min, S. K., Kim, W. Y., Cho, Y. & Kim, K. S. Fast DNA sequencing with a graphene-based nanochannel device. Nat. Nanotechnol. 6, 162-165 (2011).
(185) Postma, H. W. C. Rapid Sequencing of Individual DNA Molecules in Graphene Nanogaps. Nano Lett. 10, 420-425 (2010).
(186) Prasongkit, J., Grigoriev, A., Pathak, B., Ahuja, R. & Scheicher, R. H. Transverse Conductance of DNA Nucleotides in a Graphene Nanogap from First Principles. Nano Lett. 11, 1941-1945 (2011).
(187) Luan, B. et al. Base-By-Base Ratcheting of Single Stranded DNA through a Solid-State Nanopore. Phys. Rev. Lett. 104, 238103 (2010). IBM's DNA transistor architecture and proposed approach to nanopore based single molecule DNA sequencing are presented here.
(188) Huang, S. et al. Identifying single bases in a DNA oligomer with electron tunnelling. Nat. Nanotechnol. 5, 868-873 (2010).
(189) Zwolak, M. & Di Ventra, M. Colloquium: Physical approaches to DNA sequencing and detection. Rev. Mod. Phys. 80, 141-165 (2008).
(190) Tanaka, H. & Kawai, T. Partial sequencing of a single DNA molecule with a scanning tunnelling microscope. Nat. Nanotechnol. 4, 518-522 (2009).
(191) Tsutsui, M., Taniguchi, M., Yokota, K. & Kawai, T. Identifying single nucleotides by tunnelling current. Nat. Nanotechnol. 5, 286-290 (2010). This is the first report of a nanofabricated gap junction being used to successfully discriminate individual nucleotides through electron tunneling measurements.
(192) Taniguchi, M., Tsutsui, M., Yokota, K. & Kawai, T. Fabrication of the gating nanopore device. Appl. Phys. Lett. 95, 123701-123703 (2009).
(193) Ivanov, A. P. et al. DNA Tunneling Detector Embedded in a Nanopore. Nano Lett. 11, 279-285 (2010).
(194) Asmann, Y. W., Kosari, F., Wang, K., Cheville, J. C. & Vasmatzis, G. Identification of Differentially Expressed Genes in Normal and Malignant Prostate by Electronic Profiling of Expressed Sequence Tags. Cancer Res. 62, 3308-3314 (2002).
(195) Feldman, A. L. et al. Discovery of recurrent t (6;7)(p25.3; q32.3) translocations in ALK-negative anaplastic large cell lymphomas by massively parallel genomic sequencing. Blood 117, 915-919 (2010).
(196) Meller, A., Nivon, L., Brandin, E., Golovchenko, J. & Branton, D. Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl Acad. Sci. USA 97, 1079-1084 (2000).
(197) Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nat. Biotech. 19, 636-639 (2001).
(198) Bates, M., Burns, M. & Meller, A. Dynamics of DNA Molecules in a Membrane Channel Probed by Active Control Techniques. Biophys. J. 84, 2366-2372 (2003).
(199) Astier, Y., Braha, O. & Bayley, H. Toward Single Molecule DNA Sequencing: Direct Identification of Ribonucleoside and Deoxyribonucleoside 5′-Monophosphates by Using an Engineered Protein Nanopore Equipped with a Molecular Adapter. J. Am. Chem. Soc. 128, 1705-1710 (2006).
(200) Borsenberger, V., Mitchell, N. & Howorka, S. Chemically Labeled Nucleotides and Oligonucleotides Encode DNA for Sensing with Nanopores. J. Am. Chem. Soc. 131, 7530-7531 (2009).
(201) Chen, P. et al. Probing Single DNA Molecule Transport Using Fabricated Nanopores. Nano Lett. 4, 2293-2298 (2004).
(202) Storm, A. J., Chen, J. H., Zandbergen, H. W. & Dekker, C. Translocation of double-strand DNA through a silicon oxide nanopore. Phys. Rev. E 71, 051903 (2005).
(203) Fologea, D., Uplinger, J., Thomas, B., McNabb, D. S. & Li, J. Slowing DNA Translocation in a Solid-State Nanopore. Nano Lett. 5, 1734-1737 (2005).
(204) Kim, Y. R. et al. Nanopore sensor for fast label-free detection of short double-stranded DNAs. Biosensors Bioelectron. 22, 2926-2931 (2007).
(205) Wanunu, M., Sutin, J., McNally, B., Chow, A. & Meller, A. DNA Translocation Governed by Interactions with Solid-State Nanopores. Biophys. J. 95, 4716-4725 (2008).
(206) Chen, Z. et al. DNA translocation through an array of kinked nanopores. Nat. Mater. 9, 667-675 (2010).
(207) Ling, X. S., Bready, B. & Pertsinidis, A. Hybridization-assisted nanopore sequencing of nucleic acids. US patent no. 2007 0190542 (2007). The HANS approach is being commercially developed by NABsys. 6-mer oligonucleotide probes are hybridized to ssDNA and current-vs-time traces are recorded as the complex translocates through the pore, spatially revealing the probe's position on the ssDNA template.
(208) Lagerqvist, J., Zwolak, M. & Di Ventra, M. Fast DNA Sequencing via Transverse Electronic Transport. Nano Lett. 6, 779-782 (2006).
(209) Heng, J. B. et al. Beyond the gene chip. Bell Labs Tech. J. 10, 5-22 (2005).
(210) Gracheva, M. E. et al. Simulation of the electric response of DNA translocation through a semiconductor nanopore-capacitor. Nanotechnology 17, 622-633 (2006).
(211) Sigalov, G., Comer, J., Timp, G. & Aksimentiev, A. Detection of DNA Sequences Using an Alternating Electric Field in a Nanopore Capacitor. Nano Lett. 8, 56-63 (2007).
(212) All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
(213) All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employed herein, if needed, to exclude (for example, to disclaim) specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the prior art, including certain compounds disclosed in the references disclosed herein (particularly in referenced patent documents), are not intended to be included in the claim.
(214) Examples of documents incorporated specifically by reference to the extent the disclosure is not inconsistent with that provided herein include: Venkatesan et al. “Stacked Graphene-Al2O3 Nanopore Sensors for Sensitive Detection of DNA and DNA-Protein Complexes.” ACS NANO 6(1): 441-450 (2012); PCT Pub. No. WO 2010/08061; U.S. Pat. Pub. Nos. 2012/0040343 and 2011/0226623.
(215) When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a number range, a voltage range, or a velocity range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.
(216) As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
(217) TABLE-US-00001 TABLE 1 Nanopore sequencing techniques and potential challenges: Sensing Modality Description of Technique Potential Challenges Ionic Current Hybridization assisted High spatial resolution required. nanopore sequencing Complex algorithms needed for analysis Sequencing by exonuclease Requires sequential passage of digestion mononucleotides in order in which they are cleaved Sequencing by synthesis Retaining processing enzymes (DNA polymerase) at the pore, Achieving long read lengths and maintaining enzyme activity under a voltage load Duplex Interrupted (DI) Converting large genomic ssDNA fragments to DNA sequencing DI structure Optical Readout Optical recognition of Complex and error-prone DNA conversion steps, converted DNA High density <2 nm nanopore arrays needed Transverse election Tunneling detector Precisely controlling orientation and position tunneling on a nanopore of nucleotides in the gap, (Metal, Graphene, Slow translocation rates required to sufficiently Carbon Nanotubes) sample over noise, Nucleotide dependent tunneling currents need to be measured in solution Capacitive Sensing Metal-Oxide-Semiconductor Must operate in high ionic strength solution with nanopore capacitor negligible drift and leakage, DNA translocation rates need to be substantially reduced