Patent Description:
There is a need in the art for systems and methods capable of precise control over the electrical properties in and around the nanopore to better control biomolecule transit and/or electrical parameter measurement in and around the nanopore, particularly during biomolecule transit or interaction with the nanopore. The methods and devices disclosed herein are configured to characterize a wide range of biomolecules, including different aspects of the biomolecule as desired, that are not readily achieved by conventional systems known in the art.

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. Specially configured membranes containing a plurality of graphene/dielectric layers in a stack configuration, with a nanopore through the layers, to facilitate improved control of biomolecule transit through the nanopore as well as measuring or monitoring of an electrical parameter generated during biomolecule transit through the nanopore.

The invention relates to a method for characterizing a biomolecule parameter, said method comprising the steps of: providing a 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 is graphene and said nanopore is a hybrid biological/ solid-state nanopore; 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, thereby characterizing said biomolecule parameter wherein said conductor-dielectric stack comprises: a plurality of conductor layers, wherein adjacent conductor layers are separated by a dielectric layer; wherein one or more of said conductor layers comprises a conductor nanoribbon, through which said nanopore traverses in a direction that is transverse to a longitudinal direction of said conductor nanoribbon; and said method further comprises 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.

In some embodiments, 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.

In some embodiments, the method comprises independently electrically biasing one or more of said graphene layers to provide electrical gating of said hybrid biological/solid-state nanopore, wherein said biasing is by electrically connecting an electrode to an individual graphene 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.

In some embodiments, said dielectric layer comprises Aluminum Oxide, Tantalum Oxide, Silicon Dioxide, or Silicon Nitride.

In some embodiments, said electrical parameter is selected from one or more of the group consisting of: current or current blockade through the nanopore; conductance; resistance; impedance; electric potential; and translocation time of said biomolecule through said nanopore.

The invention also relates to 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; a conductor / dielectric/ conductor / dielectric stack positioned between said first surface and said second surface; and a nanopore through said membrane that fluidically connects said first compartment and said second compartment and said nanopore is a hybrid biological/solid-state nanopore to a surface of the nanopore; a power supply in electrical contact with said membrane to provide an electric potential difference between said first fluid compartment and said second fluid compartment; and a detector 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 the conductor is graphene.

In some embodiments, the device further comprises one or more gate electrodes, wherein each of said one or more gate electrodes is a graphene layer in said stack, wherein the gate electrode is electrically connected to a source electrode powered by said power supply.

In some embodiments, said dielectric is deposited by atomic layer deposition.

In some embodiments, said graphene layer has a thickness that is less than or equal to <NUM> at the nanopore, and said electrical contact comprises a Ti/Au pad in electrical contact with said graphene layer and an electrically conductive wire in electrical contact with said Ti/Au pad, wherein said Ti/Au pad is electrically isolated from any of said first and second fluid compartment.

In some embodiments, one of said conductor layers comprises a nanoribbon through which said nanopore transits in a transverse direction to said nanoribbon longitudinal axis.

Said nanoribbon may further comprise electrical contacts for measuring a transverse current along said nanoribbon during transit of a biomolecule through said nanopore.

In some embodiments, said nanopore has a diameter that is greater than <NUM>% of the nanoribbon width or is selected from a range between <NUM>% and <NUM>% of the nanoribbon width.

In some embodiments, 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 nanopore.

In some embodiments, the device comprises two or more independently biased gate electrodes.

"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.

"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 <CIT> (<NUM>-<NUM>).

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.

"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 <NUM> times that of silicon dioxide. Useful high-k dielectrics include, but are not limited to Al<NUM>O<NUM>, HfO<NUM>, ZrO<NUM>, HfSiO<NUM>, ZrSiO<NUM> and any combination of these. In an aspect, any of the methods and devices provided herein have a dielectric that is Al<NUM>O<NUM>.

"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 <NUM> or more layers, or a range that is greater than or equal to <NUM> layers and less than or equal to <NUM> layers. 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. All of the methods and devices of the invention use graphene conducting layers.

"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. "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.

The invention may be further understood by the following non-limiting examples. 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. The scope of the invention should be determined by the appended claims rather than by the examples given. <CIT>, <CIT> and <CIT> are cited herein 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.

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 <NUM>-<NUM> 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<NUM>O<NUM> 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.

Fabrication of Graphene-Al<NUM>O<NUM> Nanopores. A <NUM> diameter pore is first formed using a focused ion beam (FIB) tool in a free standing Al<NUM>O<NUM> membrane (<FIG>). Graphene, grown via chemical vapor deposition (CVD) is next transferred onto this substrate spanning over the <NUM> Al<NUM>O<NUM> pore (<FIG>). This layer is referred to as graphene <NUM> or g1. Graphene growth conditions are as follows: CVD graphene is grown on <NUM> mil copper foils. The foils are annealed under Ar/H<NUM> flow for <NUM> mins and graphene is grown under a CH<NUM>/H<NUM> flow at <NUM>, ≈ <NUM> mTorr for <NUM> mins. The resulting Cu/graphene substrates are cooled to room temperature under Ar flow at a rate of ~<NUM>/min. Transfer to the receiving substrate proceeds as follows: graphene is coated with a bilayer of PMMA (<NUM> and <NUM>), backside graphene on the copper foil is removed in an O<NUM> plasma, and then the backside copper is etched in a <NUM> FeCl<NUM> solution. The resultant PMMA/graphene film is wicked onto a glass slide, rinsed in DI water, rinsed in <NUM>% HCl in DI to remove residual metal particles and wicked onto the receiving substrate. After the graphene dries on the receiving substrate, PMMA is removed in a <NUM>:<NUM> Methylene Chloride:Methanol solution. The transferred film is annealed in a CVD furnace at <NUM> under Ar/H<NUM> flow to remove any residual PMMA. Following the annealing step, electron diffraction imaging and Raman Spectroscopy are used to evaluate the quality of the graphene (<FIG> right column). Next, <NUM> of metallic Aluminum is evaporated onto the graphene to form an adhesion layer followed by <NUM> of Al<NUM>O<NUM> (dielectric layer <NUM> or d1) deposited via atomic layer deposition (ALD). Process steps 1b and 1c are repeated once more i.e. growth and transfer of a second graphene layer (g2) and repeat Al/Al<NUM>O<NUM> deposition (d2) resulting in a graphene/ Al<NUM>O<NUM>/ graphene/ Al<NUM>O<NUM> stack as shown in <FIG>. Note, a gold pad is used to contact the g2 layer at its edge allowing the application of gate potentials to the conductive g2 layer. Finally, a field emission gun TEM is used to form a nanopore in this stack as shown in <FIG>.

Electrical Characterization of Graphene-Al<NUM>O<NUM> Nanopores. The current-voltage characteristics of graphene-Al<NUM>O<NUM> nanopores are shown in <FIG> for pores of varying size in <NUM> KCI, <NUM> Tris, <NUM> EDTA, pH <NUM>. Linear IV curves are generally observed suggesting a symmetric nanopore structure as previously reported for Al<NUM>O<NUM> nanopores. The IV characteristics of four pores of varying diameter are shown in <FIG>. Also shown are fits to the data constructed using numerical simulations. <FIG> also shows the conductance stability of these same pores as a function of time. Stable conductance values are obtained for over <NUM> minutes, confirming the stability of these pores in ionic fluid. Conductance values after drilling a nanopore are several orders of magnitude higher than the conductance of a graphene-Al<NUM>O<NUM> membrane with no pore as seen in <FIG> (solid squares).

Detection of dsDNA using graphene-Al<NUM>O<NUM> Nanopores. To study the transport properties of graphene-Al<NUM>O<NUM> nanopores, experiments are performed involving the translocation of λ-DNA, a <NUM> kbp long, dsDNA fragment extracted and purified from a plasmid. Given the relatively small persistence length of dsDNA (<NUM> ± <NUM>), λ-DNA is expected to assume the shape of a highly coiled ball in high salt solution with a radius of gyration, <MAT> as shown in <FIG>. Upon capture in the nanopore, the elongation and threading process occurs as shown in part (ii). <FIG> illustrates the corresponding current blockades induced by λ-DNA as it translocates through an <NUM> diameter pore at an applied voltage of <NUM> mV in <NUM> KCI, <NUM> Tris, <NUM> EDTA pH <NUM>. The λ-DNA concentration used in these experiments is <NUM> ng/µl. High pH buffer is used to minimize electrostatic interactions between the bottom graphene surface of the nanopore and the negatively charged dsDNA molecule. Also, it is important to note that Al<NUM>O<NUM> is negatively charged at this pH value (isoelectric point of Al<NUM>O<NUM> is <NUM>-<NUM>) and thus will not electrostatically bind DNA. Thus, these experimental conditions yield repeatable DNA translocation through grahene-Al<NUM>O<NUM> nanopores.

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 <FIG> and the current blockage histogram of <FIG>. Note that ΔI here represents the current blockage induced by dsDNA relative to the baseline current at a particular voltage (<NUM> mV in this case). The current histogram of <FIG> is constructed from <NUM> individual DNA translocation events. To confirm that these events are indeed due to DNA translocation and not simply interactions with the pore surface, the effect of voltage on translocation time is probed. Voltage dependent DNA transport is observed, translocation times, to, decreasing with increasing voltage, corresponding to an increased electrophoretic driving force. Measured values for translocation time are tD = <NUM> ± <NUM> at <NUM> mV (<FIG>) and to = <NUM> ± <NUM> at <NUM> mV from n=<NUM> events (<FIG> inset). The broad distribution of translocation times is representative of translocations involving significant interactions with the pore surface.

The λ-DNA translocation experiments described in this example show that the graphene-Al<NUM>O<NUM> 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.

The translocation of protein-DNA complexes through a graphene/Al<NUM>O<NUM> nanopore with the resolution of a single protein is shown in <FIG>. The model DNA-protein system used in these studies is ERα bound to a <NUM> bp long probe containing a single ERE, the cognate binding sequence for the ERα protein. DNA-bound ERα primarily serves as a nucleating factor for the recruitment of protein complexes and is involved in key biological processes including oxidative stress response, DNA repair, and transcription regulation. A schematic showing the binding of ERα to dsDNA containing a single ERE and the ERE sequence itself are shown in <FIG> respectively. <FIG> shows a gel shift assay, ERα/ERE binding being observed exclusively at low salt concentrations. The detection of protein-DNA complexes using a nanopore is analogous to dsDNA detection as shown in <FIG>. Notably, the transport of the ERα/ERE complex through a -<NUM> diameter pore in <NUM> KCI results in current enhancements (<FIG>), likely due to counterion condensation on the complex locally increasing pore conductance during transport as previously reported in DNA transport studies at low salt. A translocation time versus current enhancement scatter plot is shown in <FIG>. The most probable translocation time for this <NUM> bp long DNA probe at <NUM> mV with a single bound ERα protein is ~<NUM>, two orders of magnitude slower than the estimated translocation time for a <NUM> bp dsDNA alone.

Another system examines recombination protein A, known to form stable nucleoprotein filaments on double-stranded DNA in the presence of magnesium and ATPyS. 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, Rl, USA) using a documented process (<NPL>). The transport of this protein-DNA complex through a graphene-Al<NUM>O<NUM> nanopore should induce significantly deeper current blockades relative to native dsDNA, as the effective diameter of this nucleoprotein filament is <NUM> ± <NUM>. <FIG> shows nanopore current versus time for the transport of <NUM> kbp long RecA-coated dsDNA molecules through a <NUM> diameter graphene- Al<NUM>O<NUM> nanopore in <NUM> KCI, <NUM> Tris, <NUM> EDTA, pH <NUM> electrolyte at an applied voltage of <NUM> mV. Deep current blockades are observed during the translocation of the nucleoprotein filament through the pore with significantly higher signal-to-noise ratio (SNR) relative to native dsDNA (higher temporal resolution traces are shown in <FIG>). <FIG> shows an event density plot of current blockage versus translocation time (tD) constructed from <NUM> individual RecA-related translocation events; the corresponding event amplitude histogram is shown in <FIG>. Two categories of transport events are clearly distinguishable: fast, low-amplitude events corresponding to the transport of unbound or free RecA protein as previously shown in SiN nanopores, and slower, higher amplitude current blockage events corresponding to the transport of single RecA-coated DNA molecules. The translocation time scales for the two event categories described are consistent with that reported in RecA-DNA translocation experiments in SiN nanopores (Smeets et al. Interestingly, a third high-amplitude peak at a current blockage value of about <NUM> nA is also observed in <FIG>. This may correspond to the simultaneous transport of multiple RecA-coated DNA molecules through the nanopore.

This example confirms that a multilayered graphene/Al<NUM>O<NUM> 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.

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 (<NUM>) detect target molecules at extremely low concentrations from minute sample volumes, (<NUM>) detect a combination of methylation aberrations across a variety of genes (important in monitoring disease progression and prognosis), (<NUM>) detect subtle variations in methylation patterns across alleles that would not be detected using bulk ensemble averaging methods such as PCR and gel-electrophoresis, (<NUM>) perform rapid methylation analysis (hundreds of copies of the same gene analyzed in minutes), (<NUM>) reduce cost (small reagent volumes needed), (<NUM>) simplify experimental and analysis steps by eliminating cumbersome PCR, DNA sequencing and bisulfite conversion steps.

Analysis Protein bound Methylated DNA using Electrical Current Spectroscopy. The nanopore based methylation analysis process is illustrated in <FIG>. First, methylated DNA molecules are combined with methyl-CpG binding proteins to form protein bound DNA complexes (<FIG>). The methyl-CpG-binding protein family (MBD) consists of five proteins, MeCP2, MBD1, MBD2, MBD3 and MBD4, each containing a methyl-CpG-binding domain (MBD) that allows them to bind to methylated DNA. Any of these are used to label methylated CpG dinucleotides.

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 <FIG>. Under an applied potential, these protein bound, methylated DNA fragments translocate through the pore resulting in characteristic current blockades, representative of the methylation status of the molecule.

Methylation Determination: A single methylated DNA molecule from an unmethylated DNA fragment of equal length using nanopore based current spectroscopy methods (<FIG>). The passage of unmethylated DNA through the pore produces only a slight deviation in the baseline current as illustrated in <FIG>. The passage of an MBD protein bound DNA fragment through the pore, however, results in a very different current signature (<FIG>). As the drop in pore current is related to the cross section of the translocating molecule, deeper blockades are observed when the large, bound protein traverses the pore. Two distinct blockade levels occur, the first corresponding to regions of DNA that do not contain bound proteins (IDNA), and the second corresponding to regions containing the MBD protein (IMBD). Gel shift assays have shown that fragments with multiple bound MBD proteins corresponding to multiple methylated CpG dinucleotides migrate slower through the gel and can be resolved with single protein resolution. Furthermore, each additional bound protein significantly reduces the mobility of the complex in the gel. This is attributed to two factors; (<NUM>) the high molecular weight of MBD2 relative to the short DNA fragments, (<NUM>) the positive charge of MBD2 in pH <NUM> buffer (isoelectric point of <NUM>). Thus, under normal pore operating conditions (pH <NUM>-<NUM>), MBD bound DNA translocation is expected.

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 <FIG>. The presence of multiple fully methylated CpG dinucleotides along a single DNA molecule facilitates the binding of multiple MBD proteins per DNA, each of which produces a deep current blockade during translocation. The translocation of fragments with multiple bound proteins results in an electrical readout as shown in <FIG> that resembles the spatial distribution of proteins along that fragment. This can then be used to determine the distribution of methylated CpG dinucleotides along the interrogated DNA fragment. The current signature can also be used to quantify the extent of methylation based on the number of deep current blockades per event.

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 <NUM>-<NUM> nucleotides surrounding a single methylated CpG pair. As the MBD of MeCP2 and MBD2 are homologous, we expect that MBD2 will cover approximately <NUM>-<NUM> bp of DNA upon binding also. Additional methyl CpG dinucleotides within this <NUM>-<NUM> 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.

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<NUM>O<NUM> nanopores in particular can help facilitate a sequencing by synthesis approach by monitoring local changes in pH through the release of H+ 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 KCI 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<NUM>O<NUM> (~pH <NUM>-<NUM>), 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 <FIG>. Pore conductance as a function of KCI concentration and pH is shown in <FIG> respectively for <NUM> ± <NUM> diameter and <NUM> ± <NUM> diameter graphene-Al<NUM>O<NUM> nanopores. Clearly the conductance of a graphene-Al<NUM>O<NUM> nanopore is strongly influenced by local pH.

Conductance saturation is clearly observed at pH <NUM> 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 <NUM> even at very low KCI concentrations (<FIG>), suggesting that the pore is only weakly charged at this pH. The pH <NUM> response more closely resembles bulk behavior where the effects of surface charge on channel conductance are minimal. <FIG> illustrates the pH response of a smaller <NUM> ± <NUM> diameter pore. Similar trends are seen as in <FIG> with lower pore conductance being observed at lower pH. Interestingly, saturation/plateauing in the conductance at pH <NUM> is observed at KCI concentrations starting at <NUM>, an order of magnitude higher than in <FIG>. This result is expected as Debye layer overlap and surface effects will begin to dominate at higher salt concentrations in smaller pores. The Debye screening length is approximately <NUM> in <NUM> KCI and thus is comparable to the <NUM> diameter of the pore in <FIG>. Thus, surface charge effects are expected to be significant at this relatively high salt concentration.

The pH response of graphene-Al<NUM>O<NUM> nanopores is significantly more pronounced than the pH response of SiN and TiO<NUM> nanopores as well as SiO<NUM> nanochannels. This may in part be due to the presence of graphene in conjunction with the high surface charge density of Al<NUM>O<NUM>. 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.

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 (<NUM> 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 <FIG> (also shown in <FIG>). These modifications include the contact of graphene layer <NUM> (g2) in <FIG> with a <NUM> evaporated Ti/Au pad prior to atomic layer deposition of dielectric <NUM> (d2), as shown in <FIG>. The nanopore is next drilled in the contacted stack. After drilling the pore, the nanopore chip is epoxied to a custom designed PCB and the Ti/Au pads contacting the graphene gate are connected using indium wires to external PCB pads (<NUM> and <NUM>) as shown in <FIG>. <FIG> illustrates and independently energizable and detectable graphene gate electrode via the Ti/Au pad connected to a power supply and detector. The resistance across pads <NUM> and <NUM> after connecting the chip is in the range of <NUM>-<NUM> kΩ typically, confirming the presence of a conductive graphene sheet on the nanopore chip after fabrication. The PCB mounted nanopore chip is next inserted into a custom designed fluidic setup as shown in <FIG>. Care is taken to ensure that the Ti/Au pads are isolated from the fluid to prevent leakage currents.

Nanopore measurements with the graphene gate are conducted by tying the gate node to the source electrode, as shown in the schematic of <FIG>. The source and gate are tied to prevent leakage currents from flowing between the source and gate nodes. Even though graphene technically should act as a non-Faradaic electrode with very little electron exchange occurring in an ionic solution under low applied biases, the presence of defects and grain boundaries, characteristic of CVD grown graphene, may give rise to such a leakage current. <FIG> illustrates the effect of connecting the graphene gate (tied gate-source) in graphene-Al<NUM>O<NUM> nanopores versus leaving the gate floating, under a variety of salt conditions and pH values.

A higher conductance level is seen at pH <NUM> and pH <NUM> with the gate connected relative to the floating case. In contrast, lower conductance is observed at pH <NUM> 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 <NUM> KCI, pH <NUM> conditions even though the Debye screening length at this concentration is only - <NUM>. This is consistent with the notion that at high pH, OH- can disrupt the sp<NUM> 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 (-<NUM> mV to <NUM> 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.

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. <FIG> shows a series of SEM images showing the fabrication a GNR and the formation a nanopore directly in the ribbon. Note the GNR is formed by patterning the g2 layer shown in <FIG>. <FIG> and <FIG> shows the approach to ssDNA sequencing. By measuring transverse current through the ribbon <NUM> during the passage of ssDNA, the GNR may serve as a nucleotide reader. The embedded graphene gate (layer g1) <NUM> provides a means to bias the GNR for either p-type or n-type behavior and can slow down DNA translocation electrostatically.

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 <FIG> and comprises three electrodes placed within a nanochannel, which passes either above or below the electrodes. An AC signal is applied to the center electrode (electrode <NUM>) and the left (electrode <NUM>) and right (electrode <NUM>) electrodes are grounded. The impedance between electrodes <NUM> and <NUM> is given by Z1 and the impedance between electrodes <NUM> and <NUM> is given by Z2. In solution in the absence of DNA and other species, Z1 and Z2 are balanced due to the symmetry of the architecture, resulting in an output potential of Vout = V1 - V2 = <NUM>. The equivalent circuit is shown in <FIG>. The introduction of DNA or protein results in the following: As a DNA molecule or protein passes through the region between electrodes <NUM> and <NUM>, it modulates/changes Z1 and is detected as a voltage spike in the output voltage. Similarly, as the molecule passes by the region between electrodes <NUM> and <NUM>, it modulates Z2 and results in a voltage spike of opposite polarity. This allows for a double count of a translocating molecule as it passes through the nanochannel. Also by comparing the amplitudes of spikes and controlling electrode separations at the nanoscale, it is possible to resolve sensitive topographic information along the length of a DNA molecule, for example, aptamers or bound proteins. Such an architecture can also be arrayed along the length of the channel to provide multiple independent counts on a single molecule for error checking purposes as well as providing length wise topographic information.

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 <FIG>. Referring to <FIG>, the membrane <NUM> comprises a multilayer stack <NUM> of conducting layers (e.g., grapheme) <NUM> and dielectric <NUM> layers, in this example Al<NUM>O<NUM>. A first surface <NUM> and second surface <NUM> define the top and bottom of the stack <NUM> that face first fluid compartment and second fluid compartment, respectively. A nanopore <NUM> transits the membrane <NUM> from the first surface <NUM> to the second surface <NUM>. Each graphene layer is patterned as shown in <FIG> with an overlap region into which a nanopore is drilled (dielectric not shown in <FIG>). Referring to <FIG>, the nanopore <NUM> defines a nanopore transit direction <NUM>. Each graphene layer <NUM> is patterned as desired, including patterned as a nanoribbon graphene layer <NUM> oriented along a longitudinal axis <NUM> (corresponding to the direction of a long edge of each nanoribbon) and having a nanoribbon width as indicted by the arrow labeled <NUM>. From <FIG>, it is appreciated that the longitudinal direction of any nanoribbon may be characterized as transverse or orthogonal with respect to nanopore transit direction <NUM>. The graphene layers may have an angular offset with respect to each other, in this embodiment adjacent layers have an angular offset relative to each other of <NUM>°. The overlap region is of the order of a <NUM> x <NUM> in area and the patterned graphene layers will again be separated by a thin dielectric layer deposited via ALD. The graphene layers are biased as shown in <FIG> such as to achieve a vertical Wheatstone Bridge architecture versus the horizontal structure shown in <FIG>. The central electrode again has an applied AC signal to it and the output potential is again measured across electrodes <NUM> and <NUM>. By measuring modulations in Z1 (impedance across electrodes <NUM> and <NUM>) and Z2 (impedances across electrodes <NUM> and <NUM>), the detection and spatial mapping of molecules using electrical impedance as the molecule passes through the nanopore should be possible. The vertical structure shown here has the added advantage of precise control over the interelectrode spacing at the Angstrom scale enabling more sensitive topographical measurements along a molecule with respect to the planar architecture shown in <FIG>.

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 -<NUM> diameter nanopores are fabricated using electron beam lithography and reactive ion etch steps in SiN/Al2O3 membranes, facilitating high throughput analysis of single DNA molecules. The fabrication of single nanopores in ultra-thin graphene/Al2O3 membranes is also reported for detection of DNA-protein complexes. Single protein resolution at low salt concentrations is demonstrated.

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 $<NUM>. 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 -<NUM> diameter nanopore arrays in suspended Al2O3/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 <NUM>-<NUM> 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/Al2O3 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 <NUM> 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.

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 <NUM> 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.

Experimental. Nanopore Array Fabrication. Free-standing Al<NUM>O<NUM>/SiN membranes are formed using a fabrication process documented previously. The membrane comprises a <NUM>Å thick Al<NUM>O<NUM> layer deposited via atomic layer deposition (ALD) followed by a capping <NUM>Å thick SiN layer deposited via plasma enhanced chemical vapor deposition (PECVD). First, ZEP <NUM> e-beam resist dissolved in Anisole in a ratio of ZEP520:Anisole (<NUM>:<NUM>) is spun onto the free standing membrane (<NUM> rpm for <NUM>), optimized to a final thickness of <NUM>Å for -<NUM> feature definition. The ZEP520 coated chips are next baked at <NUM> for <NUM> minutes, followed by electron beam exposure using a JEOL JBX-6000FS Electron Beam Lithography (dose = <NUM>,<NUM>µC/cm<NUM>). The array patterns are developed in Xylenes for <NUM> followed by IPA for <NUM>. A reactive ion etching (RIE) step is next used to transfer the array pattern in ZEP520 into the SiN. Etching is done in <NUM> sccm CF<NUM>: <NUM> sccm CHF<NUM> at a power of <NUM> W and pressure of <NUM> mTorr. Etch rates of ~<NUM>Å/min versus -<NUM>Å/min for ZEP520 are measured under these conditions. The ZEP520 and SiN etch windows serve as the mask for dry etching Al<NUM>O<NUM>, done in a PlasmaTherm SLR-<NUM> Inductively Coupled Plasma (ICP) Reactive Ion Etcher. Etching is done in <NUM> sccm BCl<NUM>: <NUM> sccm Ar at an ICP power of <NUM> W, platen power of <NUM> W at a DC Bias -65V. An Al<NUM>O<NUM> etch rate of -<NUM>Å/min versus <NUM>Å/min for SiN and <NUM>Å/min for ZEP520 is observed under these conditions.

Fabrication of single nanopores in graphene/Al<NUM>O<NUM> membranes. Free-standing Al<NUM>O<NUM>/SiN membranes are again formed using the membrane fabrication process documented previously. Large -<NUM> 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 <NUM> mil copper foils purchased from Basic Copper. The foils are annealed under Ar/H<NUM> flow for <NUM> minutes and graphene is grown under a CH<NUM>/H<NUM>/Ar flow at <NUM>, -<NUM> mTorr for <NUM>. The resulting Cu/graphene substrates are cooled to room temperature under Ar flow at a rate of -<NUM>/min. Graphene transfer to the receiving substrate proceeds as follows: graphene is coated with a bilayer of PMMA (<NUM> and <NUM>), backside graphene on the copper foil is removed in an O<NUM> plasma, and then the backside copper is etched in a <NUM> FeCl<NUM> solution. The resultant PMMA/graphene film is wicked onto a glass slide, rinsed in DI water, rinsed in <NUM>% 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 <NUM>:<NUM> Methylene Chloride : Methanol solution. The transferred film is annealed in a CVD furnace at <NUM> under Ar/H<NUM> flow to remove any residual PMMA. Next, a seed layer, such as a metal seed layer comprising <NUM>Å of metallic Al is evaporated on the graphene coated chip using a CHA SEC-<NUM> E-Beam Evaporator. This layer completely oxidizes in air and serves as a seed layer for ALD Al<NUM>O<NUM>. <NUM>Å of Al<NUM>O<NUM> is next deposited using ALD. A nanopore is drilled in the graphene/Al<NUM>O<NUM> 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<NUM>O<NUM> membranes. Results and Discussion. The nanopore array fabrication process is shown in <FIG>. Arrays are formed by first patterning ZEP520 e-beam resist using electron beam lithography and then transferring this pattern into the SiN and Al<NUM>O<NUM> layers using a series of reactive ion etch steps. The optimized process allows the parallel fabrication of ~<NUM> diameter nanopores with a pitch of <NUM> as shown in <FIG>. Similarly, larger arrays can be fabricated with relative ease as shown in <FIG>. ZEP520 electron beam resist is selected for this application due to its high dry etch selectivity relative to PMMA and its ~10x lower electron dose required for clearance, providing rapid wafer scale fabrication of nanopore arrays. Prior to array fabrication in suspended membranes, electron dose is optimized using a series of test exposures on planar substrates to achieve sub-<NUM> resolution. The process provided herein facilitates the highly anisotropic dry-etching of sub-<NUM> wide features in SiN using a mixture of CF<NUM>/CHF<NUM> with an etch selectivity for SiN:ZEP520 of <NUM>:<NUM>. Al<NUM>O<NUM> serves as a robust etch stop for this recipe. Pattern transfer into Al<NUM>O<NUM> is achieved using a BCl<NUM>:Ar etch recipe with a measured selectivity for Al<NUM>O<NUM>:SiN of <NUM>:<NUM>. A tapered etch profile in Al<NUM>O<NUM> is observed as schematically shown in <FIG> (iv) with a cone angle of ∼<NUM>°, permitting the final pore diameter in Al<NUM>O<NUM> to be significantly less than the patterned pore diameter in ZEP520. This feature is potentially very useful as it may facilitate the fabrication sub-<NUM> diameter nanopore arrays in Al<NUM>O<NUM>. Integrating nanoscale electrodes into such an architecture to form arrays of individually addressed nanopores allows for high throughput detection of individual DNA molecules for sequencing applications.

The fabrication of individual nanopores in ultra-thin graphene/Al<NUM>O<NUM> membranes is shown in <FIG>. A single, -<NUM> pore is first formed in a free-standing SiN/Al<NUM>O<NUM> membrane using a focused ion beam tool. CVD grown graphene is next transferred to the substrate containing the FIB pore as detailed in the experimental section. Following transfer, the integrity of the graphene at both the nano-scale and micro-scale is inspected using TEM diffraction imaging and Raman Spectroscopy. The hexagonal symmetry seen in the diffraction pattern (<FIG>) from the graphene membrane spanning the FIB pore suggests likely monolayer graphene, further supported by the peak intensities from the Raman spectrum of <FIG> where I(G')/I(G) > <NUM>.

The growth of primarily monolayer graphene using the CVD process employed here has been reported. Note the D peak seen in <FIG> is characteristic of CVD graphene and results from defects. A <NUM>Å thick Al seed layer is next evaporated onto the graphene layer followed by ALD deposition of <NUM>Å of Al<NUM>O<NUM> giving a total membrane thickness of less than <NUM>. A single nanopore is formed in this ultra-thin graphene/Al<NUM>O<NUM> membrane using a focused convergent electron beam as shown in <FIG> (TEM image). These nanopores are remarkably robust and exhibit linear IV characteristics.

The translocation of protein-DNA complexes through a graphene/Al<NUM>O<NUM> nanopore is shown in <FIG>. The model DNA-protein system in these studies is ERα bound to a <NUM> bp long probe containing a single ERE, the cognate binding sequence for the ERα protein. DNA-bound ERα primarily serves as a nucleating factor for the recruitment of protein complexes and is involved in key biological processes including oxidative stress response, DNA repair, and transcription regulation. A schematic showing the binding of ERα to dsDNA containing a single ERE and the ERE sequence itself are shown in <FIG> respectively. <FIG> shows a gel shift assay, ERα/ERE binding being observed exclusively at low salt concentrations. The detection of protein-DNA complexes using a nanopore is analogous to dsDNA detection as shown in <FIG>. Notably, the transport of the ERα/ERE complex through a -<NUM> diameter pore in <NUM> KCI results in current enhancements (<FIG>), likely due to counter-ion condensation on the complex locally increasing pore conductance during transport as previously reported in DNA transport studies at low salt. A translocation time versus current enhancement scatter plot is shown in <FIG>. The most probable translocation time for this <NUM> bp long DNA probe at <NUM> mV with a single bound ERα protein is ~<NUM>, two orders of magnitude slower than the estimated translocation time for a <NUM> bp dsDNA alone. The work presented here confirms that a graphene/Al<NUM>O<NUM> nanopore can spatially resolve a single protein bound to dsDNA.

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 -<NUM> diameter nanopores and greater, fabricated using electron beam lithography and reactive ion etch steps in suspended SiN/Al<NUM>O<NUM> 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<NUM>O<NUM> 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.

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 $<NUM>. The emerging role of nanopores in sequencing, genomic profiling, epigenetic analysis and medical diagnostics is described in this example.

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 $<NUM> genome challenge proposed by the National Institute of Health in <NUM>, are expected to revolutionize genomic medicine. Nanopore DNA sequencing is one such technology that is currently poised to meet this grand challenge.

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 <NUM>'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.

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 (<FIG>) and novel sensing architectures and modalities that add functionality to the nanopore are also examined (Table <NUM>). The application of these new techniques to sequencing and the associated challenges are briefly presented. Finally, the application of nanopores to areas outside sequencing are discussed, particularly the emerging role of this technology in medical diagnostics.

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.

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 <FIG>. The channel is comprised of a <NUM>-nm vestibule connected to a transmembrane β-barrel ~<NUM> in length, containing a <NUM>-nm constriction that permits the passage of single stranded DNA but not double stranded DNA (dsDNA). Kasianowicz first demonstrated the electrophoretic transport of individual ssDNA and ssRNA molecules through α-hemolysin. In particular, early results demonstrated the ability of native α-hemolysin to distinguish between freely translocating RNA homopolymers of cytidylic and adenylic acid, as well as poly(dA) and poly(dC) strands of ssDNA, suggesting the potential emergence of α-hemolysin as a next-generation DNA sequencing tool. The realization of such a tool however has proven challenging, primarily due to the remarkably high velocity with which ssDNA moves through the pore under typical experimental conditions (estimated at -<NUM> nucleotide/µs) as seen in <FIG>. At these rapid timescales, as few as -<NUM> ions are available in the pore to correctly identify a translocating nucleotide, a daunting proposition given thermodynamic fluctuations (statistical variations in the number of charge carriers and position of the nucleotide in the pore) and the subtle chemical differences that exist amongst nucleotides. It has, therefore, proven impossible to sequence freely translocating ssDNA using α-hemolysin.

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: (<NUM>) the enzyme-DNA complex forms in bulk solution enabling its electrophoretic capture in the pore and, (<NUM>) 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 <NUM>% 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).

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 ~<NUM> long cylindrical β barrel that accommodates up to ~<NUM> 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 <NUM> 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 ~<NUM> with a channel length of -<NUM>, forming a tapered funnel shape (structural cross section shown in <FIG>), as opposed to the cylindrical structure of α-hemolysin. Derrington et al. demonstrated the ability of genetically engineered MspA to discriminate between tri-nucleotide sets (AAA, GGG, TTT, CCC) with an impressive <NUM> fold enhancement in nucleotide separation efficiency over native α-hemolysin. Interestingly, in experiments involving immobilized ssDNA, as few as three nucleotides within or near the constriction of MspA were seen to contribute to the pore current, a significant improvement over the -<NUM> nucleotides known to modulate ionic current in native α-hemolysin. The authors hypothesize that this could be further improved to perhaps a single nucleotide through site specific mutagenesis, an obvious goal of future MspA mutants. The application of MspA to de novo sequencing is not without challenges either. The speed of unimpeded ssDNA translocation through MspA still remains too fast to sequence ssDNA 'on the fly'. To overcome this limitation, duplex interrupted (DI) nanopore sequencing was recently proposed. DI sequencing involves the insertion of a 'short' double-stranded DNA segment between each nucleotide in an analyte DNA molecule. As duplex converted DNA is driven through an MspA nanopore, each duplex sequentially halts the translocation process, exposing a single analyte nucleotide to the confining nanopore aperture for identification using ionic current. Upon duplex dissociation, the DNA advances until the next duplex where the next analyte nucleotide is determined and so forth. Such a method could ultimately enable fast and long sequential reads; however, the ability to convert and read large genomic fragments with high fidelity using this approach still remains to be seen. An alternative approach to DI sequencing is to couple an enzyme-motor to MspA to controllably step ssDNA through the pore with nucleotide identification occurring at each step, a method referred to as strand sequencing. Candidate enzymes suited for this application include T7 DNA Polymerase, Klenow fragment of DNA Polymerase <NUM> and phi29 DNA polymerase, the latter known to be remarkably stable and highly efficient at catalyzing sequential nucleotide additions at the α-hemolysin orifice under a high <NUM> mV applied load. It is plausible therefore that phi29 DNA polymerase coupled to MspA could enable the sequencing of long strands of DNA, though the experimental realization of such a system has not yet been demonstrated.

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 ~<NUM>. Interestingly, the open channel conductance of this nanopore is ~<NUM> 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.

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.

The first reports of DNA sensing using solid-state nanopores emerged from the Golovchenko lab in early <NUM>. 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 <NUM>. 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 (-<NUM>) 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<NUM>O<NUM> membranes using atomic layer deposition (ALD) is proposed, a process that yields angstrom level control over membrane thickness. The fabrication of nanopores in Al<NUM>O<NUM> membranes using a focused electron beam revealed two interesting phenomena, the dose-dependent conversion of Al<NUM>O<NUM> 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 <NUM>/f noise and DNA transport velocities. Interestingly, slower DNA transport was observed in Al<NUM>O<NUM> nanopores relative to SiN pores of similar diameter, attributed to strong electrostatic interactions between the positively charged Al<NUM>O<NUM> 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: <NUM>) the formation of membranes and nanopores in a variety of other high-k dielectric materials including TiO<NUM> and HfO<NUM>, each with unique material properties and <NUM>) the integration of metallic contacts and graphene layers directly into the membrane due to the low temperature nature of the ALD process (typically < <NUM>). 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.

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 <NUM>-<NUM> 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 <NUM>. 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 <NUM>-<NUM> diameter nanopores in suspended graphene films, prepared through either chemical vapor deposition (CVD) or exfoliation from graphite. Nanopores were formed in as few as <NUM>-<NUM> 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, dpore, in monolayer thin membranes as opposed to the dpore<NUM> scaling typically observed with pores in thick SiN membranes. An effective membrane thickness, heff, of ~<NUM> was extracted for nanopores formed in a graphene monolayer. This result agrees with the theory in the limit as heff → <NUM> where the dominant resistance is not the pore resistance itself (Rpore) but rather the access resistance (Raccess attributed to the potential drop in the electrolyte from the electrode to the nanopore), where Raccess scales inversely with dpore. In contrast, the Delft studies showed that the conductance of nanopores in a graphene monolayer scales as a function of dpore <NUM>, an intriguing result that suggests a cylindrical nanopore geometry of non-negligible thickness (Rpore > Raccess). The origin of this dpore <NUM> scaling may be due to a polymer coating (<NUM>-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 <NUM> 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 (<FIG>). This effect was confirmed recently using TEM image analysis and is also visible in earlier studies. A terraced nanopore architecture could prove very useful for two reasons: <NUM>) it potentially relaxes the constraint of growing and transferring a large area monolayer in order to fabricate a graphene monolayer nanopore and <NUM>) a multilayered support may increase the stability and longevity of a graphene nanopore sensor.

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 <NUM>-<NUM> 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 <NUM>-nm diameter graphene pore of thickness -<NUM> revealed a resolution of -<NUM>, 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 ~<NUM> 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 (<NUM>-<NUM> 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.

Nanopore applications outside DNA sequencing. The more immediate application for solid-state nanopores is likely in medical diagnostics. A nanopore based diagnostic tool could: (<NUM>) detect target molecules at very low concentrations from minute sample volumes (perhaps shed DNA from tumor cells in patient serum); (<NUM>) simultaneously screen panels of biomarkers/genes (important in diagnosis, monitoring progression and prognosis); (<NUM>) provide rapid analysis at relatively low cost; and (<NUM>) 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 (<FIG>). Another exciting prospect is the use of solid-state nanopores for epigenetic analysis, more specifically the detection of aberrant DNA methylation, an early and frequently observed event in carcinogenesis. Hypo- and hypermethylation in the promoter sequences of specific genes serve as both robust cancer biomarkers (e.g. GSTP1 promoter hypermethylation observed in over <NUM>% of prostate cancer cases), as well as indicators of disease severity and metastatic potential in many tumor types. Preliminary progress towards nanopore based methylation analysis has been demonstrated by the Timp and Drndic labs involving the detection of methylated and hydroxymethylated DNA.

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 ~<NUM> 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 (<FIG>). This approach could be extended to screen mutations in the cognate sequences of various other DNA binding proteins, including transcription factors, nucleases and histones. The Meller lab, using solid-state nanopores, is actively pursuing another direction; the rapid genotyping of viruses and human pathogens. An innovative approach involving the introduction of highly invasive Peptide Nucleic Acid (PNA) probes was used to label target genomes with high affinity and sequence specificity, creating local bulges (P-loops) in the molecule. Translocation of this labeled molecule resulted in secondary DNA-PNA blockade levels (<FIG>), effectively barcoding a target genome. While further studies are needed to determine the ultimate spatial resolution of this technique, this methodology could potentially enable the rapid, accurate and amplification free, identification of small <NUM>-<NUM> kb viral genomes including Hepatitis C, Dengue and West Nile Virus.

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<NUM> nanopores, higher flux and smaller translocation times were observed for the passage of perfect complementary (PC) ssDNA versus single base mismatched probes (<NUM>), a highly sensitive strategy for the detection of SNPs (<FIG>). Functionalized biomimetic nanopores in SiN have furthermore enabled the study of nucleocytoplasmic transport phenomena at the single-molecule level. Altering the surface chemistry of a pore can also facilitate the sensitive detection and discrimination of proteins. Drawing inspiration from the lipid coated olfactory sensilla of insect antennae, the Mayer lab recently demonstrated the identification of proteins using fluid lipid bilayer coated SiN nanopores (<FIG>). The incorporation of mobile ligands into the bilayer introduced chemical specificity into the pore, slowed the translocation of target proteins, prevented pores from clogging and eliminated nonspecific binding, thereby resolving many issues inherent to solid-state nanopores. A lipid bilayer coated nanopore architecture of this nature (in either SiN or Al<NUM>O<NUM>) also allows for future integration with biological nanopores to form robust nanopore sequencing elements.

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 <NUM>-<NUM> 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 <FIG>, the entry of this engineered α-hemolysin channel into a SiN nanopore could be electrophoretically guided to form a coaxially aligned structure. Hybrid pore conductance and ssDNA translocation event durations were in good agreement with α-hemolysin embedded in lipid bilayers. Interestingly, ssDNA blockade amplitudes through hybrid pores were significantly less than in α-hemolysin-bilayer systems, attributed to both deformation of the biological pore, and leakage currents around its body when inserted into a solid-state pore. Also, an increase in electrical noise was observed in hybrid structures. These parameters will likely need to be optimized in order to match the single nucleotide sensitivity of aminocyclodextrin modified α-hemolysin. Nevertheless, this hybrid architecture opens up the exciting possibility of high throughput sequencing by coupling the single nucleotide recognition capabilities of either α-hemolysin or MspA, with wafer-scale arrays of individually addressed solid-state nanopores.

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+ 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 ~<NUM> Alms (length of a single nucleotide moving in a millisecond through a sensor region with spatial resolution of ~<NUM>Å), and if nucleotides could be identified uniquely with an electronic signature, a <NUM> million base long molecule could be sequenced in less than <NUM> minutes. Scaling this technology to an array of <NUM>,<NUM> individually addressed nanopores operating in parallel could enable the sequencing of a <NUM> billion bp human genome with <NUM> fold coverage in less than <NUM> hour.

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 <FIG>, with electrical contacts <NUM> to the embedded graphene layer to measure transverse current via the electrically connected power source and detector (<FIG> illustrates a nanoribbon graphene layer <NUM> and an embedded gate electrode <NUM> that is an embedded graphene layer.

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 <NUM> 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 <NUM> bases might not need to be uniquely identified. Investigators have been using binary conversion of nucleotide sequences (A/T=<NUM>, and G/C=<NUM>), 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.

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.

Exemplary embodiments of certain devices and methods are provided in <FIG>, <FIG> and <FIG>. Referring to <FIG>, membrane <NUM> comprises a stack <NUM> formed by a plurality of graphene layers <NUM> separated from each other by dielectric layers <NUM>. A first surface <NUM> forms one surface of first fluid compartment <NUM> and a second surface <NUM> forms one surface of second fluid compartment <NUM>. Nanopore <NUM> fluidically connects first and second fluid compartments <NUM> and <NUM> through membrane <NUM>. Power supplies <NUM> and detectors <NUM> are used to energize the system and to measure an electrical parameter in the nanopore, including independently with embedded gate electrode <NUM> connected to a power supply and detector via gate electrode <NUM>. Source <NUM> and drain <NUM> electrodes may bias the first and second fluid compartments relative to each other. Electrically conductive wire <NUM> may connect the various electrical components.

Referring to <FIG>, a tunneling detector <NUM> is formed by a pair of electrodes <NUM> and <NUM> that face each other in the nanopore and oriented in a direction <NUM> that is transverse or orthogonal to the nanopore axial direction <NUM>, as indicated by the dashed arrows. In an aspect, the pair of electrodes <NUM> and <NUM> are formed from a graphene layer <NUM>. Gate electrode <NUM>, such as Ti/Au pad, is connected to the tunneling detector to provide electrical contact to the tunneling detector corresponding to embedded graphene layer that is electrically isolated from the other layers. In this manner, a biomolecule interacting or transiting nanopore from first fluid compartment <NUM> to second fluid compartment <NUM> is characterized via monitoring of an electrical parameter that reflects a biomolecule parameter. For clarity, <FIG> is not drawn to scale, as the pair of facing electrodes may be positioned so that single stranded DNA passes between tips in a base-by-base translocation so that the tunneling detector measures an electrical parameter for individual bases within the biomolecule, thereby providing sequence information for the biomolecule (see also <FIG>).

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.

Examples of documents include: <NPL>); <CIT>; <CIT> and <CIT>.

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.

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 as defined by the claims and illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

Claim 1:
A method for characterizing a biomolecule parameter, said method comprising the steps of:
providing a 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 is graphene and said nanopore is a hybrid biological/solid-state nanopore;
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, thereby characterizing said biomolecule parameter;
wherein said conductor-dielectric stack comprises:
a plurality of conductor layers, wherein adjacent conductor layers are separated by a dielectric layer; wherein one or more of said conductor layers comprises a conductor nanoribbon, through which said nanopore traverses in a direction that is transverse to a longitudinal direction of said conductor nanoribbon; and said method further comprises 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.