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    • 专利摘要:
    Graphene Nanopore Sensors and Method for Evaluating a Polymer Molecule The present invention relates to a substantially exposed single layer graphene membrane including a nanopore extending through a graphene membrane thickness from a first and second surface membrane opposite a first surface of the graphene membrane. a connection from the first surface of the graphene membrane to a first reservoir provided on the first surface of the graphene membrane, a species in an ion solution for nanopore, and a connection from the second surface of the graphene membrane to a second A reservoir is provided to collect species and ionic solution after translocation of species and ionic solution through the nanopore from the first surface of the graphene membrane to the second surface of the graphene membrane. An electrical circuit is connected at opposite sides of the nanopore to measure the flow of ionic current through the nanopore in the graphene membrane.
    公开号:BR112012005888B1
    申请号:R112012005888
    申请日:2010-09-17
    公开日:2019-10-22
    发明作者:Branton Daniel；Slaven Garaj；A Golovchenko Jene
    申请人:Harvard College；
    IPC主号:
    专利说明:

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    Descriptive Report of the Invention Patent for GRAPHEN NANOPORE SENSORS AND METHOD FOR EVALUATING A POLYMER MOLECULE.
    Cross Reference for Related Orders [001] This order claims the benefit of United States Provisional Order No. 61/243, 607, filed on September 18, 2009, all of which is incorporated by reference. This application also claims the benefit of United States Provisional Application No. 61/355, 528, filed on June 16, 2010, all of which is incorporated herein by reference.
    Federally Sponsored Research Statement [002] This invention was made with government support under contract No. 2R01HG003703-04 granted by NIH. The Government has certain rights in the invention.
    Background [003] This invention relates generally to the detection and molecular analysis, and more particularly it relates to configurations of a nanopore arranged to detect molecules that translocate through the nanopore.
    [004] The detection, characterization, identification and sequencing of molecules, including biomolecules, for example, polynucleotides, such as DNA, RNA and peptide nucleic acid (PNA) nucleic acid molecules, as well as proteins, and others biological molecules, is a broad and important field. There is currently a great need for processes that can determine the state of hybridization, the configuration, the stacking monomer, and the sequence of polymer molecules in a fast, reliable, and low cost manner. Advances in the manufacture of synthetic polymers and advances in biological development and medicine, in particular in the field of gene therapy, development of new pro
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    2/29 pharmaceutical products and the combination of appropriate therapy for the patient are largely dependent on such processes.
    [005] In a process for molecular analysis, it has been shown that molecules such as nucleic acids and proteins can be transported through a natural or solid state nanoscale pore, or nano pore, and that the characteristics of the molecule, including its identification, its state of hybridization, its interaction with other molecules, and its sequence, that is, the linear order of the monomers of which a polymer is composed, can be discerned by and during transport through the nanopore. The transport of a molecule through a nanopore can be accomplished by, for example, electrophoresis, or another translocation mechanism.
    [006] In a particularly popular configuration for molecular analysis with a nanopore, the flow of ionic current through a nanopore is monitored as an ionic liquid solution, and the molecules to be studied that are supplied in the solution, travel through the nanopore. As molecules in the ionic solution translocate through the nanopore, the molecules at least partially block the flow of the liquid solution, and the ions in the solution, through the nanopore. This blockage of the ionic solution can be detected as a reduction in the measurement of the ionic current through the nanopore. With a configuration that imposes the passage of a single molecule in the nanopore, this technique of measuring ion block has been demonstrated to successfully detect individual molecular translocation events in the nanopore.
    [007] Ideally, this technique of measuring ion block for molecular analysis, like others that have been proposed, should allow molecular characterization with high sensitivity and resolution on the resolution scale of a single monomer. Unambiguous resolution of individual monomer characteristics is critical for
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    3/29 reliable applications, such as biomolecular sequencing applications. But this capability has been difficult to achieve in practice, particularly for solid-state nanopore configurations. It has been found that the length of a solid-state nanopore, determined by the thickness of a layer of material or layers in which the nanopore is formed, impacts the nature of the molecular passage of the nanopore, and directly limits the sensitivity and resolution with which molecules in the nanopore can be detected and analyzed.
    Summary of the Invention [008] A nanopore sensor is provided that overcomes the sensitivity and resolution limitations of conventional sensors. In one example, a nanopore sensor including a solid state membrane having a thickness between the first membrane surface and the second membrane surface opposite the first membrane surface which is less than about 1nm is provided. A nanopore extends through the thickness of the membrane between the first and second surfaces of the membrane and has a diameter that is greater than the thickness of the membrane. There is a connection from the first membrane surface to a first reservoir to provide, on the first membrane surface, a species in an ion solution for the nanopore, and a connection from the second membrane surface to a second reservoir to collect the species and ionic solution after translocation of species and ionic solution through the nanopore from the first membrane surface to the second membrane surface. An electrical circuit is connected to monitor the translocation of species in the ionic solution through the nanopore in the membrane.
    [009] This nanopore sensor can be supplied as a graphene nanopore sensor. Here, a grain membrane is provided
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    4/29 substantially exposed single-layer hay including a nanopore that extends through a thickness of the graphene membrane from a first surface of the graphene membrane to a second surface of the graphene membrane opposite the first surface of the graphene membrane. A connection from the first surface of the graphene membrane to a first reservoir provides, on the first surface of the graphene membrane, a kind of an ion solution for the nanopore, and a connection from the second surface of the graphene membrane to a second reservoir is provided to collect the species and the ionic solution after translocation of the species and ionic solution through the nanopore from the first surface of the graphene membrane to the second surface of the graphene membrane. An electrical circuit is connected on opposite sides of the nanopore to measure the flow of ionic current through the nanopore in the graphene membrane.
    [0010] In an additional graphene nano-pore sensor a substantially exposed single-layer graphene membrane includes a nano-pore that extends across a thickness of the graphene membrane from a first surface of the graphene membrane to a second surface of the graphene membrane opposite the first graphene surface and which has a diameter that is less than about 3 nm and greater than the thickness of graphene. An electrical circuit is connected on opposite sides of the nanopore to measure the flow of ionic current through the nanopore in the graphene membrane.
    [0011] These configurations allow a method for evaluating a polymer molecule in which the polymer molecule to be evaluated is supplied in an ionic solution. The polymer molecule in the ionic solution is translocated through a nanopore on a substantially exposed single-layer graphene membrane to
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    5/29 from a first surface of the graphene membrane to a second surface of the graphene membrane opposite the first surface of graphene and the flow of the ionic current through the nanopore in the graphene membrane is monitored.
    [0012] These methods of sensor placement and detection allow high resolution, detection and molecular analysis of high sensitivity, thus achieving the detection of closely spaced monomers in a polymer and therefore sequentially resolve the different ion blocks caused by each monomer in, for example, a strand of a DNA polymer. Other features and advantages of the invention will be evident from the following description and figures that follow, and from the claims.
    Brief Description of the Drawings [0013] Figure 1 is a schematic perspective view of an example graphene nanopore device for detecting molecules by measuring ionic flux through the nanopore;
    Figures 2A-2E are schematic side views of six theoretical nanopores in the membranes, each nanopore 2.4 nm in diameter and varying in nanopore length from 0.6 nm, 1 nm, 2 nm, 5 nm and 10 nm , respectively, with the average density of the ionic current in various regions through each nanopore representing the lengths of arrows shown in the nanopores;
    figure 3 is an ion current blocking graph, defined as the absolute value of the difference between the ion current through an unblocked nanopore and the ion current through the same nanopore when blocked with a molecule of the indicated diameter, for an ion solution of 3M KCI and a nanopore offset of 160mV, for nanopores having a diameter of 2.5 nm and effective length of 0.6 nm, 2 nm, 5 nm, and 10 nm;
    figure 4 is an X-ray diffraction image of a
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    6/29 experimental graphene membrane, exhibiting the required hexagonal pattern that arises from the hexagonal packaging of carbon atoms in a single layer of graphene;
    figure 5 is a graph of Raman displacement measurements for an experimental graphene membrane indicating a single layer graphene membrane;
    figure 6 is a graph of experimentally measured data of the ion current as a function of the voltage polarization applied between the 3M KCI ion solutions on cis and trans sides of an experimental graphene membrane;
    figure 7 shows the graph of figure 6 and a graph of ionic current as a voltage function for an experimental graphene membrane including a nanopore 8 nm wide;
    figure 8 is a graph of ionic conductivity as a function of nanopore diameter for nanopores having a length of 0.6 nm, 2 nm, and 10 nm;
    figure 9 is a graph of the ionic current measured as a function of time for a 2.5 nm nanopore on an experimental graphene membrane as DNA fragments translocated through the nanopore;
    Figures 10A-10C are graphs of the ionic current measured as a function of the time taken from the graph in Figure 9, showing in detail the current profile for the translocation of DNA in the nanopore in the form of a single file, in the form of partially folded and folded in half;
    figure 11 is an ion current block plot as a function of translocation of DNA into a nanopore on a graphene membrane for 400 translocation events; and.
    figure 12 is a graph of the percent change in ion current blocking as a function of distance through a nano
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    7/29 pore, for a nanopore of 0.6 nm in length and for a nanopore of 1.5 nm in length.
    Detailed Description [0014] Figure 1 is a schematic perspective view of an example of a molecular characterization device for a graphene 10 nanopore. For clarity of the discussion, features of the device illustrated in Figure 1 are not shown to scale. As shown in figure 1, on the device, a nanoscale opening, or nano-pore 12, is provided in an exposed single-layer graphene membrane 14. The graphene membrane is self-supporting, meaning that there are no structures under the membrane extension for support the membrane. At the ends of the membrane, for example, a support structure 16 can be provided, which in turn can be provided on a support substrate or another structure 18. The self-supporting exposed graphene membrane is configured in a fluidic cell such that on the first, or cis, side of the graphene membrane is a connection to the first liquid reservoir or liquid stock containing a liquid solution including 20 molecules to be characterized, and on the second, or trans, side of the graphene membrane is a connection to a second liquid reservoir, in which characterized molecules are transported by translocation through the graphene nanopore 12.
    [0015] In an application of the graphene nanopore, shown in the figure, the molecules 20 to be characterized comprise a simple chain of DNA molecules (ssDNA) having a sequence of nucleoside bases 22 to be characterized, for example, by determining the base sequence identity along each ssDNA backbone. For clarity of the discussion this example of sequence will be used in the following description, but this is not the exclusive application of the characterization of the nanopore device of
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    8/29 graphene. In addition, the sequencing of the operation described below is not limited to the DNA example; the RNA polynucleotide can be characterized similarly. The molecular characterization enabled by the graphene nanopore device includes a wide variety of analyzes, including, for example, sequencing, hybridization detection, molecular interaction detection and analysis, configuration detection, and other molecular characterizations. The molecules 20 to be characterized can, in general, include any molecule, including polymers and biomolecules such as proteins, nucleic acids, such as DNA and RNA polynucleotides, sugar polymers, and other biomolecules. The discussion below is therefore not intended to be limiting to a particular application, but it does provide details of an example in a range of embodiments for molecular characterization.
    [0016] For the graphene nanopore of Figure 1, an arrangement of characteristics is provided to cause molecules 20 to pass through the nanopore through the exposed self-supporting exposed single-layer graphene membrane. For example, silver chloride electrodes 24, 26, immersed in solutions on both sides of the graphene membrane 14, can be provided to control the voltage of each solution across the graphene membrane. The application of a voltage deviation between the electrodes 24 in the two solutions on opposite sides of the membrane causes the molecules, for example, ssDNA molecules, supplied in the solution on the first, or cis, side of the membrane, to be conducted electrophoretically to inside and through the nanopore 12 for the solution on the second, or third, side of the membrane, because the main strand of DNA is negatively charged when in solution.
    [0017] The present inventors made a surprising discovery that ionic resistivity perpendicular to the plane of an exposed single-layer graphene membrane that separates two re
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    9/29 tanks full of ionic solutions are extremely large, which makes it possible to establish a significant voltage polarization across the graphene membrane, between the two solutions, as described above. As explained in more detail in the experimental discussion below, this discovery allows the configuration of fig. 1, in which the electrical control of the potential through a single layer of graphene can be maintained in a manner necessary for molecular electrophoresis.
    [0018] It is further discovered that an exposed single-layer graphene membrane is sufficiently mechanically robust to operate as a structural barrier between two solution-filled reservoirs whether or not these reservoirs are in direct communication with each other through a nanopore in the membrane of graphene that is supported only at its ends by a structure, that is, that is self-supporting through its extension. As a result, an articulated nanopore membrane from a single exposed graphene layer can operate to separate two reservoirs filled with ionic solutions, using methods known to those familiar with the nanopore field, by applying a voltage deviation between the two ionic solutions on the cis and trans side of the exposed graphene membrane to electrophoretically conduct molecules through the nanopore.
    [0019] Other techniques and arrangements can be employed to design molecules through the nanopore, and no particular technique is needed. More details and examples for driving electrophoretic molecular translocation through a nanopore are provided at the atomic scale and molecular evaluation of biopolymers, USA 0 No 6627067, for Branton and others, issued on September 30, 2003, the entire which is incorporated herein by reference.
    [0020] As shown in figure 1, a circuit can be provided
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    26, 28 for the measurement of changes in the ionic current flow between the cis and trans sides of the graphene membrane, through the nanopore 12. With this configuration, the translocation of molecules through the nanopore 12 can be detected and based on the detection, it can be analyzed how the molecules are conducted through the nanopore. This molecular detection technique is just one of a wide range of detection techniques that can be used with graphene and nanopore membranes. Tunneling current between electrodes, for example, between carbon nanotubes or other probes articulated in the nanopore, changes in conductivity in probes or in the graphene membrane itself, or another molecular detection technique can be used, as described, for example, in Molecular characterization with control of carbon nanotubes, from the USA No. 7,468,271 by Golovchenko et al., Issued on December 23, 2008, all of which is incorporated by reference.
    [0021] Specifically considering the molecular detection technique by measuring the flow of ionic current, the present inventors made a surprising discovery that the ionic current through the exposed single-layer graphene membrane nanopore, when empty of a translocation species, and the flow of ionic current through the nanopore, when blocked by a molecule that is in the nanopore, are both approximately 3 times greater than the flow of ionic current through a nanopore of similar diameter on any other known membrane interface in a lipid state or solid. This significantly higher ion current flow through a nanopore on an exposed single-layer graphene membrane, compared to a biopore or nanopore of similar diameter on another solid state membrane, is understood by the inventors to be due to the thinness of the graphene membrane and correspondingly, the length of the nanopore through the membrane.
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    11/29 [0022] An exposed graphene membrane is a single atom layer of a hexagonal carbon network that is therefore atomically thin, being only about 0.3 nm thick. At this thickness, the ionic flux through a nanopore in the exposed single-layer graphene membrane can be characterized in a regime in which the length of the nanopore is much less than the diameter of the pore. In this regime, the ion conductivity of the nanopore is proportional to the nanopore diameter, d, and the ionic current density through the nanopore is clearly a peak in the peripheral nanopore, that is, at the end of the nanopore, compared to the current density in the middle of the nanopore. In contrast, nanopores having a length that is greater than the nanopore diameter is characterized by an ionic conductivity that is proportional to the nanopore area, and that is homogeneous across the nanopore diameter, with ionic conductivity uniformly flowing down through the medium nanopore as well as on the nanopore periphery.
    [0023] The clear distinction between nanopore conductivity in these two nanopore length regimes is illustrated in figures 2A-2E. Referring to these figures, the representations shown are the average current density minus ten points through each nanopore having a diameter of 2.4 nm and having a length of 0.6 nm, 1 nm, 2 nm, 5 nm and 10 nm, respectively The relative lengths of the arrows in the figures indicate the average relative current density in the region in a nanopore that is represented by the location of each arrow. As shown in figures 2A-2C, for nanopore lengths that are smaller than the 2.4nm nanopore diameter, the current density is a peak in the peripheral nanopore. As the nanopore length approaches the nanopore diameter, the conductivity through the nanopore becomes more uniform. When the length of the nanopore is greater than the diameter
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    12/29 of the nanopore, as in Figures 2D and 2E, the ionic conductivity is uniformly homogeneous throughout the nanopore, with no preference for the nanopore periphery. The density of local current within different regions of the nanopores becomes more and more homogeneous when the nanopore length is increased.
    [0024] As a consequence, a nanopore on an exposed single-layer graphene membrane where the diameter of the nanopore is greater than the length of the nanopore exhibits a total ionic conductivity, in a free state, which is significantly greater than the conductivity total of a nanopore of equal diameter in a membrane with a thickness greater than the diameter of the nanopore. Other conditions that are the same, the best conductivity results in a significant larger total ion current through an open nanopore of a given diameter of a thinner membrane than the diameter of an open nanopore of equal diameter in a thicker membrane than the diameter. The larger ionic currents through the graphene membrane facilitate the high precision of the measurement of the ionic current flow through the nanopore.
    [0025] Because the ionic current through nanopores having a length less than the diameter of the nanopore is mainly on the periphery of the nanopore instead of through the central axis of the nanopore, small changes in the diameter of the molecules that run through the nanopore have a huge effect about the change in the ionic flow current. This is due to the fact that differences in the diameter of the molecules are manifest at the end of the nanopore, where the ion current flow is greater for a short nanopore length, rather than at the center of the nanopore, while for the short nanopore length the current ionic is lower. As a result, an exposed single-layer graphene nanopore having a length less than the nanopore diameter is more sensitive to particles.
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    13/29
    Molecularly sized wires or differences in differently sized particles, molecules, or the respective components that are nanopores with a length greater than the diameter of the nanopore.
    [0026] The consequence of this consideration is shown quantitatively in Figure 3, in which the level of blocking of the ionic current computed in a nanopore is traced as a function of the diameter of polymer molecules centrally passing through nanopores having a diameter of 2.5 nm and with effective lengths of 0.6 nm, 2 nm, 5 nm and 10 nm. The computed current block is the absolute value of the difference between the ion current through an unblocked nanopore, that is, no polymer molecules in the nanopore, and the ion current through the nanopore even when blocked with a polymer of the indicated diameter. The graphs assume translocation of the molecule with an ionic solution of KCI 3M and a voltage deviation of 160mV between cis and trans sides of the nanopore. As shown in the graphs here, the ionic current through the nanopores demonstrates the increased sensitivity to changes in the diameter of the translocation molecules while the length of the nanopores is decreased.
    [0027] The inventors have further discovered that the sensitivity in the conductivity of a nanopore to changes in the diameters of the translocation molecules is maximized when the diameter of the nanopore is adjusted to be as close as possible to the diameter of the translocation molecules. This condition is true for nanopores of any length. For example, as shown in the graphs in figure 3, for nanopores of 2.5 nm in diameter, while the diameter of the translocation molecule approaches the diameter of the nanopore, the current blockage increases, even when the nanopore length is greater than the nanopore diameter. But stop
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    14/29 nanopores in which the nanopore length is less than the nanopore diameter, that is, 2 nm and 0.6 nm, in the plotted data, it is shown that such short-length nanopores are much more acutely sensitive to small changes in diameter of the translocation molecule while the diameter of the molecule approaches the diameter of the nanopore. For these nanopores the blocking currents rise exponentially with the increase in the diameter of the blocking molecule. For the 5 nm and 10 nm length of the nanopores, which are larger than the diameter of the nanopore, the blocking currents only increase in an almost linear manner, even when the diameters of the blocking molecules approximate the diameter of the nanopore.
    [0028] Thus, the resolution of closely spaced differences in translocation of molecule diameters is preferably maximized by supplying a single-layer graphene membrane, a nanopore having a diameter that is both greater than the outer thickness of the membrane, but not much larger than the expected diameter for molecules that are translocated from the nanopore, for example, no more than 5% larger. To determine this second condition for the nanopore diameter for a given application, an analysis similar to that described in the example below cannot be performed. Briefly, in such an analysis, there is determined through, for example, a Laplace equation, the density of the ionic current of the ionic solution that will be used for the translocation of the molecule, the desired sensitivity of the detection of the translocation of the molecule is defined, and the general requirements for the viable nanopore diameter are determined. Based on these factors, and the limitation of the restriction that the diameter of the nanopore is greater than the thickness of the membrane, a diameter of the nanopore that optimizes all these factors can then be selected.
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    15/29 [0029] The inventors have further discovered that the electrical noise from an exposed single-layer graphene nanopore that separates two reservoirs filled with electrically biased ionic solutions, proportionally, no greater than the electrical noise from any other state nanopore solid. As a result, once the ion current is changed, that is, the ion blockage, through a graphene nanopore is greater during the passage of a molecule of any given diameter than in other known nanopores that have a length greater than the nanopore diameter, an exposed single layer graphene nanopore can produce a better signal-to-noise ratio than other known nanopores because most of the number of ions counted per unit time, or by traversing the nucleobase, will be more accurate than the one lower count rate. These discoveries, together with the known chemical inertia and exceptionally large strength of graphene, establishes a single layer articulated nanopore graphene membrane exposed as a superior interface for molecular detection and characterization.
    [0030] As a result of these discoveries, it is preferred that the membrane be supplied as a single layer of graphene that is exposed, that is, that it is not coated on both sides with any layer of material or species that contributes to the thickness of the graphene of the membrane. In this state, the thickness of the membrane is minimized and is safe in the short-length nanopore regime in which the peripheral flow of the ionic current is maximized and in which the conductivity of the nanopore as a function of changes in the analyzed physical dimensions is maximized. The very short length of the nanopore provided by the graphene membrane also makes it possible for a graphene nanopore to detect narrowly spaced monomers in a polymer and, therefore, to resolve the blo
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    16/29 sequentially different ionic causes caused by each monomer in, for example, a strand of a DNA polymer.
    [0031] It is recognized that a single layer graphene membrane has an affinity for many molecules, such as polymer molecules, such as DNA and RNA. Therefore, DNA, RNA, and other molecules can be expected to have a tendency to adsorb onto a preferably exposed graphene membrane. It is preferred that the absorption properties of the graphene surface are at least partially inhibited with an appropriate environment and / or surface treatment, which keeps the membrane in an exposed state without aggregated surface layers.
    [0032] For example, an ionic solution can be provided which is characterized by a pH greater than about 8, for example, between about 8.5 and 11 and which includes a relatively high salt concentration, for example, greater than about 2M and in the range from 2.1M to 5M. By employing a basic solution of high ionic strength, the adhesion of the molecules to the surface of the exposed graphene membrane is minimized. Any suitable selected salt can be used, for example, KCI, NaCI, LiCI, RbCI, MgCh, or any readily soluble salt, whose interaction with the analysis molecule is not destructive.
    [0033] Furthermore, as explained in detail below, during the synthesis and manipulation of the graphene membrane, extreme care is preferred in order to keep the membrane in a primitive state such that substantially no residue or other species, which can attract molecules to the graphene surface are present. It is also recognized that, in operation, the graphene membrane can be electrically manipulated to repel molecules from the graphene surface. For example, given the translocation of negatively charged DNA molecules through a nanopore
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    17/29 in a graphene membrane, a graphene membrane can itself be electrically polarized to a negative potential, which repels the negatively charged DNA molecule. Here electrical contact can be made to the graphene membrane in any suitable way that allows the application of a selected voltage. In such a scenario, the voltage between the ionic solutions on both sides of the graphene membrane can be adjusted high enough to produce an electrophoretic force that overcomes the repulsion on the graphene surface to cause DNA translocation through the nanopore instead of adsorption on the graphene surface.
    [0034] Going for methods to produce the graphene device in the nanopore, a single exposed layer of graphene can be synthesized by any convenient and appropriate technique, and no specific synthesis technique is needed. In general, atmospheric chemical vapor deposition with methane gas on a catalyst material, for example, a nickel layer, can be used to form the graphene layer. Raman spectroscopy, transmission electron microscopy, and studies of the selected diffraction area can be used to verify whether a synthesized graphene region to be employed is truly a single layer in nature.
    [0035] The transfer of the graphene layer to a device structure for disposal as a graphene membrane can be carried out by any suitable technique, but it is preferred that any materials used in the transfer do not damage the graphene surface. In a preferable technique, a selected manipulator material is coated over the graphene layer synthesized over the catalyst material and the substrate. For many applications, it may be preferable to use a handling material that is easily removed from the graphene surface since handling the ca
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    18/29 graphene layer is complete. Copolymer of methyl methacrylitomethylacrylic acid (MMA-MAA) may be one in particularly suitable handling material. With a layer of MMA-MAA in place of the graphene layer, the input structure can be cut into pieces.
    [0036] The resulting parts can then be processed to remove the catalyst layer and the substrate material underlying the graphene layer while adhering to the manipulator layer. For example, given a Ni catalyst layer, an HCI solution can be used to etch the Ni layer away and release the graphene / MMA-MAA composite, with distilled water used for rinsing. The graphene / MMA-MAA composite, floating on water, can then be captured by, for example, a silicon wafer coated with a layer of SINx. The central region of the silicon wafer can be etched by KOH or another suitable corrosive product to produce a free SIN X membrane, for example, 50 x 50 pm 2 area . A focused ion beam (FIB) or other process can then be employed to drill a suitable hole through the SINx membrane in such a way that it forms a support for the graphene layer membrane. For example, a square window of, for example, 200 nm x 200 nm can be formed on the nitride membrane to produce a support for the graphene membrane.
    [0037] With this complete device configuration, the graphene / MMA-MAA composite can be placed over the square window on the graphene membrane, using, for example, the nitrogen wind (a gentle jet of nitrogen) to firmly press the graphene against the substrate. The MMA-MAA can then be removed, for example, under a slow drip system, of acetone, followed by immersion in acetone, dichloroethane, and isopropanol.
    [0038] It is preferable to remove any residue from the film
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    19/29 graphene to reduce the tendency of species to adhere to graphene once configured as a membrane. For example, once the MMA-MAA is removed, the resulting structure, including a graphene membrane extended through a nitride frame, as in figure 1, can be immersed in, for example, a KOH solution at room temperature briefly, for example, for 1min and then vigorously washed with, for example, water, then isopropanol, and finally ethanol. To avoid damage to the graphene membrane, the structure can be dried at the critical point. Finally, the structure can be exposed to a selected environment, for example, a rapid thermal annealing process minus about 450 ° C in a gas stream containing 4% He in it to, for example, 20 minutes, expel any remaining hydrocarbon . To avoid recontamination, the structure of preference is then immediately loaded into, for example, a TEM, for further processing.
    [0039] A nanopore can then be formed on the graphene membrane. A focused electron beam or other process can be employed to form the nanopore. The diameter of the nanopore is preferably greater than the thickness of the graphene membrane, to obtain the benefits of the unexpected discovery of the increase in the peripheral current of the ionic flux and the increase in sensitivity, the change in the molecular dimension, as described above. For ssDNA translocation, a nanopore diameter between about 1 nm and about 20 nm may be preferred, with a diameter between about 1 nm and about 2 nm more preferred. For dsDNA translocation, a nanopore diameter of between about 2nm and about 20nm may be preferred, with a diameter of between about 2nm and about 4nm more preferred. After the formation of the nanopore, it is preferred to keep the graphene structure under a clean environment, for example, a vacuum of ~ 10 5 Torr.
    [0040] To complete the nano molecular detection device
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    20/29 portion of the figure. 1, the assembled graphene membrane can be inserted between two halves of the cells in, for example, a polyether ether ketone- (PEEK) microfluidic tape or other suitable material, sealed together with, for example, poly-dimethylsiloxane (PDMS). It may be preferred that the sealing hole is smaller than the dimensions of the graphene membrane to completely seal the ends of the graphene membrane from the solutions.
    Example I [0041] This example describes an experimental demonstration of a graphene membrane exposed to a single layer. A layer of graphene was synthesized by CVD on a nickel surface. Nickel was supplied as a film by E-beam evaporation on a silicon substrate coated with a layer of SiO2. The nickel layer was thermally annealed to generate a microstructure of the Ni film with a single crystalline grain of sizes between about 1 pm and 20 pm. The surfaces of these grains have automatic flat terraces and steps, similar to the surface of single crystal substrates for epitaxial growth. With this topology, the growth of graphene in Ni grains is similar to the growth of graphene on the surface of a single crystal substrate.
    [0042] In the CVD synthesis, the Ni layer was exposed to H 2 and CH4 gases, at a temperature of about 1000 ° C, Raman spectroscopy, electron transmission microscopy and the selected diffraction area study showed the film of graphene to be of excellent quality and mostly (87%) from a mixture of one and two thick layer domains, with domain sizes of ~ 10 pm. Thicker regions of three or more layers of graphene, easily distinguished by the color contrast under an optical microscope, cover only a small fraction of the total surface. If thicker regions or domain boundaries were found, that area
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    21/29 has been discarded.
    [0043] Graphene was transferred to a Si / SiNx chip carrier by first coating the graphene with MMAMAA copolymer (MMA-MAA (8.5) EL9, Microchem Corp) and cutting into 0.5 nm x 0.5 pieces mm. These pieces were immersed for ~ 8h in HCI 1N solution etched to move away from the Ni film and release the graphene / polymer membrane, which was transferred to the distilled water in which the graphene / polymer floated, with the graphene side down . Transporting the Si chip coated with ~ 250 nm thick SIN X was used to collect graphene / floating polymer films from the pieces, taking care that the graphene / polymer films were each stretched over the central region of a chip. The central region of the chip had been microfabricated using standard anisotropic techniques etched to leave an area of ~ 50 x 50 pm 2 of the SINx coating as an autonomous SIN X membrane in which a square window, ~ 200 nm x 200 nm, was perforated using an ion focused beam (FIB). A nitrogen gas wind was used to press graphene firmly against the chip surface. This led to the expulsion of a small amount of liquid from under the graphene, which adhered strongly and irreversibly to the chip of the SIN X coating conveyor. The polymer at the top of the graphene was removed under a slow dripping system of acetone, followed by subsequent dips in acetone, dichloroethane and, finally, isopropanol.
    [0044] To remove any residues from the graphene film, each chip was then immersed in a 33% by weight KOH solution at room temperature for 1 min and then vigorously washed with isopropanol and ethanol. To avoid damage to the autonomous portion of the graphene film suspension, each chip was dry at a critical point. Finally, the chips were loaded in a thermal ring
    Petition 870190057845, of 06/24/2019, p. 24/43
    22/29 fast and heated to 450 ° C in a gas stream containing 4% H 2 in it for 20 minutes to eliminate any remaining hydrocarbons. To avoid recontamination, the chips were immediately loaded into a transmission electron microscope for further processing.
    [0045] An X-ray diffraction image of one of the graphene membranes is shown in figure 4, showing the required hexagonal pattern that arises from the hexagonal packaging of carbon atoms in a single layer of graphene. Figure 5 shows the Raman deviation measurements for the graphene layer. The very small G peak and the very sharp 2D peak, producing a G / 2D ratio of less than 1, indicate a single layer membrane. Example II [0046] This example describes an experimental determination of the conductivity of the exposed single-layer graphene membrane of Example I.
    [0047] A chip assembled from the single-layer graphene membrane from example I was inserted between the two half cells of a custom-embedded microfluidic tape made of etherketone- (PEEK) polyester. The two sides of the chip were sealed together with polydimethylsiloxane (PDMS). The opening of the joint that pressed against the graphene film on the Si / SiN x carrier chip had an internal diameter of ~ 100 pm. Consequently, the sealing hole was smaller than the dimensions of the graphene membrane (0.5x0.5 mm 2 ), and the edge of the graphene membrane was completely sealed from the electrolyte. On the opposite side of the chip, the electrolyte was in contact with graphene only through 200 nm of the square window in the SiNx membrane. Note that with this arrangement there was no large area difference between the two sides of the graphene membrane in contact with the electrolyte (a circular area of 100 pm in diameter versus a
    Petition 870190057845, of 06/24/2019, p. 25/43
    23/29 square area 200 nm x 200 nm).
    [0048] The two halves of the cells were first filled with ethanol to facilitate wetting the chip surface. The cell was then washed with deionized water, followed by 1M KCI salt solution with no buffer. To avoid any potential interaction between the graphene membrane and solutes that may affect the experimental measurements, all electrolytes used in the experiment were kept as simple as possible and were capped. All pHs of the solution varied only in 0.2 pH units, from 5.09 to 5.29, as measured before and after use in the described experiments.
    [0049] The Ag / AgCI electrodes in each half of the cell were used to apply an electrical potential across the graphene membrane and to measure ionic currents. The current traces were acquired through an Axopatch 200B amplifier (Axon Instruments), which was connected to an external low-frequency Bessel 8-pole filter (type 90IP-L8L, frequency devices, Inc.), operating at 50 kHz. The analog signal was digitized using an NI PCI-6259 DAQ (National Instruments) card, operating at 250 kHz sampling rate and 16-bit resolution. All experiments were controlled using the IGOR Pro program.
    [0050] Figure 6 is a graph of experimentally measured ion current data as a function of the voltage deviation applied between 3 M KCI ion solutions on the cis and trans sides of the graphene membrane. Applying Ohm's law to these data, it is found that an ion current resistivity is well within the range of 3-4 GO, perpendicular to the plane of the graphene membrane. This demonstrates a finding of the present invention that the ionic resistivity perpendicular to the plane of a graphene membrane is very large, and allows for a configuration in which an electrical deviation
    Petition 870190057845, of 06/24/2019, p. 26/43
    24/29 can be maintained through an exposed single-layer graphene membrane that separates two skewed voltages from reservoirs filled with ionic solutions.
    [0051] With a 100 mV deviation applied between the two Ag / AgCI electrodes, measurements of the ion current for a variety of chloride electrolytes on the cis and trans sides of the graphene membrane were conducted. Electrolyte conductivities were measured using an Accumet Research AR50 conductivity meter, which had been calibrated using standard conductivity solutions (Alfa Aesar, product # 43405, 42695, 42679). All fluidic experiments were carried out under controlled temperature laboratory conditions, at 24 ° C. Table 1 shows that the conductivity of graphene membranes is well below the nS level. The highest conductivities were observed for solutions with the largest sizes of atomic cations, Cs and Rb, correlated with a minimum hydration layer that mediates their interaction with graphene. This conductivity was attributed to the transport of ions through the defect structures in the autonomous graphene membrane.
    Table I
    Solution Graphene conductivity (os) Sol. Conductivity (IO 3 Sm ' 1 ) Hydration Energy (eV) CsCI 67 ± 2 1.42 3.1 RbCI 70 ± 3 1.42 3.4 KCI 64 ± 2 1.36 3.7 NaCI 42 ± 2 1.19 4.6 LiCI 27 ± 3 0.95 5.7
    [0052] Contributions of electrochemical currents to and from the graphene membrane were excluded by an additional experiment here, to investigate the contribution from electrochemical currents (Fárdica), a large area separated from the graphene film (~
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    25/29
    2x4 mm2) was transferred to a glass slide and contacted at one end with silver paint attached to a metal clamp on which the wax insulation was placed. The exposed end of the graphene film was immersed in a 1M KCI electrolyte with an Ag / AgCI counter electrode, and the electrochemical l-V curves of were measured in the voltage range even as used in trans-electrode experiments. After normalization to the surface area, it was concluded that any electrochemical current in the electrodostrans devices was three orders of magnitude too small to explain the measurements of ~ pA currents through the graphene membranes as developed in Table 1. The conductivities observed for different cations drop much faster than the conductivity solutions in progress from CsCI to LiCI, suggesting an influence of graphene-cation interactions. However, ionic transport through graphene that is in contact with the chip surface cannot be considered completely excluded.
    Example III [0053] This example describes an experimental determination of the conductivity of the exposed single-layer graphene membrane of example I, including a nanopore.
    [0054] A single nanopore of nanometric size was drilled through several graphene membranes of example I, using an electron beam focused on a JEOL FEG 2010 transmission electron microscope operated at an acceleration voltage of 200kV. The nanopore diameter was determined by EM visualization, in a well-distributed electron beam in order to keep the total electron exposure of the graphene membrane to a minimum. A nanometer diameter of 8 nm was determined as the average of 4 measurements along different axes of the nanopor, as determined from TE calibrated micrographs using Digital Micrograph program
    Petition 870190057845, of 06/24/2019, p. 28/43
    26/29 (Gatan, Inc.). If the chip or TEM holder has any contaminating organic waste, the disordered carbon has been seen to visibly deposit under the electron beam. Such devices have been discarded. After drilling the nanopore, the graphene nanopore chips that were not immediately investigated were kept under a clean vacuum of ~ 10 5 Torr.
    [0055] Figure 7 both shows a graph of the ionic current as a function of the voltage applied as given above in example II for a continuous graphene membrane, as well as for a graphene membrane including an 8 nm wide nanopore. These graphs demonstrate that the ionic conductivity of the graphene membrane is increased by orders of magnitude by the nanopore.
    [0056] It was found that experiments with known diameters of graphene nanopores and known conductivities of ionic solutions allow the deduction of the effective insulation thickness of the exposed single-layer graphene membrane. Ten separate graphene membranes from Example I were processed to include nanopore diameters ranging from 5 to 23 nm. Then, the ionic conductivity of each of the ten membranes was measured with a 1 M KCI solution supplied to both cis and trans solution reservoirs, with a conductivity of 11 Sm -1 . Figure 8 is a graph of the measurement of ionic conductivity as a function of the nanopore diameter for the 10 membranes. The solid curve in the figure is the modeled conductivity of a 0.6 nm thick insulating membrane, which is the best fit to the conductivity of the experimental measurement. The modeled conductivity for a 2 nm thick membrane is shown as a dotted line, and the modeled conductivity for a 10 nm thick membrane is shown as a dashed-dotted line, presented for comparison.
    [0057] The ionic conductivity, G, of a nanopore in diameter, d,
    Petition 870190057845, of 06/24/2019, p. 29/43
    27/29 in an infinitely thin insulating membrane is given by:
    Gfina = od (1) [0058] where σ = F {p K + pci) c is the conductivity of the ionic solution, F is the Faraday Constant, c is the ionic concentration, and Pi (c) is the mobility of potassium (i = K) and ion chloride (i = Cl) used for an ionic KCI solution. The linear dependence of the conductivity on the next diameter from the current density being sharply peaked at the perimeter of the nanopore for an infinitely thin membrane, as described above. For membranes thicker than the nanopore conductivity diameter it becomes proportional to the nanopore area. For finite thin membranes, computer calculations can predict conductance.
    [0059] As shown in the graph in figure 8, according to expression (1), the conductivities of exposed single-layer graphene nanopores with diameters ranging from 5 to 23 nanometers exhibited an almost linear dependence on the nanopore diameter. The modeled curve was produced based on the nanopore ionic conductivity calculations in an insulating membrane ideally discharged, as a function of the nanopore diameter and membrane thickness. Points on this curve were obtained by numerically solving the Laplace equation for the density of the ionic current, with conductivity of the appropriate solution and boundary conditions, and integrating through the nanopore area to obtain the conductivity. These numerical simulations were performed using the COMSOL multiphysics finite element solver in the appropriate 3-D geometry with cylindrical symmetry along the nanopore axis. The complete set of Poisson-Nerst-Planck equations was solved in the steady state regime. In the range of physical parameters of interest, high salt concentration and small applied voltage, the solution of the numerical simulation was en
    Petition 870190057845, of 06/24/2019, p. 30/43
    28/29 contrasted not to differ significantly from the solution of the Laplace equation with fixed conductivity, which has significantly less computational penalty. The thickness of the membrane, L, used in this idealized model is here referred to as the thickness of the insulating graphene, or Lm. ) nm, with the uncertainty determined from at least one square error analysis.
    Example IV [0060] This example describes the experimental measurement of DNA translocation through a nanopore on an exposed single-layer graphene membrane, that of example I.
    [0061] The microfluidic cell of the examples above was washed with 3M KCI salt solution, pH 10.5, containing 1 mM EDTA. As explained above, high salt concentration and high pH have been found to minimize the interaction of DNA graphene and, therefore, these solution conditions may be preferred. The restriction fragments of the 10 kbp double-lambda chain of the DNA molecule were introduced into the cis chamber of the system. The negatively charged DNA molecules were electrophoretically designed and conducted through the nanopore by the applied electrophoretic force of 160mV. Each isolation molecule that passes through the nanopore transiently reduced, or blocked, the ion conductivity of the nanopore in a way that reflects both the polymer size and configuration. Like the DNA fragments passing through the nanopore due to the applied electrophoretic force, the translocation events were analyzed with MATLAB using a fitting function that consisted of multiple convoluted square pulses with an appropriate Bessel filter function to mimic the recording conditions.
    [0062] Figure 9 is a graph of ionic current measurement through
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    29/29 through the nanopore as a function of time to one minute from the time of a voltage deviation applied between the cis and trans reservoirs. Each drop in the measurement of current in the plane corresponds to a translocation of DNA through the nanopore, and allows the characterization of two parameters, namely, the average current drop, or blockage, and the duration of the blockage, which is the time it takes. leads to the molecule translocating completely through the nanopore. Note the high number of translocation events for the exposed graphene membrane nanopore in the period of one minute, indicating successful inhibition of DNA adherence to the exposed graphene membrane surface with the high pH salt solution , and careful cleaning and handling of the graphene membrane during preparation for DNA translocation experiments.
    [0063] Figures 10A, 10B, and 10C are graphs of ionic current measurement through the nanopore for individual translocation events. Figure 10A shows the blocking of the current ion flow during a translocation of DNA in the form of a single file. Figure 10B shows the blocking of the ionic current flow during the translocation of the DNA that was partially folded. Finally, figure 10C shows the blocking of the ionic current flow during the translocation of the DNA that was folded in half. These three experimental translocation events typify the possible ionic measurements of the current flow that may occur during the translocation of DNA fragments, and demonstrate that DNA folding and configuration can occur with the graphene nanopore as with more conventional thick nanopores than solid state.
    权利要求:
    Claims (20)
    [1]
    1. Graphene nanopore sensor (10), characterized by the fact that it comprises:
    a substantially exposed, self-supporting, single-layer graphene membrane (14) including a nanopore (12) that extends through a thickness of the graphene membrane (14) from a first surface of the graphene membrane to a second surface of the membrane graphene opposite the first surface of the graphene membrane;
    a connection from the first surface of the graphene membrane to a first reservoir to provide, on the first surface of the graphene membrane, a species (20) in an ion solution for the nanopore (12);
    a connection from the second surface of the graphene membrane to a second reservoir to collect the species (20) and ionic solution after the translocation of the species (20) and the ionic solution through the nanopore (12) from the first surface of the membrane graphene for the second surface of the graphene membrane, and an electrical circuit (28) connected on the opposite sides of the nanopore (12) to measure the flow of ionic current through the nanopore (12) in the graphene membrane (14).
    [2]
    2. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that the electrical circuit (28) is connected between the first and second ionic solutions to measure the flow of ionic current through the nanopore (12) in the graphene membrane (14), or the electrical circuit (28) includes a connected electric current monitor to measure the time-dependent ionic current flow through the nanopore (12), in a particular way
    Petition 870190057845, of 06/24/2019, p. 33/43
    2/6 where the electric current monitor is connected to measure time-dependent ionic current flow blocks indicative of species translocation through the nanopore (12).
    [3]
    3. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that it also comprises an electrode (24,26) disposed in each of the first and second ionic solutions for the application of a voltage through the nanopore (12) to electrophoretically cause species translocation through the nanopore (12).
    [4]
    4. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that (I) the ionic solution has a salt content that is greater than about 2 M, or (li) the ionic solution has a pH that is greater than about 8, or (iii) the ionic solution is KCI.
    [5]
    5. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that the nanopore (12) has a diameter that is greater than a thickness of the graphene membrane between the first and second surfaces of the membrane of the graphene (14) through which the nanopore (12) extends.
    [6]
    6. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that (i) the nanopore (12) has a diameter that is between about 1 nm and about 10 nm, or (ii) the nanopore (12) has a diameter that is between about 1nm and about 5nm, or (iii) nanopore (12) has a diameter that is less than about 3nm, or (iv) the nanopore (12) has a diameter which is smaller than
    Petition 870190057845, of 06/24/2019, p. 34/43
    3/6 that about 2.5nm.
    [7]
    7. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that (i) the graphene membrane (14) has a thickness that is less than about 2nm, or (ii) the membrane graphene (14) has a thickness that is less than about 1nm, or (iii) the graphene membrane (14) has a thickness that is less than about 0.7nm.
    [8]
    8. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that the diameter of the nanopore (12) is adapted to be no more than about 5% larger than a diameter of the species (20 ) in the nanopore (12).
    [9]
    Graphene nanopore sensor (10) according to claim 1, characterized in that the graphene membrane (14) is mechanically supported at the edges of the membrane (14) by a membrane frame structure (16).
    [10]
    10. Method for evaluating a polymer molecule (20), characterized by the fact that it comprises:
    supply, in an ionic solution, a polymer molecule (20) to be evaluated;
    translocating the polymer molecule (20) in the ionic solution through a nanopore (12) on a substantially exposed self-supporting single-layer graphene membrane (14) from a first surface of the graphene membrane to a second surface of the graphene membrane opposite the first graphene surface, as defined in claims 1 to 9; and monitoring the flow of ionic current through the nanopore (12) in the graphene membrane (14).
    [11]
    Method according to claim 10, characterizing
    Petition 870190057845, of 06/24/2019, p. 35/43
    4/6 due to the fact that the monitoring of the ionic current flow comprises the measurement of ionic current flow blocking depending on the indicative time of the translocation of the polymer molecule through the nanopore (12).
    [12]
    12. Method according to claim 10, characterized by the fact that it further comprises the application of a voltage across the nanopore (12) to electrophoretically cause the polymer molecule (20) to translocate through the nanopore (12).
    [13]
    13. Method according to claim 10, characterized in that the nanopore (12) has a diameter that is greater than a thickness of the graphene membrane (14) between the first and second surfaces of the graphene membrane (14) through which the nanopore (12) extends.
    [14]
    14. Method according to claim 10, characterized by the fact that the nanopore (12) has a diameter that is no more than about 5% greater than a diameter of the polymer molecule (20) that translocates through the nanopore (12).
    [15]
    15. Method according to claim 10, characterized by the fact that the monitoring of the ionic current flow comprises the measurement of the ionic current flow blockage depending on the indicative time of the diameter of the polymer molecule (20) that translocates through the nanopore (12).
    [16]
    16. Method according to claim 10, characterized in that the species (20) in ionic solution that translocate through the nanopore comprises biomolecules, or DNA molecules, RNA molecules, or oligonucleotides, or nucleotides.
    [17]
    17. Graphene nanopore sensor (10) according to claim 1, characterized by the fact that it comprises:
    a substantially exposed, self-supporting, single-layer graphene membrane (14) including a nanopore (12)
    Petition 870190057845, of 06/24/2019, p. 36/43
    5/6 extends through a thickness of the graphene membrane (14) from a first surface of the graphene membrane to a second surface of the graphene membrane opposite the first surface of the graphene and which has a diameter that is less than about 3 nm and greater than the thickness of graphene; and an electrical circuit (28) connected on opposite sides of the nanopore to measure the flow of ionic current through the nanopore (12) in the graphene membrane (14).
    [18]
    18. Graphene nanopore sensor (10) according to claim 17, characterized by the fact that it also comprises:
    a connection from the first surface of the graphene membrane to a first reservoir to supply, on the first surface of the graphene membrane, the polymer molecules (20) in an ionic solution for the nanopore (12);
    a connection from the second surface of the graphene membrane to a second reservoir to collect the polymer molecules and ionic solution after the translocation of the polymer molecules and the ionic solution through the nanopore from the first surface of the graphene membrane to the second surface of the graphene membrane.
    [19]
    19. Graphene nanopore sensor (10) according to claim 17, characterized by the fact that the electrical circuit (28) includes a connected electrical current monitor to measure time-dependent ion current flow blockages indicative of the translocation of the polymer molecule (20) through the nanopore (12); or where the ionic solution has a salt content that is greater than about 2 M and a pH that is greater than about 8.
    [20]
    20. Graphene nanopore sensor (10) according to claim 18, characterized by the fact that it also comprises an electrode (26.28) disposed in each of the first and second solutions
    Petition 870190057845, of 06/24/2019, p. 37/43
    6/6 ionic applications for the application of a voltage across the nanopore (12) to cause electrophoretically translocation of the nanopore species (12).
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    CN102630304B|2014-11-26|
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    KR20120069720A|2012-06-28|
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    AU2010307229B2|2016-02-25|
    AU2018201010A1|2018-03-01|
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    EP3196645B1|2019-06-19|
    EP3196645A1|2017-07-26|
    JP2013505448A|2013-02-14|
    AU2016203366A1|2016-06-16|
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    CN102630304A|2012-08-08|
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    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-03-26| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2019-08-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2019-10-22| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/09/2010, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US24360709P| true| 2009-09-18|2009-09-18|
    US35552810P| true| 2010-06-16|2010-06-16|
    PCT/US2010/049238|WO2011046706A1|2009-09-18|2010-09-17|Bare single-layer graphene membrane having a nanopore enabling high-sensitivity molecular detection and analysis|
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