Patent Application
20210302410
Freestanding ultrathin membranes have attracted a lot of interest in recent years as filtration materials, [1-8] synthetic analogues of biological membranes, [9] and as microelectromechanical sensor (MEMS)-based devices. [10-13] Ultrathin membranes have also found use as substrates for high-resolution nanopores, where the nanopores are tools for single-molecule detection and next-generation DNA sequencing. [14-17] While dielectrics such as silicon nitride (SiN) [18], aluminum oxide [19,20], and silicon oxide [21], have been explored as membrane materials, there is a practical lower limit on membrane thickness for silicon-based membranes (˜5-10 nm) [22-24] because silicon oxide and SiN are structurally and chemically more susceptible to damage during [25,26] and after [27] nanopore fabrication. Since high-resolution readout of DNA requires robust ultrathin (<2 nm) membranes, other membrane materials have been explored, including graphene [28-30], boron nitride [31], DNA origami [32-34], hafnium oxide [35,36], and molybdenum disulfide (MoS2) [37]. Due to its atomic thickness, mechanical properties, and unique electrical properties [38-43], graphene in particular allows an additional sensing modality that may allow faster readout speeds and improved resolution. Finally, graphene membranes and their derivatives are impermeable to small molecules and ions, which allows nanopores in such membranes to be tailored for water desalination/purification [44] and selective molecular sieving [3,45,46].
Graphene can be obtained by mechanical exfoliation from graphite flakes [47,48], chemical oxidation/reduction [49], chemical vapor deposition (CVD) [50], and epitaxial growth [51]. In this context, CVD-assisted graphene growth on appropriate catalytic metal surfaces has garnered considerable interest due to its relative ease of synthesis, low cost of production of large-area high-quality graphene, and lack of intense mechanical and chemical treatments. While various approaches for graphene membranes have been reported for nanopore-based DNA analysis [28-30,52], these approaches require graphene transfer to an appropriately perforated substrate. Although considerable progress has been made in graphene transfer techniques [30,53-55], no process offers a scalable approach to produce a large number of membranes for use in nanopore and other membrane-related experiments. In addition, synthesize-then-transfer-graphene protocols [30] for producing graphene can degrade the quality of the membranes by introducing wrinkles, cracks, and contamination during the transfer process. Recently, freestanding graphene membranes have been produced on TEM grids by a transfer-free approach on larger (˜30 m) apertures [56], although the ionic permeability of these membranes were not studied.
Consequently, there is need for methods of making ultrathin, freestanding graphene membranes. In particular, methods are needed for making intact, ion-impermeable freestanding graphene membranes and methods of introducing small (0.5-50 nm) nanopores in such membranes without otherwise affecting membrane integrity. In addition, methods for avoiding membrane transfer would be advantageous.
Described herein are devices containing freestanding, ultrathin (<10 nm thick) membranes and methods of making such devices. Also described are methods of using devices containing freestanding ultrathin membranes for determining the sequence of a polynucleotide and for desalination of aqueous solutions.
In one aspect, the invention is a device containing: a substrate having an upper surface, a lower surface, and an aperture, the aperture having one or more walls connecting the upper and lower surfaces and forming a well; and a membrane attached to the lower surface of the substrate and forming a floor of the well, the membrane having a thickness of less than 10 nm. In some embodiments, the electrical conductance across the membrane is less than 1 nS/μm2.
In one aspect, the invention is a device containing: a substrate having an upper surface, a lower surface coated with a passivating layer, and an aperture, the aperture having one or more walls connecting the upper and lower surfaces and forming a well; and a membrane attached to the passivating layer and forming a floor of the well, the membrane having a thickness of less than 10 nm and comprising a nanopore. In some embodiments, the background conductance of the membrane is less than less than 1 nS/m2.
In some embodiments, the membrane is less than about 7.5 nm, less than about 5 nm, less than about 4 nm, less than about 3 nm, less than about 2 nm, or less than about 1 nm thick.
In some embodiments, the lower surface of the substrate, the upper surface of the substrate, and/or the one or more walls of the substrate are coated with a layer of passivating material. In some embodiments, the passivating material is HfO2 or TiO2. In some embodiments, the layer of passivating material is about 5 nm, about 7.5 nm, about 10 nm, about 15 nm, about 20 nm, or about 40 nm thick.
In some embodiments, the membrane contains one nanopore. In some embodiments, the graphene membrane contains a plurality of nanopores. In some embodiments, each nanopore has a diameter of about equal length. In some embodiments, each nanopore has a diameter of about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1 nm, about 1.5 nm, about 2 nm, about 2.5, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 7.5 nm, about 8 nm, about 9 nm, about 10 nm, about 12 nm, about 15 nm, about 20 nm, about 30 nm, about 40 nm, or about 50 nm.
In some embodiments, the substrate contains silicon nitride, aluminum oxide, or hafnium oxide. In some embodiments, the substrate is about 100 nm thick.
In some embodiments, the substrate has multiple wells. In some embodiments, each well has the same diameter. In some embodiments, different wells have different diameters. In some embodiments, the substrate has a set of one or more larger wells and a set of one or more smaller wells. In some embodiments, each well has a diameter of less than 2 μm, less than 1.5 μm, less than 1.2 μm, less than 1p m, less than 750 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, or less than 100 nm. In some embodiments, the wells in one set have a diameter of about 800 nm, and the wells in another set have a diameter of about 450 nm.
In some embodiments, the membrane is made of Bi2Se3, bismuth strontium calcium copper oxide, Bi4Ti3O12, boron nitride, boron carbon nitride, (Ca,Sr)2Nb3O10, Ca2Ta2TiO10, carbon nitride, Cu oxide, Cu2O, CuO, Cu2O3, Eu(OH)2, fluorographene, GaSe, GaTe, graphene, graphene oxide, InSe, LaNb2O7, MnO, MoO3, MoSe2, MoS2, MoTe, Ni(OH)2, NiSe2, NbSe2, NbS2, RuO2, TaO3, TaS2, TiO, TiS2, VO, WO3, WSe2, WS2, WTe, ZrSe, or ZrS. In some embodiments, the membrane is made of graphene. In some embodiments, the graphene membrane is less than about 5 atomic layers, less than about 4 atomic layers, less than about 3 atomic layers, or less than about 2 atomic layers thick.
In some embodiments, the graphene at each nanopore has been stabilized to minimize chemical reactivity of the graphene with molecules, ions, and solutes that pass through the nanopore. In some embodiments, that graphene has been stabilized by hydrogenation, hydroxylation, treatment with amphiphilic molecules, biological peptides, or other surface active agents (surfactants).
In some embodiments, the graphene membrane is coated with one or more amphiphilic molecules non-covalently bound to its upper and lower surfaces. In some embodiments, the amphiphilic molecule contains a hydrophobic portion comprising pyrene. In some embodiments, the amphiphilic molecule contains a hydrophilic portion containing an ethylene glycol.
In some embodiments, the device contains a hydrophilic layer attached to the surface of each graphene membrane external to the well and to the lower surface of the substrate. In some embodiments, the hydrophilic layer contains Al2O3, SiO2, TiO2, or HfO2. In some embodiments, the hydrophilic layer is less than about 30 nm, less than about 20 nm, less than about 15 nm, less than about 10 nm, or less than about 5 nm thick.
In some embodiments, the device has a nanopore in the hydrophilic layer that is aligned with a nanopore in the membrane, e.g., a graphene membrane. In some embodiments, the device has a lipid bilayer non-covalently bound to the hydrophilic layer, the lipid bilayer covering the nanopore in the hydrophilic layer and the graphene membrane. In some embodiments, device contains a biological nanopore situated in the region of the lipid bilayer covering the nanopore.
In some embodiments, the device contains a first lipid monolayer non-covalently bound to the surface of the graphene membrane internal to the well and a second lipid monolayer non-covalently bound to the surface of the graphene membrane external to the well. In some embodiments, the device has one or more nanopores in the graphene membrane to which the lipid monolayers are bound, the lipid monolayers forming a bilayer in the region of the nanopore. In some embodiments, the device contains a biological nanopore disposed in the lipid bilayer.
In some embodiments, the biological nanopore is alpha-hemolysin, MspA porin, or ClyA porin.
In some embodiments, the device contains a supporting structure attached to the upper surface of the substrate, the supporting structure containing a window that provides access to the well. In some embodiments, the device contains an insulating layer attached to the upper surface of the substrate and a supporting structure attached to the insulating layer. In some embodiments, the supporting structure is made of silicon, silicon dioxide, glass, quartz, or mica. In some embodiments, the insulating layer is made of silicon, silicon dioxide, glass, quartz, or mica. In some embodiments, the supporting structure and the insulating layer, if present, contain a plurality of windows, each window providing access to at least one well. In some embodiments the supporting structures contain one or more scored lines between two or more windows, the scored lines enabling the division of the device into two or more pieces, each piece containing one or more windows.
In some embodiments, the device has at least 5, at least 10, at least 20, at least 50, at least 100, at least 150, or at least 200 windows.
In one aspect, the invention includes an apparatus for the study of polynucleotides, the apparatus having: a nanopore membrane device of the invention, wherein the membrane is positioned between a first fluid reservoir and a second fluid reservoir; an electrode pair having a first electrode disposed in the first fluid reservoir and a second electrode disposed in the second fluid reservoir; and a circuit capable of detecting an electrical signal that correlates with the sequence of the polynucleotide.
In one aspect, the invention includes an apparatus for the study of polynucleotides, the apparatus having: a graphene nanopore membrane device of the invention, wherein the graphene membrane is positioned between a first fluid reservoir and a second fluid reservoir; a first electrode pair having a first electrode disposed in the first fluid reservoir and a second electrode disposed in the second fluid reservoir, wherein the first electrode pair is capable of applying an electrical field between the fluid reservoirs across the graphene membrane; a second electrode pair having a first electrode and second electrode on opposite sides of the nanopore, wherein the second electrode pair is capable of applying an electrical field laterally through the graphene membrane and across the nanopore of said device; and a circuit capable of detecting an electrical signal from the second electrode pair that correlates with the sequence of the polynucleotide.
In some embodiments, the apparatus includes a processor for processing signals from the electrodes. In some embodiments, the processor is capable of identifying a polynucleotide, a component of a polynucleotide, or a precursor of a polynucleotide from a signal provided by the electrical sensor.
In some embodiments, the apparatus contains a plurality of devices of the invention.
In another aspect, the invention includes an apparatus for removal of one or more ionic solutes from an aqueous solution, the apparatus having a plurality of devices containing graphene membranes having a plurality of nanopores.
In another aspect, the invention includes a method of making a membrane device of the invention, the method including the steps of: providing a substrate having an upper surface, a lower surface, and an aperture, the aperture having one or more walls connecting the upper and lower surfaces and forming a well; depositing a passivating layer on the lower surface of the substrate; and forming on the lower surface of the substrate a membrane that extends across the aperture, thereby forming a floor of the well.
In another aspect, the invention includes a method of making a membrane device of the invention, the method including the steps of: providing a substrate having an upper surface, a lower surface, and an aperture, the aperture having one or more walls connecting the upper and lower surfaces and forming a well; depositing a passivating layer on the lower surface of the substrate; depositing a sacrificial layer having an upper surface and a lower surface on the passivating layer and across the aperture, thereby forming a floor of the well; forming on the upper surface of the sacrificial layer and/or the passivating layer a membrane that extends across the aperture; and removing the sacrificial layer, leaving the membrane as the floor of the well.
In some embodiments, the sacrificial layer contains Cu, Fe, Ni, Pd, or Pt. In some embodiments, the sacrificial layer contains Cu. In some embodiments, the sacrificial layer is 200 nm thick. In some embodiments, the Cu-containing sacrificial layer is formed by thermal evaporation. In some embodiments, the Cu-containing sacrificial layer is removed by dissolving the Cu in ammonium persulfate.
In some embodiments, the membrane is made of graphene and is made by carbon vapor deposition of methane and hydrogen gases.
In some embodiments, the method includes making a plurality of membrane devices.
In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the membranes produced by the method are intact. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the membranes produced by the method have a conductance of less than 1 nS/μm2.
In some embodiments, the method entails creating one or more nanopores in the membrane. In some embodiments, the nanopores are created by an electron beam or an ion beam.
In another aspect, the invention includes a method of determining the sequence of bases of a polynucleotide using the apparatus of the invention, the method including the steps of: providing an apparatus that includes (1) a device with a freestanding ultrathin membrane having one or more nanopores positioned between two fluid reservoirs, (2) an electrode pair including one electrode disposed in the first fluid reservoir and a second electrode disposed in the second fluid reservoir, and (3) a circuit capable of detecting an electrical signal; adding an aqueous solution comprising the polynucleotide to the first reservoir and adding an aqueous solution to the second reservoir; applying an electrical field between the first and second electrodes, wherein the electrical field causes one or more charged molecules to transit through the nanopore; measuring a signal due to the transit of the one or more charged molecules through the nanopore; and determining the sequence of bases of the polynucleotide from a previously determined correlation between identities of the bases and the signal.
In some embodiments, the signal is optical. In some embodiments, the signal is electrical. For example, the signal may be a change in current through the nanopore.
In some embodiments, the charged molecules that transit through the nanopore are copes of the polynucleotide whose sequence is being determined. In some embodiments, the polynucleotide that transits through the nanopore is single-stranded. In some embodiments, the polynucleotide that transits through the nanopore is double-stranded. In some embodiments, the polynucleotide that transits through the nanopore contains both single-stranded and double-stranded portions.
In some embodiments, the charged molecules that transit through the nanopore include four synthetic molecules that correspond to each of the four bases that naturally occur in a given polynucleotide, for example, DNA or RNA, wherein each synthetic molecule confers a change in current as it passes through the nanopore, the change in current due to each synthetic molecule being distinct from the change in current due to each of the other three synthetic molecules.
In some embodiments, the first electrode is the anode and the second electrode is the cathode. In some embodiments, the first electrode is the cathode and the second electrode is the anode. In some embodiments, the first electrode alternates between being the anode and the cathode and the second electrode alternates between being the cathode and the anode.
In some embodiments, the polynucleotide transits through the nanopore in a direction determined by an electromotive force provided by the electrical field. In some embodiments, the polynucleotide transits through the nanopore against an electromotive force provided by the electrical field. In some embodiments, the polynucleotide transits through the nanopore alternately with and against an electromotive force provided by the electrical field.
In some embodiments, the force that drives transit of the polynucleotide against the electromotive force is provided by a magnetic field. In some embodiments, the polynucleotide is bound to a magnetic bead. In some embodiments, the force that drives transit of the polynucleotide against the electromotive force is provided by a polynucleotide polymerase.
In some embodiments, the first reservoir contains an enzyme. In some embodiments, the enzyme is DNA polymerase, RNA polymerase, DNA exonuclease, RNA exonuclease, DNA helicase, or RNA helicase. In some embodiments, the enzyme is bound to the membrane adjacent to the nanopore.
In some embodiments, the first reservoir contains an oligonucleotide primer capable of hybridizing with the polynucleotide. In some embodiments, the oligonucleotide primer is bound to the membrane adjacent to the nanopore.
In another aspect, the invention includes a method of determining the sequence of bases of a polynucleotide using the apparatus of the invention, the method including the steps of: providing an apparatus that includes (1) a device with a freestanding ultrathin graphene membrane having one or more nanopores positioned between two fluid reservoirs, (2) a first electrode pair including one electrode disposed in the first fluid reservoir and a second electrode disposed in the second fluid reservoir, and (3) a second electrode pair including electrodes in contact with the graphene membrane on opposite sides of the nanopore, wherein the second electrode pair is capable of applying an electrical field laterally through the graphene membrane, and (4) a circuit capable of detecting an electrical signal from the second electrode pair; adding an aqueous solution comprising the polynucleotide to the first fluid reservoir and adding an aqueous solution to the second fluid reservoir; applying an electrical field between the first pair of electrodes, wherein the electrical field causes one or more charged molecules to transit through the nanopore; applying an electrical field between the second pair of electrodes; measuring a signal between the second pair of electrodes due to the transit of the one or more charged molecules through the nanopore; and determining the sequence of bases of the polynucleotide from a previously determined correlation between identities of the bases and the signal.
In another aspect, the invention includes a method of deionizing an aqueous solution comprising water and unwanted ions, said method including the steps of: providing a device of the invention; applying a pressure to forcibly flow the aqueous solution into the well, thereby causing molecules of water to flow through the nanopores of the membrane and exit the well and causing the unwanted ions to be retained in the well; and collecting the molecules of water that have exited the well.
The invention provides freestanding, crack-free ultrathin (<10 nm thick) membranes. Also provided is a novel approach for in-situ fabrication of large arrays of freestanding ultrathin membranes. Ultrathin graphene membranes are grown directly onto an array of sub-micrometer apertures in a scalable method. The membranes grow precisely above the apertures to yield crack-free membranes, as determined by ionic current measurements. Because the method does not involve transfer of membranes after they have been synthesized, the approach is more practical for obtaining freestanding ultrathin membranes with high yield. The present invention also provides methods of using freestanding ultrathin membranes for determining the sequence of bases in a polynucleotide and for removal of ions from an aqueous solution.
The invention includes a method of making devices containing freestanding, crack-free ultrathin membranes.
In alternate embodiments, a passivating layer is not used. In such embodiments, the periphery of the upper membrane extends beyond the floor of the well to become sandwiched between the sacrificial layer and substrate. When the sacrificial layer and lower membrane, if present, are removed, the upper membrane remains attached to the substrate, leaving a freestanding ultrathin membrane as the floor of the well.
In other embodiments, a sacrificial layer is not used. In such embodiments, a passivating layer may or may not be used. If a passivating layer is used in such embodiments, the membrane is formed directly on the passivating layer and extends laterally to cover the aperture to create a floor of the well formed by the aperture, thereby forming a freestanding ultrathin membrane. If a passivating layer is not used in such an embodiment, the membrane is formed directly on the lower surface of the substrate and extends laterally to cover the aperture to create a floor of the well formed by the aperture, thereby forming a freestanding ultrathin membrane.
The membrane may be made of any material capable of forming ultrathin membranes impermeable to ionic solutes. For example, the membrane may be made of Bi2Se3, bismuth strontium calcium copper oxide, Bi4Ti3O12, boron nitride, boron carbon nitride, (Ca,Sr)2Nb3O10, Ca2Ta2TiO10, carbon nitride, Cu oxide, Cu2O, CuO, Cu2O3, Eu(OH)2, fluorographene, GaSe, GaTe, graphene, graphene oxide, InSe, LaNb2O7, MnO, MoO3, MoSe2, MoS2, MoTe, Ni(OH)2, NiSe2, NbSe2, NbS2, RuO2, TaO3, TaS2, TiO, TiS2, VO, WO3, WSe2, WS2, WTe, ZrSe, or ZrS.
The substrate may be made of a low-dielectric material. For example, the substrate may be made of silicon nitride, silicon oxide, aluminum oxide, or hafnium oxide. The substrate may be, for example, about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 200 nm, or about 500 nm thick.
The substrate may contain one well or multiple wells. For example, the substrate may contain at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 40, at least 60, at least 80, at least 100, at least 150, or at least 200 wells. The wells in the substrate may be uniform in size, or the substrate may have sub-populations of wells of different sizes. For example and without limitation, the substrate may have a single larger central well surrounded by four smaller wells.
The wells may take different shapes. For example, the wells may be cylindrical, having a diameter at the floor of the well equal to the diameter of the opening of the well. The wells also may be conical, having a diameter at the floor of the well that is greater than or less than the diameter of the opening of the well. The wells may have both cylindrical and conical regions. The depth of the well may vary, depending on the thickness of the substrate.
The freestanding membranes form the floor of the well, and the well and membrane may vary in size. For example, the freestanding membranes at the floor of the well may have a diameter of about 200 nm, about 300 nm, about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 m, about 2 m, about 4 m, about 6 m, about 8 m, or about 10 m.
The passivating material may be any material that prevents the substrate from interfering with formation of the membrane, or improves the ability to form membranes on the substrate material. For example, the passivating material may be HfO2 or TiO22. The layer of passivating material may about 5 nm, about 7.5 nm, about 10 nm, about 15 nm, about 20 nm, or about 40 nm thick. The passivating layer may be applied by any method known in the art. For example, the passivating layer may be applied by atomic layer deposition.
The sacrificial layer may be made of any material that supports formation of the membrane and can be selectively removed by chemical etching without affecting the membrane integrity. For example, the sacrificial layer may be Cu, Fe, Ni, Pd, or Pt. The sacrificial layer may be made by any suitable method known in the art. For example, the sacrificial layer may be made using sputtering or using a vacuum thermal evaporation system.
The membranes may be formed by any method known in the art. For example, graphene membranes may be formed by chemical vapor deposition (CVD) using methane and hydrogen gases. The membranes may be less than 10 nm, less than 9 nm, less than 8 nm, less than 7 nm, less than 6 nm, 5 nm, less than 4 nm, less than 3 nm, less than 2 nm, or less than 1 nm thick. The membranes may be, for example, less than 5 atomic layers, less than 4 atomic layers, less than 3 atomic layers, or less than 2 atomic layers thick.
The sacrificial layer may be removed by any method that removes the sacrificial layer without affecting the integrity of the upper membrane. For example, a Cu sacrificial layer may be etched using 10% ammonium persulfate.
The method can be used to make intact membranes that are essentially free of defects. Defects, such as cracks and holes, allow ions to pass through the membrane, resulting in increased ionic conductance. Therefore, the presence of an intact membrane can be inferred from low ionic conductance across the membrane. For example, while testing using a 1M solution of KCl at room temperature, the ionic conductance across the membrane may be less than 0.1 nS/μm2, less than 0.3 nS/μm2, less than 0.5 nS/μm2, less than 0.7 nS/μm2, less than 1 nS/μm2, less than 1.5 nS/μm2, less than 2 nS/μm2, less than 5 nS/μm2, less than 10 nS/μm2, or less than 20 nS/m2.
The method reliably produces intact membranes that are free of cracks. For example, the percentage of intact membranes produced by the method may be greater than 60%, greater than 70%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. Whether a membrane is intact may be determined, for example, from its ionic conductance. Thus, a membrane may considered intact if the ionic conductance across the membrane is less than 0.1 nS/μm2, less than 0.3 nS/μm2, less than 0.5 nS/μm2, less than 0.7 nS/μm2, less than 1 nS/μm2, less than 1.5 nS/μm2, less than 2 nS/μm2, less than 5 nS/μm2, less than 10 nS/μm2, or less than 20 nS/μm2. The percentage of intact membranes also can be determined by inspection using electron microscopy, in which cracks are identified by their structure.
As shown in
As used herein, “background conductance” is the conductance of the membrane due to cracks and holes in the membrane prior to the creation of nanopores. The creation of nanopores in a membrane increases its conductance. Therefore, background conductance is the conductance of a membrane before nanopores have been created in the membrane.
Pores in the membrane may be made by any method that allows the nanopores size to be precisely controlled and that does not damage the remaining portions of the membrane. For example, electron beams or ion beams can be used to create nanopores in the membrane.
The shape and size of the nanopores in the membrane will vary depending on the application of the membrane. For some applications, the nanopores are circular or nearly circular. For some applications, the circular or nearly circular nanopores have a diameter of about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1 nm, about 1.5 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 7.5 nm, about 8 nm, about 10 nm, about 12 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm. In various embodiments, nanopores may have a size in the range from about 0.5 nm to about 3 nm, from about 0.5 nm to about 5 nm, from about 1 nm to about 10 nm, or from about 0.5 nm to about 50 nm. In membranes that have multiple nanopores, the nanopores may be uniform or nearly uniform in size and shape. Alternatively, in membranes that have multiple nanopores, the nanopores may vary in size and/or shape.
The carbon atoms at the edge of the nanopores in graphene membranes may be passivated to strengthen or stabilize the nanopore or to change its chemical properties. For example, the edges of the nanopores, particularly with graphene membranes, may hydrogenated, hydroxylated, or treated with a functionalized peptide or surfactant. Hydrogenation of carbon atoms at nanopore edges can be used to create more hydrophobic nanopores, and hydroxylation of carbon atoms or treatment with a surfactant at nanopore edges can be used to create more hydrophilic nanopores.
In membranes that have multiple nanopores, the nanopores may be regularly or randomly spaced. For example, the average nanopore spacing may be, for example, about 10 nm, about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 400 nm, about 600 nm, about 1 m, about 2 m, or about 3 m apart.
For some applications, it may be necessary to coat the membranes with other materials to prevent analytes from binding to the membrane. For example, DNA may stick to graphene due to hydrophobic interactions between graphene and the nucleotide bases. Thus, graphene membranes may be coated with other materials to prevent unwanted interactions with the membranes, such as materials that decrease the hydrophobicity and increase the hydrophilicity of the membranes. Graphene membranes may be coated by non-covalent binding of amphiphilic polymers to the membrane surfaces. A two-step method for coating graphene membranes has been described by Schneider et al. [68] In the first step, aminopyrene is adsorbed onto graphene. In the second step, tetraethyleneglycol monomethyl ether N-hydroxysuccinimide ester is added, causing the formation of stable peptide bond between the amine group of aminopyrene and the carbonyl group of N-hydroxysuccinimide ester. Other methods that achieve comparable effects may also be used. Alternatively, graphene membranes may be coated with hydrophilic metallic compounds. For example, coating of graphene membranes with Al2O3 has been described by Connelly et al. [69] TiO2, SiO2, and HfO2 can also be used. The amphiphilic or hydrophilic coating may be less than 20 nm, less than 15 nm, less than 10 nm, or less than 5 nm thick. The amphiphilic or hydrophilic coating may be applied to a membrane before or after nanopores have been created in the membrane. Consequently, it may be necessary to create nanopores in the amphiphilic or hydrophilic coating as well as the membrane so that the nanopore extends through both the coating and the membrane.
The membranes may also be coated with lipid layers, for example, lipid monolayers and lipid bilayers. The lipid bilayer may interact with the membrane directly or indirectly. For example, the membrane can be coated on one side with a hydrophilic layer, e.g., Al2O3, and the polar head groups of one layer of the lipid bilayer can bind to the hydrophilic layer. Because the lipid bilayer forms a continuous sheet, the nanopores in the membrane and hydrophilic layer remain covered with a region of freestanding lipid bilayer. Alternatively, each side of a hydrophobic membrane, e.g., graphene, may bind directly to the hydrophobic acyl chains of a lipid monolayer. Consequently, the regions of intact membrane form a three-layered structure in which the membrane is sandwiched between two lipid monolayers. Over the nanopores, however, the acyl chains of one monolayer interact directly the acyl chain from the other monolayer, resulting in a freestanding lipid bilayer over the nanopore. In both of these arrangement, the freestanding lipid bilayers over the nanopores prevent ionic solutes from passing through the nanopores.
For some applications, biological nanopores are integrated into the regions of freestanding lipid bilayer over the nanopores of a membrane to allow passage of charged molecules across the membrane. The biological nanopores may be proteins, for example, nanopore proteins or ion channels. The nanopore proteins may be proteins useful for sequencing polynucleotides. For example, the nanopore protein may be alpha-hemolysin, MspA porin, ClyA porin, or another nanopore protein.
The substrate is attached to a supporting structure and, optionally, an insulating layer. As shown in
The method of making devices is scalable so that multiple wells and membranes can be made on a single supporting structure, i.e., the devices can be manufactured as “chips.” One chip may have, for example, 1, 2, 3, 4, 5, 10, 20, 40, 60, 80, 100, 150, 200, 400, 600, 800, 1000, 2000, 4000, 6000, 8000, 10,000 or more windows, each window containing one or more wells. A chip with multiple nanopores may be designed for use as such, or it may be designed to be divided into smaller devices, each containing one or more windows that contain one or more wells, for separate use, as shown in
The invention includes composite devices that can be used to determine the base sequence of a polynucleotide. In addition to the substrate and membranes having one or more nanopores as described above, such composite devices may include one or more of the following: a pair a reservoirs capable of holding conductive fluid, the two reservoirs in contact with, but on opposite sides of, the membrane; a first pair of electrodes capable of applying a an electrical field between the two fluid reservoirs, each of the two electrodes in electrical contact with one reservoir; an electrical sensor capable of detecting an electrical signal between the two reservoirs; an electrical sensor capable of detecting a lateral electrical signal in the plane of the membrane; and a processor attached to the electrical sensor, the processor capable of identifying a polynucleotide, a component of a polynucleotide, or a precursor of a polynucleotide from a signal provided by the electrical sensor.
The invention also encompasses methods of using the devices described herein to determine the sequence of bases in a polynucleotide. Several methods for determining the sequence of a polynucleotide using nano-scale nanopores in other materials have been have been described previously, and these methods can be adapted for use with the devices containing freestanding ultrathin membranes described herein. See, for example, WO 2013/185137; U.S. Pat. No. 8,652,779; US 2013/0264207; WO 2012/088339; US 2007/0190542; WO 2013/119784; and US 2013/0256118. The methods involve an apparatus having the following: a device with a freestanding membrane having one or more nanopores positioned between two fluid reservoirs; and an electrode pair including one electrode in electrical contact with the one fluid reservoir and another electrode in contact with the other fluid reservoir, the electrodes capable of applying an electrical field; and a circuit capable of detecting an electrical signal. The methods involve the following steps: adding an aqueous solution containing the polynucleotide to one reservoir and adding an aqueous solution to the other reservoir; applying an electrical field between the two electrodes, causing one or more charged molecules to transit through the nanopore; measuring a signal due to the transit of the one or more charged molecules through the nanopore; and determining the sequence of bases of the polynucleotide from a previously determined correlation between identities of the bases and the signal.
The signal measured may be optical. See, for example, US 2013/0256118. Alternatively, the signal measured by be electrical. See, for example, WO 2013/185137; U.S. Pat. No. 8,652,779; US 2013/0264207; WO 2012/088339; US 2007/0190542; WO 2013/119784.
The electrode in contact with the reservoir that initially contains the polynucleotide may function as the anode or the cathode, or it may alternate between functioning as the anode and cathode.
The charged molecules that transit through the nanopore may be the polynucleotides. See, for example, WO 2013/185137; WO 2012/088339; US 2007/0190542; WO 2013/119784; and US 2013/0256118. The polynucleotide that passes through the nanopore may be single-stranded, double-stranded, or have a combination of single-stranded and double-stranded portions. See, for example, WO 2013/185137; WO 2012/088339; US 2007/0190542; WO 2013/119784; and US 2013/0256118. Alternatively, the charged molecules that transit through the nanopore may be synthetic molecules that correspond to the four naturally-occurring bases of the polynucleotide. For example, they may be derived from dNTP analogs in which each of the four naturally-occurring nucleotides has been modified by the addition of a synthetic tag via three or more phosphates. See, for example, U.S. Pat. No. 8,652,779; US 2013/0264207. Such analogs are used for synthesis of a new polynucleotide strand by a polymerase situated near the nanopore. In some methods, the synthetic molecule includes a linker and a current blockade label. See U.S. Pat. No. 8,652,779. While dNTP base is paired with its complement on the template strand, the blockade label becomes temporarily lodged within the nanopore. See U.S. Pat. No. 8,652,779. Cleavage of the phosphate linkage by the polymerase allows charged molecule composed of the linker and blockade label to pass through the nanopore. Alternatively, in some methods, the synthetic molecule includes a tag that is unable to enter the nanopore while the dNTP base is paired with its complement on the template strand. See US 2013/0264207. Cleavage of the phosphate linkage by the polymerase allows the charged molecule composed of the tag and one or more phosphates to pass through the nanopore.
The polynucleotides that transit through the nanopores may have modified bases. See, for example, WO 2013/185137. The polynucleotide may have modified bases on only one strand. In some methods, only one strand of the polynucleotide passes through the nanopore, and the strand passing through the nanopore may be the strand with modified bases or the strand without modified bases. If the strand with the modified bases passes through the nanopore, the modified bases may block or alter the flow of ions through the nanopore and thus be identified by measuring the current through the nanopore. Alternatively, the modified bases may alter the kinetics of transit of the polynucleotide through the nanopore. See, for example, WO 2013/185137. For example, transit of a strand of a polynucleotide may be coupled with an enzymatic reaction, such as the synthesis of a new strand of DNA or RNA, the exonucleolytic degradation of a strand or DNA or RNA, or the separation of two strands of DNA or RNA, and the modified base may slow the rate of the enzymatic reaction. See, for example, WO 2013/185137.
The polynucleotides that transit through the nanopores may move with the electromotive force or against it, or they may alternate between moving with and against the electromotive force. The force moving the polynucleotides through the nanopore against the electromotive force may be magnetic. For example, the polynucleotide may be covalently bound to a magnetic bead. See, for example, WO 2013/119784. Alternatively, the polynucleotide may move through a nanopore against an electromotive force due to the pulling forces of an enzyme, for example, a polymerase. See, for example, WO 2013/185137.
The reservoir that initially contains the polynucleotide may also contain an enzyme. For example, it may contain a DNA polymerase, RNA polymerase, DNA exonuclease, RNA exonuclease, DNA helicase, and RNA helicase. See, for example, WO 2013/185137. The enzyme may be bound to the membrane located precisely above or directly adjacent to the nanopore. See, for example, WO 2013/185137; and U.S. Pat. No. 8,652,779.
The reservoir that initially contains the polynucleotide may also contain a primer that is capable of hybridizing with the polynucleotide. The primer is a short, usually chemically synthesized oligonucleotide, of appropriate length, for example about 18-24 bases, sufficient to hybridize to a target DNA (e.g. a single stranded DNA) and permit the addition of a nucleotide residue thereto, or oligonucleotide or polynucleotide synthesis therefrom, under suitable conditions well-known in the art. In an embodiment the primer is a DNA primer, i.e. a primer consisting of, or largely consisting of, deoxyribonucleotide residues. The primers are designed to have a sequence which is the reverse complement of a region of template/target DNA to which the primer hybridizes. The addition of a nucleotide residue to the 3′ end of a primer by formation of a phosphodiester bond results in a DNA extension product. The addition of a nucleotide residue to the 3′ end of the DNA extension product by formation of a phosphodiester bond results in a further DNA extension product. The primer may be bound to the membrane adjacent to the nanopore. See, for example, US 2013/0264207.
Substrates for nanopore fabrication were 5 mm×5 mm Si chips with a 100-nm-thick SiN film deposited on a 2.5-μm-thick thermal SiO2 layer, which helps to reduce electrical noise. SiN was protected with a 950 PMMA etch mask, and a small region (2 m×2 m square region with a pattern of four 450 nm-diameter holes and a central 800 nm hole) was exposed using Nabity NPGS e-beam writing software on a Hitachi S-4800 scanning electron microscope. Exposed PMMA was developed with 3:1 isopropyl alcohol and methyl isobutylketone, and AFM- and ellipsometry-calibrated thicknesses of SiN were etched in a Technics Micro-RIE Series 800 etcher using sulfur hexafluoride (SF6) at 300 mTorr and 150 W. PMMA was removed using 30 min treatment with acetone and chips were cleaned with hot piranha followed by warm water to remove the residual PMMA. HfO2 films were deposited at 150° C. using a benchtop ALD system (Arradiance Gemstar), with tetrakis(ethylmethylamino) hafnium and H2O used as a precursor and oxidizer, respectively. A calibrated thickness of Cu was evaporated on the membrane side of the chip using a vacuum thermal evaporation system. Graphene membranes were synthesized using a low-pressure CVD technique in a split tube furnace with a 35 mm O.D. quartz tube as follows: Following Cu deposition, the chips were placed in the center of the furnace and the vacuum system was turned on. Once the pressure inside the furnace became ˜11 mTorr, 35 sccm of H2 were allowed to flow and the tube was heated to growth temperature of 1000° C. Upon reaching 1000° C. the H2 flow rate was reduced to 2 sccm, and 35 sccm of CH4 gas were flowed for 3 h for graphene growth. During the growth process, the vacuum of the whole system was kept under 1.5 Torr. Following deposition, the furnace was allowed to naturally cool down to room temperature under a 35 sccm flow of H2. After removal of the chips from the furnace the Cu was etched using 10% ammonium persulfate, and then the chips were rinsed with DI water and isopropyl alcohol and dried under a gentle flow of dry N2. Raman spectroscopy was carried out using a Jobin Yvon LabRam HR800 spectrometer attached to an Olympus BH2 microscope. Graphene nanopores were fabricated and imaged at Northeastern University using a JEOL 2010FEG transmission electron microscope operating in bright-field mode at 200 kV.
A process of membrane fabrication is shown in
The ionic conductance of transfer-free freestanding graphene membranes were studied by mounting graphene nano-membrane devices into a custom-made CTFE holder that allows 1 M KCl electrolyte solution to be placed on either side of the membrane. Ag/AgCl electrodes immersed in each electrolyte bath were used to apply voltage in the range of 300 mV across the membrane, and ion currents were measured using an Axopatch 200B patch-clamp amplifier. A current-voltage curve for a typical graphene nano-membrane device without a nanopore (red curve), as well as for a 7.5 nm (black curve) and a 20 nm (blue curve) diameter nanopores are shown in
G=σd (1)
where σ=0.096 S/cm is the measured specific conductance of buffer at 25° C. The calculated G values for transfer-free graphene membranes from known a and d, indicated in
The noise characteristics of ion current signals through nanopores have been studied profusely. Uncoated graphene nanopores exhibit a high noise as compared with silicon nitride and other hydrophilic nanopores in inorganic dielectrics. The inset to
Scatter plots of ΔI vs. td for 225 mV and 300 mV are shown in
Analysis of the most probable dwell time populations (td) at four voltages in the range 225-300 mV is shown in
Finally, histograms of ΔI at each DNA translocation experimental voltage are shown in
Mean DNA dwell times, as shown in
This is a divisional application of U.S. application Ser. No. 15/037,863, filed 19 May 2016, which is the U.S. national phase of PCT Application No. PCT/US14/67280, filed 25 Nov. 2014. This application also claims the benefit of U.S. Provisional Application No. 61/908,695, filed Nov. 25, 2013. Each of the aforementioned priority applications is hereby incorporated by reference in its entirety.
This invention was made with government support under Grant No. HG006873 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61908695 | Nov 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15037863 | May 2016 | US |
| Child | 17344035 | US |
