Devices, systems, and methods for processing samples

Abstract

The present disclosure provides a nanopore device for processing a fluid sample, comprising a substrate, an upper cavity including a first dielectric, a lower cavity including a second dielectric, a first dielectric membrane supported by the substrate and separating the upper and lower cavities, and a first set of electrodes and a second set of electrodes positioned in close proximity to the first dielectric, wherein the first set of electrodes and the second set of electrodes are configured to apply a first electric field to generate a corona discharge, thereby forming one or more nanopores in the first dielectric membrane.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 419,559, filed October 26, 2022, and U.S. Provisional Application No. 63 / 419,551, filed October 26, 2022, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Existing biomolecular size separation instruments are generally based on gel electrophoresis, which can rely on the migration of biomolecules within a gel matrix under the influence of an electric field. Several technical issues can exist with gel electrophoresis. For example, gel electrophoresis may be unable to resolve biomolecules at very low or very high concentrations; observed bands of biomolecules of the same size may be blurred and broadened, leading to poor resolution and poor size distribution profiles; accurate quantification of concentrations may be difficult to achieve; the use of fluorophores or single-use gel cassettes may increase analysis costs; and analysis may be time-consuming. Furthermore, gel electrophoresis may raise environmental concerns due to the use of plastics and / or toxic chemicals. Summary of the Invention

[0003] In one aspect, the present disclosure provides a nanopore device for processing a fluid sample, the nanopore device comprising: a substrate; an upper cavity comprising a first dielectric; a lower cavity comprising a second dielectric; a first dielectric membrane supported by the substrate and separating the upper and lower cavities; and first and second sets of electrodes positioned proximate to the first dielectric membrane, the first and second sets of electrodes configured to apply a first electric field to generate a corona discharge, thereby forming one or more nanopores in the first dielectric membrane.

[0004] In some embodiments, the first and second sets of electrodes are configured to apply a first electric field before contacting the device with a fluid sample. In some embodiments, the first or second dielectric comprises a gas, water, an electrolyte solution, or a solid. In some embodiments, the first or second dielectric comprises a fluid sample. In some embodiments, the fluid sample comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, lipids, or carbohydrates suspended in a gas, water, an electrolyte solution, or a porous solid. In some embodiments, the first and second sets of electrodes are configured to electrically couple to the first or second dielectric. In some embodiments, the first and second sets of electrode pairs are configured to apply a first electric field that facilitates the formation of a corona discharge, thereby forming one or more nanopores between the first and second sets of electrode pairs. In some embodiments, the device further comprises a first electric field generator configured to apply a first electric field sufficient to generate a corona discharge by applying a pulsed or continuous wave AC or DC voltage signal between the first and second sets of electrode pairs. In some embodiments, the first electric field generator is configured to generate an electric field having a field strength of about 1 kilovolt per meter (kV / m), 10 kV / m, 100 kV / m, or more. In some embodiments, the first electric field is configured to generate a corona discharge near the ends of the first set of electrodes or near the ends of the second set of electrodes. In some embodiments, the first electric field is configured to generate a corona discharge near the ends of the first set of electrode pairs, near the ends of the second set of electrode pairs, or a combination of both. In some embodiments, the ends of the first and second sets of electrode pairs are configured or shaped as substantially sharp convex tips. In some embodiments, the corona discharge is configured to increase the temperature of the device by no more than about 10, 1, or 0.1° C. In some embodiments, the corona discharge is configured to increase the temperature near the first or second set of electrodes by no more than about 10, 1, or 0.1° C.In some embodiments, the corona discharge is configured to form one or more nanopores having a diameter ranging from about 0.1 nanometers (nm) to about 10 micrometers (μm). In some embodiments, the substrate comprises one or more combinations of silicon, glass, quartz, silicon-on-insulator (SOI), gallium arsenide, gallium nitride, silicon carbide, ceramic, aluminum nitride, aluminum oxide, silicon nitride, or silicon dioxide. In some embodiments, the first dielectric film comprises one or more combinations of silicon nitride, silicon dioxide, graphene, boron nitride, tungsten disulfide, MXene, or molybdenum disulfide. In some embodiments, the first dielectric film is configured to have a thickness ranging from about 0.1 nanometers (nm) to about 100 nm.

[0005] In some embodiments, the device further comprises a third set and a fourth set of electrodes configured to apply a second electric field, the third set being located proximate to the first dielectric and the fourth set being located proximate to the second dielectric. In some embodiments, the third and fourth sets of electrodes are configured to apply the second electric field to generate an electric arc discharge, thereby forming one or more nanopores in the first dielectric membrane. In some embodiments, a combination of the corona discharge and the electric arc discharge is configured to form one or more nanopores in the first dielectric membrane. In some embodiments, the third and fourth sets of electrodes are configured to generate an electric arc discharge through the first dielectric, the second dielectric, or the first dielectric membrane. In some embodiments, the third and fourth sets of electrodes are configured to apply the second electric field before contacting the device with a fluid sample or before generating a corona discharge. In some embodiments, the second electric field is configured to exceed a dielectric breakdown of the first dielectric, the second dielectric, or the first dielectric membrane, thereby generating an electric arc discharge. In some embodiments, the corona discharge is configured to concentrate a second electric field near the corona discharge to improve the positional accuracy of the one or more nanopores by at least about 1%, 5%, 10%, or more compared to not using the corona discharge. In some embodiments, the third set of electrodes is configured to be electrically coupled to the first dielectric, and the fourth set of electrodes is configured to be electrically coupled to the second dielectric.

[0006] In some embodiments, the third and fourth sets of electrode pairs are configured to apply a second electric field that facilitates the formation of an electrical arc discharge, thereby forming one or more nanopores in the first dielectric membrane. In some embodiments, the third and fourth sets of electrodes are configured to be integrated into a device, and the electrodes are made of a material including one or more combinations of gold, chromium, aluminum, platinum, iridium, or titanium. In some embodiments, the device further includes a second electric field generator configured to apply a pulsed or continuous wave AC or DC voltage signal between the third and fourth sets of electrode pairs to apply a second electric field sufficient to generate an electrical arc discharge. In some embodiments, the second electric field generator is configured to generate a second electric field having a strength of about 1 kV / m, 10 kV / m, 100 kV / m, or more. In some embodiments, the combination of the corona discharge and the electric arc discharge is configured to increase the temperature of the device by no more than about 1000, 100, 10, 1, or 0.1° C. In some embodiments, the combination of the corona discharge and the electric arc discharge is configured to increase the temperature near the first, second, third, or fourth set of electrodes by no more than about 1000, 100, 10, 1, or 0.1° C. In some embodiments, the combination of the corona discharge and the electric arc discharge is configured to form one or more nanopores having a diameter in a range from about 0.1 nm to about 10 μm.

[0007] In some embodiments, the device further comprises one or more sensors configured to measure a tunneling current between the first and second sets of electrode pairs or an ionic current between the third and fourth sets of electrode pairs. In some embodiments, the first and second sets of electrode pairs are configured to measure a tunneling current. In some embodiments, the third and fourth sets of electrode pairs are configured to measure an ionic current. In some embodiments, the one or more sensors are configured to be integrated into the device.

[0008] In some embodiments, the device further comprises a second dielectric film, the second dielectric film forming a layer between the first dielectric film and the substrate or the second dielectric, hi some embodiments, the second dielectric film is configured to support the first dielectric film during formation of the one or more nanopores and to be removed by a chemical etching process after formation of the one or more nanopores.

[0009] In some embodiments, the one or more nanopores comprise at least about 1, 10, 100, 1,000, 10,000, 100,000, 1,000,000, or more nanopores. In some embodiments, the device is configured to electrically couple the one or more nanopores, thereby forming one or more nanopore cells. In some embodiments, the device is configured to electrically couple the one or more nanopore cells, thereby forming one or more nanopore arrays. In some embodiments, the one or more nanopore arrays are configured for high-throughput processing of fluid samples. In some embodiments, the high-throughput processing comprises sequencing DNA at a rate of at least about 1, 10, 100, 1,000, 10,000 kilobases per second (kb / s) or more. In some embodiments, the device is configured to re-form one or more nanopores in the first membrane. In some embodiments, the device further comprises a top layer coupled to the first dielectric and a bottom layer coupled to the second dielectric, the top and bottom layers configured to contain or seal a fluid sample or direct the flow of the fluid sample through the device. In some embodiments, the device further comprises a fifth electrode and a sixth electrode configured to generate a dielectrophoretic (DEP) force in conjunction with the first and second sets of electrodes. In some embodiments, the DEP force is used to concentrate biomolecules contained in the fluid sample.

[0010] In another aspect, the present disclosure provides a method for manufacturing a nanopore device for processing a fluid sample, the method comprising: (a) providing a device comprising a substrate, an upper cavity, a lower cavity, a first dielectric membrane, a first set and a second set of electrodes, and a third set and a fourth set of electrodes; (b) applying a first electric field between the first and second sets of electrodes to generate a corona discharge near the ends of the first and second sets of electrodes; and (c) applying a second electric field between the third and fourth sets of electrodes to generate an electrical arc discharge due to the corona discharge, wherein the combination of the corona discharge and the electrical arc discharge forms one or more nanopores in the first dielectric membrane.

[0011] In some embodiments, (a) includes (i) depositing a first dielectric film on a substrate; (ii) forming a lower cavity in the substrate below the first dielectric film, the lower cavity including a dielectric; (iii) depositing a first set and a second set of electrodes above the first dielectric film; (iv) forming an upper cavity above the first dielectric film and above the first and second sets of electrodes, the upper cavity including an additional dielectric; (v) depositing a third set of electrodes above the upper cavity; and (vi) depositing a fourth set of electrodes below the lower cavity.

[0012] In some embodiments, the depositing of (iii) comprises depositing first and second sets of electrodes proximate the dielectric, further dielectric, or first dielectric film, hi some embodiments, the first and second sets of electrodes comprise a material selected from the group consisting of one or more of gold, chromium, aluminum, platinum, iridium, and titanium.

[0013] In some embodiments, the depositing of (vi) comprises depositing a third set of electrodes proximate the first dielectric and a fourth set of electrodes proximate the second dielectric, hi some embodiments, the third and fourth sets of electrodes comprise a material selected from the group consisting of one or more of gold, chromium, aluminum, platinum, iridium, or titanium.

[0014] In some embodiments, the method further includes generating a first electric field by applying a pulsed or continuous wave AC or DC voltage signal between the first and second sets of electrodes. In some embodiments, the first electric field comprises a level of at least about 1 kV / m, 10 kV / m, 100 kV / m, or more. In some embodiments, the method further includes generating a second electric field by applying a pulsed or continuous wave AC or DC voltage signal between the third and fourth sets of electrodes. In some embodiments, the second electric field comprises a level of at least about 1 kV / m, 10 kV / m, 100 kV / m, or more. In some embodiments, the device further comprises a second dielectric film. In some embodiments, (a) further comprises depositing a second dielectric film between the first dielectric film and the substrate or the second dielectric. In some embodiments, the device further comprises a top layer and a bottom layer. In some embodiments, (a) further comprises forming a top layer over the first dielectric and a bottom layer under the second dielectric, in some embodiments, the top and bottom layers are configured to contain or encapsulate a fluid sample or direct the flow of a fluid sample through the device.

[0015] In some embodiments, (a) further comprises: (1) fabricating a plurality of nanopore devices; (2) electrically connecting at least one nanopore device of the plurality of nanopore devices to at least one other nanopore device of the plurality of nanopore devices to form a plurality of nanopore cells; and (3) electrically connecting at least one nanopore cell of the plurality of nanopore cells to at least one other nanopore cell of the plurality of nanopore cells to form a plurality of nanopore arrays.

[0016] In some embodiments, the plurality of nanopore arrays are configured for high-throughput processing of fluid samples. In some embodiments, the device further comprises a fifth electrode and a sixth electrode configured to generate a dielectrophoretic (DEP) force in conjunction with the first and second sets of electrodes.

[0017] In another aspect, the present disclosure provides a method for processing a fluid sample using one or more nanopores, the method comprising: (a) providing a device comprising a substrate, an upper cavity, a lower cavity, a first dielectric film, first and second sets of electrodes configured to generate a corona discharge, and third and fourth sets of electrodes configured to generate an electrical arc discharge, wherein the one or more nanopores are formed in the first dielectric film using a combination of the corona discharge and the electrical arc discharge; (b) flowing a fluid sample through the one or more nanopores; and (c) processing the fluid sample as it flows through the one or more nanopores.

[0018] In some embodiments, the method further includes forming one or more nanopores using the first, second, third, or fourth set of electrodes. In some embodiments, the first and second sets of electrodes are further configured to process one or more events related to processing a fluid sample. In some embodiments, the fluid sample includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, lipids, or carbohydrates suspended in a gas, water, an electrolyte solution, or a porous solid. In some embodiments, the one or more events include events related to biomolecule analysis, biomolecule measurement, biomolecule purification, biomolecule enrichment, or biomolecule sequencing. In some embodiments, the first and second sets of electrodes are further configured to measure tunneling current. In some embodiments, the third and fourth sets of electrodes are further configured to measure ion current. In some embodiments, the device further includes fifth and sixth electrodes configured to generate a dielectrophoretic (DEP) force in conjunction with the first and second sets of electrodes. In some embodiments, the method further includes concentrating biomolecules contained in the fluid sample using the DEP force. In some embodiments, (b) comprises flowing the fluid sample through one or more nanopores via a capillary, pressure-driven, electroosmotic flow, or electrowetting mechanism.

[0019] Citation by reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the present specification is intended to supersede and / or supersede such conflicting material.

[0020] Where reference is made to a URL or other such identifier or address, such identifiers may change and specific information on the Internet may shift, but equivalent information may be found by searching the Internet, and reference thereto is evidence of the availability and public dissemination of such information.

[0021] As used herein, abbreviations for any protecting groups, amino acids, and other compounds shall follow their common usage or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9):1726-1732), unless otherwise indicated. [Brief explanation of the drawings]

[0022] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0023] [Figure 1A] FIG. 1 is a diagram of an exemplary nanopore unit cell, according to some embodiments. [Figure 1B] FIG. 1 is a diagram of another exemplary nanopore unit cell, according to some embodiments. [Figure 2] 1 is a top view schematic of an exemplary nanopore chip comprising multiple nanopore unit cells (e.g., nanopore unit cell blocks), according to some embodiments. [Figure 3] FIG. 1 is a diagram of an exemplary nanopore array, according to some embodiments. [Figure 4] FIG. 1 is a diagram of an exemplary system for sample processing or biomolecular analysis, according to some embodiments. [Figure 5A] FIG. 1 illustrates an exemplary configuration for generating a corona discharge, according to some embodiments. [Figure 5B] FIG. 1 illustrates an exemplary configuration for generating a corona discharge, according to some embodiments. [Figure 6] 1A-1C are diagrams of exemplary configurations for forming nanopores, according to some embodiments. [Figure 7A] 1A-1C are diagrams of exemplary configurations for forming nanopores, according to some embodiments. [Figure 7B] 1A-1C are diagrams of exemplary configurations for forming nanopores, according to some embodiments. [Figure 7C] 1A-1C are diagrams of exemplary configurations for forming nanopores, according to some embodiments. [Figure 7D] 1A-1C are diagrams of exemplary configurations for forming nanopores, according to some embodiments. [Figure 8] FIG. 1 is a diagram of an exemplary configuration for corona discharge assisted controlled breakdown nanopore fabrication, according to some embodiments. [Figure 9] FIG. 10 is a diagram of another exemplary configuration for corona discharge assisted controlled breakdown nanopore creation, according to some embodiments. [Figure 10] FIG. 1 is a diagram of an exemplary configuration for arc discharge nanopore creation, according to some embodiments. [Figure 11] FIG. 1 is a diagram of another exemplary configuration for creating an arc discharge nanopore, according to some embodiments. [Figure 12] FIG. 1 is an exemplary size distribution diagram, according to some embodiments. [Figure 13] FIG. 1 is a diagram of a biomolecule translocating from the upper chamber to the lower chamber of a nanopore unit cell, according to some embodiments. [Figure 14] 1 shows an exemplary measurement of current versus time in a nanopore, according to some embodiments. [Figure 15] FIG. 1 is a diagram of a computer system according to some embodiments. [Figure 16] FIG. 1 is a diagram of an exemplary nanopore device for sample processing or biomolecular analysis. DETAILED DESCRIPTION OF THE INVENTION

[0024] While various embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may be made without departing from the present disclosure. It will be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0025] Whenever the terms "at least," "greater than," or "greater than or equal to" appear before the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0026] Whenever the terms "no more than," "less than," or "less than or equal to" appear before the first number in a series of two or more numbers, the terms "no more than," "less than," or "less than or equal to" apply to each number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0027] As used in this specification and claims, the singular forms "a," "an," and "the" can include plural references unless the context clearly dictates otherwise. For example, the term "a biomolecule" can include a plurality of biomolecules.

[0028] The terms "about" and "approximately," when used interchangeably herein, generally refer to being within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within one or more standard deviations, in accordance with practice in the art. Alternatively, "about" may mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of a value, such as within 5-fold or within 2-fold. When a particular value is described, unless otherwise specified, the term "about" may mean being within an acceptable range of error for the particular value.

[0029] Unless otherwise indicated, all numerical values ​​expressing properties such as quantities of ingredients, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the properties sought to be obtained by the present disclosure.

[0030] As used herein, "comprising" is generally open-ended and means the recited components or their structural or functional equivalents, plus any other component(s) not recited. The terms "having" and "including" should also be construed as open-ended unless the context suggests otherwise.

[0031] As used herein, "optional" or "optionally" generally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur. For example, an optional variant moiety means that the moiety is variant or non-variant.

[0032] The terms "polynucleotide" and "nucleic acid," when used interchangeably herein, generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be deoxyribonucleic acid (DNA). A polynucleotide can be ribonucleic acid (RNA). A polynucleotide can assume any three-dimensional structure and can perform any function, known or unknown. A polynucleotide can contain one or more analogs (e.g., backbone, sugar, or nucleobase analogs). Non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, complementary DNA (cDNA, e.g., double-stranded cDNA (dd-cDNA) or single-stranded cDNA (ss-cDNA)), circulating tumor DNA (ctDNA), damaged DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes (e.g., fluorescent in situ hybridization (FISH) probes), and primers.

[0033] The term "sequencing" as used herein generally refers to the procedure of determining the order in which nucleotides occur in a target nucleotide sequence. Sequencing methods can include, for example, high-throughput sequencing, such as next-generation sequencing (NGS). Sequencing can be whole genome sequencing or targeted sequencing. Sequencing can be single molecule sequencing or massively parallel sequencing. Next-generation sequencing methods can be useful for obtaining millions of sequences in a single run. In some cases, sequencing can be performed using one or more nanopore sequencing methods, such as sequencing by synthesis, sequencing by ligation, or sequencing by cleavage.

[0034] The term "nanopore," as used herein, generally refers to a pore, channel, or flow path formed or otherwise provided in a membrane. The nanopore may be a biological nanopore, a solid-state nanopore, a hybrid biological-solid nanopore, variations thereof, or combinations thereof. The membrane may be an organic membrane, e.g., a lipid bilayer, or a synthetic membrane, e.g., a membrane formed from a polymeric material, such as a protein nanopore. The membrane may be a solid membrane (e.g., a silicon substrate). The nanopore may be positioned adjacent to or in close proximity to a detection circuit, such as, for example, a complementary metal-oxide semiconductor (CMOS) circuit or a field-effect transistor (FET) circuit, or an electrode coupled to the detection circuit. The nanopore may be part of the detection circuit.

[0035] As used herein, the term "nanopore site" generally refers to a location where a nanopore can be created, where a nanopore is located, or where a nanopore was previously located.

[0036] Solid-state nanopores may be fabricated by at least one of the following methods: 1) transmission electron microscopy (TEM), 2) controlled dielectric breakdown (CDB), 3) laser etching (LE), 4) laser-assisted controlled dielectric breakdown (LCDB), and 5) electron beam lithography (EBL). Technical challenges may be associated with these methods. For example, these methods may require expensive equipment and complex alignment. In addition, these methods may only produce a single nanopore. Therefore, there remains an unmet need for a scalable fabrication process for building devices with nanopore arrays (e.g., nanopore devices) capable of high-throughput biomolecular analysis.

[0037] The present disclosure provides devices, systems, and methods for processing fluid samples. The present disclosure further provides methods for manufacturing or fabricating nanopore devices for processing fluid samples. Scalable manufacturing or fabrication processes provided herein, such as massively parallel nanopore fabrication processes using corona discharge and / or arc discharge, can create nanopores on a large scale (e.g., millions or more) to be useful for high-throughput biomolecular analysis.

[0038] In some embodiments, the devices, systems, and methods provided herein can be used to analyze biomolecules, such as nucleic acids, polynucleotides, and polypeptides. In some embodiments, the devices, systems, and methods disclosed herein can be used for characterizing, analyzing, and / or sequencing biomolecules. In some embodiments, the devices, systems, and methods described herein can be used to determine or measure multiple characteristics of biomolecules. In some embodiments, the biomolecules can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, lipids, or carbohydrates. In some embodiments, the characteristics can include sizing and / or enrichment of the biomolecule. In some embodiments, the characteristics can include sequencing of the biomolecule (e.g., DNA, RNA, or protein).

[0039] Device for processing samples In some embodiments, the present disclosure provides a device for processing a fluid sample. In some embodiments, the device may include a nanopore device. In some embodiments, the nanopore device may include a plurality of nanopore unit cells, a nanopore chip, and / or a nanopore chip array.

[0040] In some embodiments, a nanopore device may include a substrate, an upper cavity comprising a first dielectric, a lower cavity comprising a second dielectric, a first dielectric membrane supported by the substrate and configured to separate the upper and lower cavities, and first and second sets of electrodes positioned proximate to the first dielectric membrane, the first and second sets of electrodes configured to apply a first electric field to generate a corona discharge, thereby forming one or more nanopores in the first dielectric membrane.

[0041] In some embodiments, the first and second sets of electrodes may be configured to apply a first electric field prior to contacting the nanopore device with a fluid sample.

[0042] In some embodiments, the nanopore device may include a second dielectric film between the first dielectric film and the substrate. In some embodiments, the second dielectric film may have the same composition as the first dielectric film. In some embodiments, the second dielectric film may have a different composition than the first dielectric film. In some embodiments, the second dielectric film may comprise silicon dioxide. In some embodiments, the second dielectric film may be configured to support the first dielectric film during the formation of one or more nanopores. In some embodiments, the second dielectric film may have a thickness of about 20 nm to about 10 μm. In some embodiments, the second dielectric film may remain within the nanopore device. In some embodiments, at least a portion of the second dielectric film may be removed, for example, by a chemical etching process, after the formation of one or more nanopores. In some embodiments, a portion of the second dielectric film underlying the nanopore site may be removed. In some embodiments, after the formation of the nanopores, the second dielectric film may only contact the substrate and not the second dielectric. In some embodiments, after formation of the nanopore, the portion of the second dielectric film that is not in contact with the substrate may be removed.

[0043] In some embodiments, the nanopore device may include additional external or embedded electrodes.

[0044] In some embodiments, the substrate may comprise silicon, glass, quartz, silicon-on-insulator (SOi), gallium arsenide, gallium nitride, silicon carbide, ceramic, aluminum nitride, aluminum oxide, silicon nitride, silicon dioxide, or a combination thereof. In some embodiments, the substrate may comprise a silicon wafer. In some embodiments, the substrate may comprise a crystalline silicon wafer. In some embodiments, the silicon wafer may comprise a dopant, such as phosphorus, arsenic, antimony, or boron. In some embodiments, the doped silicon wafer may have a resistivity of about 0.001 ohm-cm to about 100 ohm-cm. In some embodiments, the silicon wafer may be undoped. In some embodiments, the undoped silicon wafer may have a resistivity greater than 20,000 ohm-cm. In some embodiments, the substrate may comprise glass. In some embodiments, the substrate may comprise doped glass. In some embodiments, the substrate may comprise undoped glass. In some embodiments, undoped silicon wafers or undoped glass wafers can reduce the noise level when measuring signals (e.g., tunneling signals), and in some embodiments, undoped silicon wafers or undoped glass wafers can increase the signal-to-noise ratio in such measurements.

[0045] In some embodiments, the first dielectric may comprise a fluid, such as a gas or a liquid (e.g., water), or a combination thereof, or a solid (porous or non-porous). In some embodiments, the first dielectric may comprise air, nitrogen, oxygen, argon, tetrafluoromethane (CF), nitrogen trifluoride (NF), trifluoromethane (CHF), or sulfur hexafluoride (SF). In some embodiments, the first dielectric may comprise porcelain, glass, agarose gel, hydrogel, or plastic. In some embodiments, the first dielectric may comprise an electrolyte liquid. In some embodiments, the first dielectric may comprise a solution, such as a KCl solution or a LiCl solution. In some embodiments, the first dielectric may comprise an electrolyte solution (buffer), such as phosphate buffered saline (PBS), (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (HEPES), low-conductivity Tris-EDTA, or water of a specific conductivity or resistivity. In some embodiments, the water may have a resistivity of about 10 kiloohm-meters (kΩ·m) to about 200 kΩ·m, hi some embodiments, the water may have a resistivity of about 180 kΩ·m.

[0046] In some embodiments, the second dielectric may comprise a fluid, such as a gas or a liquid (e.g., water), or a combination thereof, or a solid (porous or non-porous). In some embodiments, the second dielectric may comprise air, nitrogen, oxygen, argon, tetrafluoromethane (CF), nitrogen trifluoride (NF), trifluoromethane (CHF), or sulfur hexafluoride (SF). In some embodiments, the second dielectric may comprise porcelain, glass, agarose gel, hydrogel, or plastic. In some embodiments, the second dielectric may comprise an electrolyte liquid. In some embodiments, the second dielectric may comprise a solution, such as a KCl solution or a LiCl solution. In some embodiments, the second dielectric may comprise an electrolyte solution (buffer), such as phosphate buffered saline (PBS), (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (HEPES), low-conductivity Tris-EDTA, or water of a specific conductivity or resistivity. In some embodiments, the water may have a resistivity of about 10 kiloohm-meters (kΩ·m) to about 200 kΩ·m, hi some embodiments, the water may have a resistivity of about 180 kΩ·m.

[0047] In some embodiments, the first dielectric and / or the second dielectric may comprise a fluid sample. In some embodiments, the fluid sample may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, lipids, or carbohydrates suspended in a gas, water, an electrolyte solution, or a porous solid (e.g., a gel). In some embodiments, the porous solid may slow down the movement of biomolecules, such as DNA, through the nanopore.

[0048] In some embodiments, the first dielectric film may comprise any suitable dielectric material. In some embodiments, the first dielectric film may comprise a two-dimensional material. In some embodiments, the first dielectric film may comprise silicon nitride (Si3N4), silicon dioxide (SiO2), graphene, boron nitride (e.g., hexagonal boron nitride), tungsten disulfide, MXene, molybdenum disulfide (MoS2), silicon carbide (SiC), titanium dioxide (TiO2), zirconium dioxide (ZrO2), aluminum oxide (Al2O3), polytetrafluoroethylene (PTFE), polyacrylonitrile (PAN), high-density polyethylene (HDPE), or a combination thereof. In some embodiments, the dielectric film may have a thickness of about 1 angstrom (Å) to about 5 Å, about 1 Å to about 1 nanometer (nm), about 1 Å to about 5 nm, about 1 Å to about 10 nm, about 1 Å to about 50 nm, about 1 Å to about 100 nm, about 5 Å to about 1 nm, about 5 Å to about 5 nm, about 5 Å to about 10 nm, about 5 Å to about 50 nm, about 5 Å to about 100 nm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, or about 50 nm to about 100 nm.

[0049] In some embodiments, the first and second sets of electrodes may be configured to be electrically coupled to the first dielectric or the second dielectric. In some embodiments, the first and second sets of electrode pairs may be configured to apply a first electric field that facilitates the formation of a corona discharge, thereby forming one or more nanopores between the first and second sets of electrode pairs. In some embodiments, the electrodes may comprise platinum, gold, chromium, aluminum, titanium, tungsten, molybdenum, niobium, hafnium, iridium, osmium, zirconium, palladium, iron, or a combination thereof. In some embodiments, the electrodes may comprise platinum. In some embodiments, at least one electrode of the first and second sets of electrode pairs may have a pointed end or may be configured or shaped as a substantially sharp convex tip.

[0050] In some embodiments, the nanopore device may include a first electric field generator configured to apply a first electric field sufficient to generate a corona discharge by applying a pulsed or continuous wave alternating current (AC) or direct current (DC) voltage signal between the first and second sets of electrode pairs. In some embodiments, the first electric field generator may be configured to generate an electric field having a field strength of at least about 1 kilovolt / meter (kV / m), at least about 5 kV / m, at least about 10 kV / m, at least about 20 kV / m, at least about 30 kV / m, at least about 40 kV / m, at least about 50 kV / m, at least about 60 kV / m, at least about 70 kV / m, at least about 80 kV / m, at least about 90 kV / m, at least about 100 kV / m, at least about 1 megavolt / meter (MV / m), at least about 10 MV / m, at least about 100 MV / m, or more.

[0051] In some embodiments, the first electric field may be configured to generate a corona discharge near the ends of the first set of electrodes or near the ends of the second set of electrodes. In some embodiments, the first electric field may be configured to generate a corona discharge near the ends of the first set of electrode pairs, near the ends of the second set of electrode pairs, or a combination of both. In some embodiments, the ends of the first and second set of electrode pairs may be configured or shaped as substantially sharp convex tips.

[0052] In some embodiments, the corona discharge may be a positive corona discharge (e.g., near the end of the positive electrode). In some embodiments, the corona discharge may be a negative corona discharge (e.g., near the end of the negative electrode).

[0053] In some embodiments, the corona discharge may increase the temperature of the nanopore device by at least about 0.1° C., at least about 0.5° C., at least about 1° C., at least about 5° C., at least about 10° C., or more. In some embodiments, the corona discharge may increase the temperature of the nanopore device by up to about 10° C., up to about 5° C., up to about 1° C., up to about 0.5° C., up to about 0.1° C., or less.

[0054] In some embodiments, the corona discharge may increase the temperature near the first or second set of electrodes by at least about 0.1° C., at least about 0.5° C., at least about 1° C., at least about 5° C., at least about 10° C., or more. In some embodiments, the corona discharge may increase the temperature near the first or second set of electrodes by up to about 10° C., up to about 5° C., up to about 1° C., up to about 0.5° C., up to about 0.1° C., or less.

[0055] In some embodiments, the corona discharge has a diameter of about 0.1 nanometers (nm) to about 1 nm, about 0.1 nm to about 5 nm, about 0.1 nm to about 10 nm, about 0.1 nm to about 50 nm, about 0.1 nm to about 100 nm, about 0.1 nm to about 500 nm, about 0.1 nm to about 1 micrometer (μm), about 0.1 nm to about 10 μm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm to about 500 nm, about 1 nm to about 1 μm, about 1 nm to about 10 μm, about 5 nm to about 10 nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 The nanopore may be configured to form one or more nanopores having a diameter of about 500 nm, about 5 nm to about 1 μm, about 5 nm to about 10 μm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 500 nm, about 10 nm to about 1 μm, about 10 nm to about 10 μm, about 50 nm to about 100 nm, about 50 nm to about 500 nm, about 50 nm to about 1 μm, about 50 nm to about 10 μm, about 100 nm to about 500 nm, about 100 nm to about 1 μm, about 100 nm to about 10 μm, about 500 nm to about 1 μm, about 500 nm to about 10 μm, or about 1 μm to about 10 μm.

[0056] In some embodiments, the nanopore device may include a third set and a fourth set of electrodes configured to apply a second electric field. In some embodiments, the third set of electrodes may be located proximate to a first dielectric (e.g., in the upper cavity), and the fourth set of electrodes may be located proximate to a second dielectric (e.g., in the lower cavity). In some embodiments, the third set of electrodes may be configured to be electrically coupled to the first dielectric. In some embodiments, the fourth set of electrodes may be configured to be electrically coupled to the second dielectric. In some embodiments, the third and fourth sets of electrodes may be configured to apply a second electric field to generate an electrical arc discharge (or arc discharge, as used interchangeably herein), thereby forming one or more nanopores in the first dielectric membrane.

[0057] In some embodiments, the third and fourth sets of electrodes may provide additional electrical intensity to form one or more nanopores in the first dielectric film. In some embodiments, the combination of the corona discharge and the electric arc discharge may be configured to form one or more nanopores in the first dielectric film.

[0058] In some embodiments, the third and fourth sets of electrodes may be configured to generate an electrical arc discharge through the first dielectric, the second dielectric, and / or the first dielectric film. In some embodiments, the third and fourth sets of electrodes may be configured to generate an electrical arc discharge through the first dielectric, the second dielectric, and the first dielectric film.

[0059] In some embodiments, the combination of corona discharge and electric arc discharge may increase the temperature of the device by at least about 0.1° C., at least about 0.5° C., at least about 1° C., at least about 5° C., at least about 10° C., at least about 100° C., at least about 500° C., at least about 1000° C., at least about 10,000° C., at least about 50,000° C., or more. In some embodiments, the combination of corona discharge and electric arc discharge may increase the temperature of the nanopore device by up to about 50,000° C., up to about 10,000° C., up to about 1000° C., up to about 100° C., up to about 10° C., up to about 5° C., up to about 1° C., up to about 0.5° C., up to about 0.1° C., or less.

[0060] In some embodiments, the combination of corona discharge and electric arc discharge may increase the temperature near the first, second, third, or fourth set of electrodes by at least about 0.1° C., at least about 0.5° C., at least about 1° C., at least about 5° C., at least about 10° C., at least about 100° C., at least about 500° C., at least about 1000° C., at least about 10,000° C., at least about 50,000° C., or more. In some embodiments, the combination of corona discharge and electric arc discharge may increase the temperature near the first, second, third, or fourth set of electrodes by up to about 50,000° C., up to about 10,000° C., up to about 1000° C., up to about 100° C., up to about 10° C., up to about 5° C., up to about 1° C., up to about 0.5° C., up to about 0.1° C., or less.

[0061] In some embodiments, the combination of corona discharge and electric arc discharge may result in a particle size of about 0.1 nanometers (nm) to about 1 nm, about 0.1 nm to about 5 nm, about 0.1 nm to about 10 nm, about 0.1 nm to about 50 nm, about 0.1 nm to about 100 nm, about 0.1 nm to about 500 nm, about 0.1 nm to about 1 micrometer (μm), about 0.1 nm to about 10 μm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm to about 500 nm, about 1 nm to about 1 μm, about 1 nm to about 10 μm, about 5 nm to about 10 nm, about 5 nm to about 50 nm, about 5 nm to about The nanopores may be configured to form one or more nanopores having a diameter of 100 nm, about 5 nm to about 500 nm, about 5 nm to about 1 μm, about 5 nm to about 10 μm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 500 nm, about 10 nm to about 1 μm, about 10 nm to about 10 μm, about 50 nm to about 100 nm, about 50 nm to about 500 nm, about 50 nm to about 1 μm, about 50 nm to about 10 μm, about 100 nm to about 500 nm, about 100 nm to about 1 μm, about 100 nm to about 10 μm, about 500 nm to about 1 μm, about 500 nm to about 10 μm, or about 1 μm to about 10 μm.

[0062] In some embodiments, the third and fourth sets of electrodes may be configured to apply the second electric field before contacting the nanopore device with a fluid sample or before generating a corona discharge. In some embodiments, the third and fourth sets of electrodes may be configured to apply the second electric field after contacting the nanopore device with a fluid sample or after generating a corona discharge. In some embodiments, the third and fourth sets of electrodes may be configured to apply the second electric field simultaneously with generating a corona discharge.

[0063] In some embodiments, the second electric field may be configured to overcome a breakdown of the first dielectric, the second dielectric, or the first dielectric film, thereby generating an electrical arc. In some embodiments, the corona discharge may be configured to concentrate the second electric field near the corona discharge to improve the positional accuracy of the one or more nanopores by at least about 1%, at least about 5%, at least about 10%, or more, compared to not using the corona discharge.

[0064] In some embodiments, the third and fourth sets of electrodes may include a pair of electrodes configured to apply a second electric field that facilitates the formation of an electrical arc discharge, thereby forming one or more nanopores in the first dielectric film.

[0065] In some embodiments, the third and fourth sets of electrodes may be external to the nanopore device. In some embodiments, the third and fourth sets of electrodes may be integrated into the device (e.g., embedded in the device). In some embodiments, the third and fourth sets of electrodes may be made from a material including one or more combinations of gold, chromium, aluminum, platinum, iridium, or titanium.

[0066] In some embodiments, the nanopore device may include a second electric field generator configured to apply a second electric field sufficient to generate an electrical arc discharge by applying a pulsed or continuous wave AC or DC voltage signal between the third and fourth sets of electrode pairs.

[0067] In some embodiments, the second electric field generator may generate a second electric field having a strength of at least about 1 kV / m, at least about 5 kV / m, at least about 10 kV / m, at least about 50 kV / m, at least about 100 kV / m, at least about 1 MV / m, at least about 10 MV / m, at least about 100 MV / m, at least about 1000 MV / m, at least about 5000 MV / m, or more.

[0068] In some embodiments, the nanopore device may further comprise a fifth electrode and a sixth electrode. In some embodiments, the nanopore device may be configured to generate a dielectrophoretic (DEP) force, for example, by applying an AC electric field (or a third electric field). In some embodiments, the first set of electrodes, the second set of electrodes, the fifth electrode, and the sixth electrode may be configured to generate a DEP force, for example, by applying an AC electric field. In some embodiments, the first and second sets of electrodes, the fifth electrode, and the sixth electrode may be configured to generate a DEP force, for example, by applying an AC electric field. In some embodiments, the DEP force may be used to concentrate biomolecules contained in a fluid sample after the fluid sample containing the biomolecules has been delivered to the nanopore device.

[0069] In some embodiments, the nanopore device may include a third electric field generator configured to apply a third electric field by applying a pulsed or continuous wave alternating current (AC) or direct current (DC) voltage signal between the fifth and sixth electrode pairs. In some embodiments, the third electric field generator may be configured to generate an electric field having a field strength of at least about 1 kV / m, at least about 5 kV / m, at least about 10 kV / m, at least about 20 kV / m, at least about 30 kV / m, at least about 40 kV / m, at least about 50 kV / m, at least about 60 kV / m, at least about 70 kV / m, at least about 80 kV / m, at least about 90 kV / m, at least about 100 kV / m, at least about 1 MV / m, at least about 10 MV / m, at least about 100 MV / m, or more.

[0070] FIG. 16 is a diagram of an exemplary nanopore device for sample processing or biomolecular analysis. The nanopore device may include a pair of electrodes 1601 and 1602 positioned proximate to nanopore site 1600 and configured to generate a corona discharge during nanopore formation. For sample processing or biomolecular analysis (in fluid 1613), the pair of electrodes 1601 and 1602 may be configured to detect tunneling current (or tunneling current, as used interchangeably herein). An electric field generator 1611 (e.g., an AC power source) can generate an electric field between electrodes 1601 and 1602. The nanopore device may include another pair of electrodes 1603 and 1604 positioned proximate to nanopore site 1600. Electrodes 1601, 1602, 1603, and 1604 may be configured to generate a DEP force. An additional electric field generator 1612 (eg, an AC power supply) can generate an additional electric field between electrodes 1603 and 1604 .

[0071] In some embodiments, the nanopore device may further comprise one or more sensors configured to measure a tunneling current between the first and second sets of electrode pairs or an ionic current between the third and fourth sets of electrode pairs. In some embodiments, the one or more sensors may be integrated into the nanopore device. In some embodiments, the one or more sensors may be operably coupled to any one of the electrodes.

[0072] In some embodiments, the first and second sets of electrode pairs may be configured to measure tunneling current, and in some embodiments, the third and fourth sets of electrode pairs may be configured to measure ionic current.

[0073] In some embodiments, a nanopore device may comprise at least about 1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000, at least about 1,000,000, or more nanopores.

[0074] In some embodiments, a nanopore device may be configured to electrically couple one or more nanopores, thereby forming one or more nanopore cells or nanopore unit cells.

[0075] In some embodiments, the nanopore device may be configured to electrically couple one or more nanopore unit cells, thereby forming one or more nanopore arrays or nanopore blocks.

[0076] In some embodiments, one or more nanopore arrays may be configured for high-throughput processing of fluid samples or analysis of biomolecules. In some embodiments, high-throughput processing or analysis may include sequencing DNA at rates of at least about 1, at least about 10, at least about 100, at least about 1000, at least about 10,000 kilobases per second (kb / s), or more. In some embodiments, high-throughput processing or analysis may include processing or analyzing at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000, or more samples simultaneously.

[0077] In some embodiments, the nanopore may become damaged or clogged after use. In some embodiments, the nanopore devices provided herein may be configured to reform or regenerate one or more nanopores in the first dielectric membrane. In some embodiments, reformation or regeneration of one or more nanopores in the first dielectric membrane may be achieved by any of the manufacturing / fabrication / formation methods provided herein.

[0078] In some embodiments, the nanopore device may include a top layer bonded to a first dielectric and a bottom layer bonded to a second dielectric, and the top and bottom layers may contain or seal a fluid sample or direct the flow of the fluid sample through the nanopore device.

[0079] FIG. 1A illustrates an exemplary nanopore unit cell as disclosed herein. The nanopore unit cell 100 may comprise an upper cavity 110 and a lower cavity 120. The upper cavity 110 may comprise an upper chamber 113 containing a first dielectric, e.g., fluid 1. The lower cavity may comprise a substrate 122 and a lower chamber 123 containing a second dielectric, e.g., fluid 2. The nanopore unit cell 100 may comprise a dielectric membrane 101 supported by the substrate 122 and configured to separate the upper cavity 110 and the lower cavity 120. The dielectric membrane 101 may include a nanopore region 102. The nanopore unit cell may further comprise at least two electrodes, e.g., a positive electrode 112 and a negative electrode 111, adjacent to and associated with the nanopore region 102. In some cases, the nanopore unit cell may further comprise at least two, at least three, at least four, or more additional electrodes. The two electrodes 111 and 112 may have sharp points (or share edges or sharp convex tips, which are used interchangeably herein). In some cases, the nanopore unit cell may comprise an upper electrode 116 and a lower electrode 121, and a dielectric separator 114 separating the electrodes 111 and 112 from the electrode 116. In some cases, the nanopore unit cell 100 may further comprise an upper lid 115 and a lower lid 125. The upper and lower chambers may be enclosed by the upper and lower lids to create a seal that allows fluid to flow into and out of the upper and lower chambers.

[0080] FIG. 1B illustrates another exemplary nanopore unit cell as disclosed herein. The nanopore unit cell 140 may comprise an upper cavity 150 and a lower cavity 160. The upper cavity 150 may comprise an upper chamber 153 encapsulated with a first dielectric. The lower cavity may comprise a substrate 162 and a lower chamber 163 encapsulated with a second dielectric. The nanopore unit cell 140 may comprise a dielectric membrane 141 supported by the substrate 162 and configured to separate the upper cavity 150 and the lower cavity 160. The dielectric membrane 141 may include a nanopore region 142. The nanopore unit cell may further comprise a second dielectric membrane 143. The nanopore unit cell may further comprise at least two electrodes, e.g., a positive electrode 152 and a negative electrode 151, associated adjacent to the nanopore region 142. In some cases, the nanopore unit cell may further comprise at least two, at least three, at least four, or more additional electrodes. Two electrodes 151 and 152 may have sharp tips. In some cases, the nanopore unit cell may comprise an upper electrode 156 and a lower electrode 161, and a dielectric separator 154 separating electrodes 151 and 152 from electrode 156. In some cases, the nanopore unit cell 140 may further comprise an upper lid 155 and a lower lid 165. The upper and lower chambers may be enclosed by the upper and lower lids to create a seal that allows fluid to flow into and out of the upper and lower chambers.

[0081] In some embodiments, each nanopore unit cell may be used to process a fluid sample or to independently analyze a biomolecule.

[0082] In some embodiments, the electrodes associated with the nanopore site may be coated with a thin layer of dielectric material. For example, if the electrodes associated with the nanopore site are constructed from aluminum, a thin layer of Al2O3 passivation layer may be coated on the surface of the electrodes attached to the nanopore site. In some embodiments, the electrodes associated with the nanopore site may be used as tunneling current sensors to detect biomolecular translocation. The thin layer of dielectric material on the electrodes associated with the nanopore site may help measure tunneling current.

[0083] In some embodiments, a nanopore device may comprise a nanopore chip comprising a plurality of nanopore unit cells as disclosed herein, which nanopore unit cells arranged and fabricated in an array to form a nanopore chip can increase the throughput of sample processing or biomolecular analysis.

[0084] The nanopore chip may comprise a dielectric membrane including a plurality of nanopore sites, each nanopore site being associated with at least two electrodes, including a positive electrode and a negative electrode, disposed between an upper cavity including a first dielectric and a lower cavity including a second dielectric, and associated adjacent to and associated with the nanopore site.

[0085] The nanopore chip may be fabricated on a substrate, such as a silicon wafer, using any suitable micro / nano fabrication technique. In some embodiments, the nanopore chip may be used to analyze a single sample. In some embodiments, the nanopore chip may be used to analyze multiple samples. FIG. 2 shows a schematic top view of an exemplary nanopore chip with multiple nanopore unit cells (e.g., nanopore unit cell blocks). The multiple nanopore unit cells (e.g., 201) may be arranged in an array (e.g., a regular array) and connected to an inlet reservoir 202 and an outlet reservoir 203 through microfluidic channels (e.g., 204). In some embodiments, the microfluidic channel is between about 1 micrometer (μm) and about 10 μm, between about 1 μm and about 20 μm, between about 1 μm and about 30 μm, between about 1 μm and about 40 μm, between about 1 μm and about 50 μm, between about 1 μm and about 100 μm, between about 10 μm and about 20 μm, between about 10 μm and about 30 μm, between about 10 μm and about 40 μm, between about 10 μm and about 50 μm, The nanopore unit may have a width of about 10 μm to about 100 μm, about 20 μm to about 30 μm, about 20 μm to about 40 μm, about 20 μm to about 50 μm, about 20 μm to about 100 μm, about 30 μm to about 40 μm, about 30 μm to about 50 μm, about 30 μm to about 100 μm, about 40 μm to about 50 μm, about 40 μm to about 100 μm, or about 50 μm to about 100 μm. In some embodiments, each nanopore unit of the plurality of unit cells may be connected to an inlet-side reservoir to receive a portion of the sample from the inlet-side reservoir. In some embodiments, each nanopore unit of the plurality of unit cells may be connected to an outlet-side reservoir to guide a portion of the sample from the nanopore unit cell to the outlet-side reservoir. Alternatively, a first nanopore unit cell of the plurality of unit cells may be connected to the inlet reservoir for receiving a portion of the sample from the inlet reservoir, and a second nanopore unit cell of the plurality of unit cells may be connected to the first nanopore unit cell instead of the inlet reservoir for receiving a portion of the sample from the first nanopore unit cell.In some embodiments, a fluid sample to be analyzed (e.g., a sample containing biomolecules) may be introduced into the inlet reservoir 202 and transported to multiple nanopore unit cells via pressure-driven, capillary, electrowetting, or electroosmotic flow. After the sample is analyzed in the nanopore unit cells, it may be transported to the outlet reservoir 203. Electrodes within each nanopore unit cell may be wired to allow independent access, similar to a DRAM chip.

[0086] In some embodiments, the nanopore chip may comprise 10 to 100, 10 to 500, 10 to 1,000, 10 to 10,000, 10 to 100,000, 10 to a million, 10 to 500, 10 to 1,000, 10 to 10,000, 10 to 100,000, 10 to a million, 100 to 500, 100 to 1,000, 100 to 10,000, 100 to 100,000, 100 to a million, 500 to 1,000, 500 to 10,000, 500 to 100,000, 500 to a million, 1,000 to 10,000, 1,000 to 100,000, 1,000 to a million, 10,000 to 100,000, 10,000 to a million, or 100,000 to a million nanopore unit cells.

[0087] In some embodiments, nanopore unit cells may be arranged in an array, e.g., a nanopore array. In some embodiments, the nanopore array may be used to analyze multiple samples in a single analytical run. FIG. 3 shows an exemplary nanopore array. The nanopore array may include multiple nanopore unit cell blocks (e.g., nanopore unit cell block 300), each of which may include multiple nanopore unit cells, an inlet reservoir 301, and an outlet reservoir 302. The nanopore array may be fabricated by a massively parallel micro / nano fabrication process. Samples may be introduced into the inlet reservoirs of different nanopore unit cells by a microfluidic network or by robotic automation.

[0088] System for processing samples or analyzing biomolecules In some embodiments, the present disclosure provides a system for sample processing, biomolecular characterization, analysis, or sequencing that includes a nanopore device as disclosed herein.

[0089] In some embodiments, the systems provided herein may be utilized in biomolecule screening (e.g., pathogen detection), diagnostics, DNA / RNA / protein fingerprinting and purification, drug discovery and development, NGS sample preparation, nanoparticle synthesis (e.g., fluids 1 and 2 may be nanoparticle source materials), food and water quality testing and purification, and / or general filtration applications using electric fields.

[0090] 4 shows an exemplary system for sample processing or biomolecular analysis. The system may include a nanopore device (e.g., a nanopore array chip).

[0091] In some embodiments, the system may include a gas reservoir, a buffer reservoir, and / or a waste reservoir. Gas may fill the upper and lower chambers of the nanopore unit cell. In some embodiments, the system may include a microfluidic interface to direct fluid (e.g., gas, sample, buffer, etc.) to the inlet of the nanopore unit cell block and direct the fluid from the nanopore unit cell block to the waste reservoir. Samples to be analyzed may be introduced into the system in various formats, such as, for example, 1) a 96-well plate, 2) a 384-well plate, 3) an 8-well strip tube, or 4) a single-well centrifuge tube.

[0092] In some embodiments, the system may include a fluid pumping system configured to pump sample into and out of the nanopore array chip, pump gas or buffer from a gas or buffer reservoir into and out of the chip, and pump waste from the chip into a waste reservoir. In some embodiments, the nanopore array chip may include one or more microfluidic channels interfaced with the fluid pumping system.

[0093] In some embodiments, the system may include a robotics system that loads the sample and adjusts the interface between the fluid pumping system and the sample.

[0094] In some embodiments, the system may further include voltage and current control circuits, a CPU, memory, and data storage. In some embodiments, the system may include a sample and input system, an optical system, a heating / cooling system, peripherals, communication / networking, and a display system.

[0095] In some embodiments, the system may include a nanopore voltage control circuit configured to provide voltages to the upper and lower electrodes (e.g., 116 and 121 in FIG. 1A and 156 and 161 in FIG. 1B) for biomolecular translocation and / or ionic current measurements. In some embodiments, the nanopore voltage control circuit may be configured to provide voltages to the positive and negative electrodes (e.g., 111 and 112 in FIG. 1A and 151 and 152 in FIG. 1B) for tunneling current measurements.

[0096] In some embodiments, the system may include a nanopore ion / tunneling current measurement circuit configured to measure an electrical signal, e.g., current, as a sample or biomolecule translocates through the nanopore. In some embodiments, the nanopore ion / tunneling current measurement circuit may report the measured electrical signal to an accelerator (e.g., FPGA / ASIC / GPU) for executing a machine learning model that converts the electrical signal into biomolecular information. Alternatively, or in addition, the nanopore ion / tunneling current measurement circuit may send the measured electrical signal to a CPU for analysis by the CPU.

[0097] The CPU acts as the central control unit for the data generated by the system and may interface with a display, user interface, other components (e.g., memory, data storage, networking, etc.), and / or peripherals (I / O). The system may be controlled via a touchscreen user interface or computer using a wired interface (e.g., USB, Ethernet) or a wireless interface. In some embodiments, the wireless interface may comprise a mobile device. Software may be used for biomolecule analysis and generation of size distribution information. The software may interface with an automation framework, such as a LIMS, to automate workflows.

[0098] In some embodiments, the system may include an optical / fluorescence detection system configured to detect biomolecules by optical or fluorescent methods, and in some embodiments, the optical / fluorescence detection system may interface with a CPU / accelerator for signal processing.

[0099] In some embodiments, the nanopores in the nanopore array chip may be generated in situ: when the nanopore array chip is loaded into the system, a fluid pumping system pumps a dielectric composition (e.g., gas or liquid) into the upper and lower chambers of the nanopore unit cells, and then the nanopore voltage control circuitry and current measurement circuitry, under the influence of the CPU, drive the chip to create the nanopores.

[0100] In some embodiments, biomolecular sequencing may be performed with this system using signals obtained from the ion / tunneling current sensor. In some embodiments, the nanopore array chip may have a membrane a few atoms thick. In some embodiments, the membrane may be a two-dimensional material (e.g., graphene, MoS2) or an ultrathin film. This may enable measurement of current fluctuations at the single base / amino acid level. In some embodiments, the measured ion / tunneling current (e.g., raw current signal data) may be stored locally or in the cloud. The measured ion / tunneling current may be passed through a detection circuit and accelerator to perform base calling. The CPU may store the base calls in a file or upload the base calls to the cloud.

[0101] In some embodiments, the systems disclosed herein may be capable of maintaining a nanopore free of clogging. For example, if biomolecules or other debris clog the nanopore, corona and / or arc discharge can be used to unclog and regenerate the nanopore.

[0102] Methods for fabricating nanopore devices In some embodiments, the present disclosure provides a method of manufacturing a nanopore device for processing a fluid sample, the method comprising: (a) providing a device comprising a substrate, an upper cavity, a lower cavity, a first dielectric membrane, and first and second sets of electrodes; and (b) applying a first electric field between the first and second sets of electrodes to generate a corona discharge near the ends of the first and second sets of electrodes, the corona discharge forming one or more nanopores in the first dielectric membrane. The corona discharge can create a high concentration of charged particles near the electrodes, which can create nanopores at the locations of the corona discharge.

[0103] In some embodiments, (a) comprises: (A) depositing a first dielectric film on a substrate; (B) forming a lower cavity in the substrate below the first dielectric film, the lower cavity comprising a dielectric (e.g., a second dielectric disclosed in the present disclosure); (C) depositing a first set and a second set of electrodes on the first dielectric film; and (D) forming an upper cavity above the first dielectric film and above the first and second sets of electrodes, the upper cavity comprising an additional dielectric (e.g., a first dielectric disclosed in the present disclosure).

[0104] In some embodiments, depositing (C) comprises depositing a dielectric, a further dielectric, and a first set of electrodes proximate to the first dielectric film, hi some embodiments, depositing (D) comprises depositing a first dielectric, a second dielectric, and a second set of electrodes proximate to the first dielectric film.

[0105] In some embodiments, the method may further include generating a first electric field by applying a pulsed or continuous wave AC or DC voltage signal between the first and second sets of electrodes. In some embodiments, the first electric field may include a level of at least about 1 kV / m, at least about 10 kV / m, at least about 50 kV / m, 100 kV / m, at least about 1 megavolt / meter (MV / m), at least about 10 MV / m, at least about 100 MV / m, or more.

[0106] 5A and 5B show an exemplary configuration for generating a corona discharge. A continuous or pulsed DC or AC voltage is applied between two electrodes 501 and 502, which have sharp edges and are positioned close to each other, and between electrodes 511 and 512, e.g., conductive metal electrodes, which are surrounded by dielectrics 503 and 513, e.g., liquid or gas. A high electric field exists at the sharp edges of the electrodes due to the electric field gradient. If the electric field is sufficiently high (e.g., greater than 30 kV / m for air), electrons escaping from the electrodes can ionize atoms in the dielectric 503 surrounding the electrodes. This ionization creates electron-ion pairs, which, when coupled with an ion current, can cause breakdown of the dielectric material. When these ions and electrons migrate toward the electrode of opposite polarity and the charged particles recombine, a glow or corona discharge occurs. In some cases, a corona discharge, e.g., a positive corona discharge 505, may form at the sharp edge of the positive electrode, as shown in FIG. 5A. In some cases, a corona discharge, e.g., a negative polarity corona discharge 515, may form at the sharp edge of the negative electrode, as shown in Figure 5B. This may result in the emission of photons and an increase in temperature. The amount of charged particles, photons (light), radius of the corona discharge, and temperature increase may depend on the applied voltage and pressure. The location where the corona forms may have a higher concentration of charged particles (e.g., electrons, ions).

[0107] 6 shows an exemplary configuration for forming a nanopore. A dielectric membrane 602 is supported by a substrate 603 and separates an upper cavity from a lower cavity. In both cavities, the dielectric may be air. Two electrodes (e.g., 611 and 612) are disposed on the dielectric membrane 602, which is supported by the substrate 603. When a voltage is applied, the resulting corona discharge 601 is used to etch or melt the dielectric membrane 602 at the corona discharge location, thereby creating a nanopore at the corona discharge location. Optionally, an additional dielectric membrane may be disposed between the dielectric membrane 602 and the substrate 603. In some embodiments, the nanopore is about 0.1 nanometers (nm) to about 1 nm, about 0.1 nm to about 5 nm, about 0.1 nm to about 10 nm, about 0.1 nm to about 50 nm, about 0.1 nm to about 100 nm, about 0.1 nm to about 500 nm, about 0.1 nm to about 1 micrometer (μm), about 0.1 nm to about 10 μm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 1 nm to about 500 nm, about 1 nm to about 1 μm, about 1 nm to about 10 μm, about 5 nm to about 10 nm, about 5 nm to about 50 nm, about 5 The diameter may be from about 100 nm, from about 5 nm to about 500 nm, from about 5 nm to about 1 μm, from about 5 nm to about 10 μm, from about 10 nm to about 50 nm, from about 10 nm to about 100 nm, from about 10 nm to about 500 nm, from about 10 nm to about 1 μm, from about 10 nm to about 10 μm, from about 50 nm to about 100 nm, from about 50 nm to about 500 nm, from about 50 nm to about 1 μm, from about 50 nm to about 10 μm, from about 100 nm to about 500 nm, from about 100 nm to about 1 μm, from about 100 nm to about 10 μm, from about 500 nm to about 1 μm, from about 500 nm to about 10 μm, or from about 1 μm to about 10 μm.

[0108] In some cases, chambers need not be structural features, but rather regions that are locations relative to one another and separated by a dielectric film.

[0109] In some cases, any dielectric material (including various gases or liquids) may be selected as the first or second dielectric, or a low-conductivity dielectric solid may be selected.

[0110] FIG. 7A shows another exemplary configuration for forming a nanopore. A dielectric membrane 702 is supported by a substrate 706 and separates an upper cavity from a lower cavity. The upper chamber of the upper cavity contains a first dielectric, e.g., fluid 701, and the lower chamber of the lower cavity contains a second dielectric, e.g., fluid 707. Fluids 701 and 707 may be any combination of gases or liquids. In some embodiments, the upper and lower chambers may contain the same fluid. In some embodiments, the upper and lower chambers may contain different fluids. The formed corona discharge 703 may be used to etch or melt the dielectric membrane at the corona discharge location, thereby creating a nanopore at the corona discharge location.

[0111] FIG. 7B shows another exemplary configuration for forming a nanopore. A dielectric membrane 712 is supported by a substrate 710. Two electrodes 714 and 715 are disposed adjacent to the dielectric membrane 712. A dielectric solid material 711 surrounds the two electrodes. A lower cavity lower chamber 717 may contain a dielectric 716, which may be any combination of gas or liquid. The dielectric solid material 711 may contain a predetermined concentration of dopant ions. In some embodiments, a corona discharge 713 may be formed. In some embodiments, the corona discharge may be invisible or absent, which may result in erosion of the dielectric membrane, referred to as partial discharge. The formed corona discharge 713 may be used to etch or melt the dielectric membrane material at the corona discharge location, thereby creating a nanopore at the corona discharge location.

[0112] In some embodiments, a second dielectric film may be used. In some embodiments, the second dielectric film may be disposed between the dielectric film and the substrate. In some cases, the dielectric film and the additional dielectric film may have substantially the same size. In some cases, the dielectric film and the additional dielectric film may have different sizes. In some embodiments, the second dielectric film may be disposed between the first dielectric film and the substrate during formation of the nanopore and may be removed after formation of the nanopore.

[0113] FIG. 7C shows another exemplary configuration for forming a nanopore. A dielectric film 722 (e.g., a first dielectric film) is supported by a further dielectric film 728 (e.g., a second dielectric film), which is supported by a substrate 720. Two electrodes 724 and 725 are disposed adjacent to the dielectric film 722 and are covered with a dielectric solid material 721. A lower chamber 727 of the lower cavity may contain a dielectric 726, which may be any combination of gas or liquid. The dielectric solid material 721 may contain a predetermined concentration of dopant ions. In some embodiments, a corona discharge 723 may be formed. In some embodiments, the corona discharge may be invisible or absent, which may result in erosion of the dielectric film, referred to as a partial discharge. The formed corona discharge 725 may be used to etch or melt the dielectric film material at the corona discharge location, thereby creating a nanopore at the corona discharge location. The further dielectric film 728 may be removed after the formation of the nanopore, for example by chemical etching. In some cases, the nanopore may be formed through both the dielectric film 722 and the further dielectric film 728.

[0114] 7D shows another exemplary configuration for forming a nanopore. Dielectric film 732 (e.g., a first dielectric film) is supported by a further dielectric film 738 (e.g., a second dielectric film), which is supported by substrate 730. Dielectric film 732 and further dielectric film 738 separate an upper cavity and a lower cavity. Two electrodes 734 and 735 may be disposed adjacent to dielectric film 732. The upper chamber of the upper cavity may include a dielectric 731 (e.g., a first dielectric, fluid, or gas). The lower chamber 737 of the lower cavity may include a dielectric 736 (e.g., a second dielectric, fluid, or gas). Dielectrics 731 and 736 may be any combination of gases or liquids. The formed corona discharge 733 may be used to remove a portion of the dielectric film material at the corona discharge location, thereby creating a nanopore at the corona discharge location. The further dielectric film 738 may be removed above the dielectric 736 after the nanopore is formed, for example by chemical etching. In some embodiments, the further dielectric film 738 located above the dielectric 736 may be etched away after the nanopore is formed. In some cases, the nanopore may be formed through both the dielectric film 732 and the further dielectric film 738.

[0115] In some embodiments, the device may further comprise a second dielectric film. In some embodiments, (a) may further comprise depositing a second dielectric film between the first dielectric film and the substrate or the second dielectric.

[0116] In some embodiments, the device may further comprise a top layer and a bottom layer. In some embodiments, (a) may further comprise forming a top layer over the first dielectric and a bottom layer under the second dielectric.

[0117] Corona discharge is a non-thermal process and can result in an increase in the temperature of the device or the temperature near the nanopore site. In some embodiments, the temperature increase can be about 0.1°C to about 10°C. In some embodiments, the charge intensity or temperature at the corona discharge location may not be high enough to etch a thick film and create a nanopore. To address this issue, a second electric field can be configured to generate an electric arc discharge, which, when applied in addition to the corona discharge, can create a localized electric field that aids in nanopore formation. In the corona discharge-assisted dielectric arc discharge nanopore generation method, the corona discharge location can function as a concentration of electric field lines, thus enabling localized nanopore generation.

[0118] In some embodiments, the present disclosure provides methods for generating nanopores using the corona discharge-assisted dielectric arc discharge provided herein.

[0119] In some embodiments, the present disclosure provides a method for manufacturing a nanopore device for processing a fluid sample, the method comprising: (i) providing a device comprising a substrate, an upper cavity, a lower cavity, a first dielectric membrane, a first set and a second set of electrodes, and a third set and a fourth set of electrodes; (ii) applying a first electric field between the first and second sets of electrodes to generate a corona discharge near the ends of the first and second sets of electrodes; and (iii) applying a second electric field between the third and fourth sets of electrodes to generate an electrical arc discharge, wherein the combination of the corona discharge and the electrical arc discharge forms one or more nanopores in the first dielectric membrane.

[0120] In some embodiments, the corona discharge generated in (ii) can produce a high concentration of charged particles near the electrodes. In some embodiments, (iii) can include applying a second electric field between the third and fourth sets of electrodes until the second electric field exceeds a threshold such that the dielectric strength of the fluid in the upper or lower cavity is broken down and begins to conduct current, thereby resulting in arcing and breakdown of the dielectric film.

[0121] In some embodiments, (i) may include: (1) depositing a first dielectric film on a substrate; (2) forming a lower cavity in the substrate below the first dielectric film, the lower cavity comprising a dielectric (e.g., a second dielectric); (3) depositing a first set and a second set of electrodes on the first dielectric film; (4) forming an upper cavity above the first dielectric film and above the first and second sets of electrodes, the upper cavity comprising a further dielectric (e.g., the first dielectric); (5) depositing a third set of electrodes above the upper cavity; and (6) depositing a fourth set of electrodes below the lower cavity.

[0122] In some embodiments, the depositing of (3) may include depositing a first dielectric and a first set and a second set of electrodes proximate the first dielectric film.

[0123] In some embodiments, depositing (5) may include depositing a third set of electrodes proximate the first dielectric. In some embodiments, depositing (6) may include depositing a fourth set of electrodes proximate the second dielectric.

[0124] In some embodiments, the method may further include generating a first electric field by applying a pulsed or continuous wave AC or DC voltage signal between the first and second sets of electrodes. In some embodiments, the first electric field may include a level of at least about 1 kV / m, at least about 10 kV / m, at least about 50 kV / m, at least about 100 kV / m, at least about 1 MV / m, at least about 10 MV / m, at least about 100 MV / m, or more.

[0125] In some embodiments, the method may further include generating a second electric field by applying a pulsed or continuous wave AC or DC voltage signal between the third and fourth sets of electrodes. In some embodiments, the second electric field may include a level of at least about 1 kV / m, at least about 10 kV / m, at least about 50 kV / m, at least about 100 kV / m, at least about 1 MV / m, at least about 10 MV / m, at least about 100 MV / m, at least about 1000 MV / m, at least about 5000 MV / m, or more.

[0126] In some embodiments, the device may further comprise a second dielectric film. In some embodiments, (i) may further comprise depositing a second dielectric film between the first dielectric film and the substrate or the second dielectric.

[0127] In some embodiments, the device may further comprise a top layer and a bottom layer. In some embodiments, (i) may further comprise forming a top layer over the first dielectric and a bottom layer under the second dielectric.

[0128] FIG. 8 shows an exemplary configuration for corona discharge-assisted dielectric arc discharge nanopore generation. Dielectric membrane 802 is supported by an additional dielectric membrane 808, which is supported by substrate 800. Dielectric membrane 802 and additional dielectric membrane 808 may separate an upper cavity from a lower cavity. In some cases, the dielectric membrane and additional dielectric membrane may have substantially the same size. In some cases, the dielectric membrane and additional dielectric membrane may have different sizes. Additional dielectric membrane 808 may be disposed between dielectric membrane 802 and substrate 800. In some embodiments, the additional dielectric membrane may not be present, and dielectric membrane 802 may be supported by the substrate and separate the upper cavity from the lower cavity. Two electrodes 804 and 805 are disposed adjacent to dielectric membrane 802. The upper chamber of the upper cavity may include a first dielectric 801. The lower chamber 807 of the lower cavity may include a second dielectric 806. Dielectrics 801 and 806 may be any combination of gases or liquids. As shown in FIG. 8 , a corona discharge 803 is created by applying a pulsed or continuous wave DC or AC electric field between electrodes 804 and 805 with sharp features (e.g., sharp edges). This can create a high concentration of charged particles near electrodes 804 and 805. When an external electric field (e.g., a second electric field) is applied using external electrodes, such as external electrodes 810 and 820, the external electric field (811 shows exemplary field lines) can be concentrated at the corona discharge location, thereby creating a much higher concentration of charge at the corona discharge location. This can lead to the creation of nanopores at the corona discharge location.

[0129] In some embodiments, external electrodes may be connected to the fluid in the upper and lower chambers (as shown in FIG. 8 ). In some embodiments, external electrodes may be embedded in the upper and lower chambers. FIG. 9 shows another exemplary configuration for corona discharge-assisted dielectric arc discharge nanopore generation. A dielectric film 902 (e.g., a first dielectric film) is supported by an additional dielectric film 908 (e.g., a second dielectric film), which is supported by a substrate 900. In some cases, the dielectric film and the additional dielectric film may have substantially the same size. In some cases, the dielectric film and the additional dielectric film may have different sizes. The additional dielectric film 908 may be disposed between the dielectric film 902 and the substrate 900. The dielectric film 902 and the additional dielectric film 908 may separate the upper and lower cavities. In some embodiments, the additional dielectric film may not be present, and the dielectric film 902 may be supported by the substrate and separate the upper and lower cavities. Two electrodes 904 and 905 are positioned adjacent to the membrane 902. The upper chamber of the upper cavity may include a first dielectric 901. The lower chamber of the lower cavity 907 may include a second dielectric 906. The dielectrics 901 and 906 may be any combination of gas or liquid. The configuration may further include an electrode 910 embedded in the upper cavity and an electrode 920 embedded in the lower cavity. As shown in FIG. 9, the corona discharge 903 is created by applying a pulsed or continuous wave DC or AC electric field (e.g., a first electric field) between electrodes 904 and 905 with sharp features (e.g., sharp edges). This can create a high concentration of charged particles near the electrodes. When an external electric field (e.g., a second electric field) is applied using external electrodes, such as external embedded electrodes 910 and 920, the electric field (911 shows exemplary electric field lines) can be concentrated at the corona discharge location, resulting in a much higher concentration of charge at the corona discharge location, which can lead to the creation of nanopores at the corona discharge location.

[0130] When external electrodes are embedded in the chamber, each of the blocks that create the nanopore can be considered a unit cell and can be mass-produced at the wafer scale to create millions of nanopores in a controlled manner. Fluids in the upper and lower chambers can be enclosed to form a microfluidic channel or network for transporting fluid to the nanopore location. In some embodiments, the microfluidic channel or network can be used to transport biomolecules to the nanopore unit cell for analysis.

[0131] FIG. 10 shows an exemplary configuration for creating an arc discharge nanopore. A dielectric membrane 1001 (e.g., a first dielectric membrane) is supported by an additional dielectric membrane 1002 (e.g., a second dielectric membrane), which is supported by a substrate 1003. In some cases, the dielectric membrane and the additional dielectric membrane may have substantially the same size. In some cases, the dielectric membrane and the additional dielectric membrane may have different sizes. The additional dielectric membrane 1002 may be disposed between the dielectric membrane 1001 and the substrate 1003. The membrane 1001 and the additional membrane 1002 may separate an upper cavity and a lower cavity. In some embodiments, the additional dielectric membrane may not be present, and the dielectric membrane 1001 may be supported by the substrate and separate the upper cavity and the lower cavity. The upper chamber of the upper cavity may contain a first fluid, and the lower chamber 1006 of the lower cavity may contain a second fluid. A first electrode 1004 with a sharp edge is positioned above the membrane 1001, a second electrode 1005 is positioned below the substrate 1003, and an electric field is applied between the first and second electrodes. When the electric field exceeds a threshold value, the dielectric strength of the fluid in the lower chamber 1006 is broken down, causing the fluid to begin conducting electrical current and creating an arc 1007. This causes breakdown of the dielectric membrane, creating a nanopore 1010 in the membrane and, if present, in any optional further membranes.

[0132] 11 shows another exemplary configuration for creating an arc discharge nanopore. A dielectric membrane 1101 (e.g., a first dielectric membrane) is supported by an additional dielectric membrane 1102 (e.g., a second dielectric membrane), which is supported by a substrate 1103. In some cases, the dielectric membrane and the additional dielectric membrane may have substantially the same size. In some cases, the dielectric membrane and the additional dielectric membrane may have different sizes. The additional dielectric membrane 1102 may be disposed between the dielectric membrane 1101 and the substrate 1103. The membrane 1101 and the additional membrane 1102 may separate an upper cavity and a lower cavity. In some embodiments, the additional dielectric membrane may not be present, and the dielectric membrane 1101 may be supported by the substrate and separate the upper cavity and the lower cavity. An upper chamber 1106 of the upper cavity a may contain a first fluid, and a lower chamber 1107 of the lower cavity a may contain a second fluid. Two electrodes 1104 and 1105 with sharp edges are disposed on top of the membrane 1101. An external electrode 1110 is embedded in the upper cavity, and a dielectric material 1111 is disposed between the external electrode 1110 and the electrodes 1104 and 1105 to separate them. Another external electrode 1120 is embedded in the lower cavity. When an electric field is applied between the electrode 1105 and the external electrode 1120, and the electric field exceeds a threshold value, the dielectric strength of the fluid in the lower chamber 1107 is broken down, causing the fluid to begin conducting current and generating an arc discharge 1108. This causes breakdown of the dielectric membrane, creating a nanopore 1112 in the dielectric membrane and, if present, in any optional additional dielectric membranes.

[0133] Unlike corona discharge-based methods, arc discharge generates intense heat (e.g., over 1000°C) that can melt dielectric films in its path. In some embodiments, arc discharge nanopore generation methods may be used to generate nanopores in thicker films, e.g., films having thicknesses of about 1 nm to 50 nm.

[0134] In some embodiments, any of the methods of fabricating a nanopore device disclosed herein may further include depositing fifth and sixth electrodes on the first dielectric film. In some embodiments, the fifth and sixth electrodes may be configured to generate a dielectrophoretic (DEP) force with the first and second sets of electrodes.

[0135] In some embodiments, any of the methods of manufacturing a nanopore device disclosed herein may further include (I) fabricating a plurality of nanopore devices, (II) electrically connecting at least one nanopore device of the plurality of nanopore devices to at least one other nanopore device of the plurality of nanopore devices to form a plurality of nanopore cells, and (III) electrically connecting at least one nanopore cell of the plurality of nanopore cells to at least one other nanopore cell of the plurality of nanopore cells to form a plurality of nanopore arrays. The plurality of nanopore arrays may be configured for high-throughput processing of fluid samples.

[0136] How to Use Nanopore Devices In some embodiments, the present disclosure provides methods for processing a fluid sample using one or more nanopores, the method including: (a) providing a device comprising a substrate, an upper cavity, a lower cavity, a first dielectric film, first and second sets of electrodes configured to generate a corona discharge, and third and fourth sets of electrodes configured to generate an electrical arc discharge, wherein the one or more nanopores are formed in the first dielectric film using a combination of the corona discharge and the electrical arc discharge; (b) flowing a fluid sample through the one or more nanopores; and (c) processing the fluid sample as it flows through the one or more nanopores.

[0137] In some embodiments, the method may include forming one or more nanopores using the first, second, third, or fourth set of electrodes, hi some embodiments, the one or more nanopores may be formed prior to contacting the first dielectric membrane with the fluid sample.

[0138] In some embodiments, the method may further include detecting one or more events associated with processing of the fluid sample using the first and second sets of electrodes.

[0139] In some embodiments, the fluid sample may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, lipids, or carbohydrates suspended in a gas, water, an electrolyte solution, or a porous solid (e.g., a gel). In some embodiments, the one or more events may comprise events related to biomolecule analysis, biomolecule measurement, biomolecule purification, biomolecule enrichment, or biomolecule sequencing. In some embodiments, (b) may comprise flowing the fluid sample through one or more nanopores via a capillary, pressure-driven, electroosmotic flow, or electrowetting mechanism.

[0140] In some embodiments, processing the fluid sample may include analyzing, synthesizing, purifying, processing biomolecules, and / or sequencing DNA, RNA, or proteins.

[0141] In some embodiments, the method may include measuring the tunneling current using the first and second sets of electrodes, hi some embodiments, the method may include measuring the ion current using the third and fourth sets of electrodes.

[0142] In some embodiments, the device may further include a fifth electrode and a sixth electrode configured to generate a dielectrophoretic (DEP) force with the first and second sets of electrodes. In some embodiments, the method may further include concentrating biomolecules contained in the fluid sample using the DEP force.

[0143] In some embodiments, the method may further include measuring the ion current and / or the tunneling current using one or more sensors.

[0144] Computer Systems The present disclosure provides computer systems programmed to implement one or more of the methods of the present disclosure, which may be used to regulate various operations, such as sequencing and / or analyzing a nanopore (e.g., a protein nanopore or a solid-state nanopore), including detecting one or more signals when a sample is present within the nanopore.

[0145] FIG. 15 illustrates a computer system 1501 programmed or otherwise configured to communicate with and regulate various aspects of the sequencing and / or analysis of the present disclosure. The computer system 1501 may communicate with, for example, one or more circuits coupled to or including a nanopore (or a membrane containing a nanopore), as well as one or more devices (e.g., machines) used to prepare, process, or maintain one or more reaction mixtures for sequencing and / or analysis. The computer system 1501 may also communicate with one or more controllers or processors of the present disclosure. The computer system 1501 may be a user's electronic device or a computer system located remotely relative to the electronic device. The electronic device may be a mobile electronic device.

[0146] The computer system 1501 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1505, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1501 also includes memory or storage locations 1510 (e.g., random access memory, read-only memory, flash memory), electronic storage 1515 (e.g., a hard disk or solid-state drive), a communications interface 1520 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage, and / or an electronic display adapter. The memory 1510, storage 1515, interface 1520, and peripheral devices 1525 communicate with the CPU 1505 via a communications bus (solid lines), such as a motherboard. The storage 1515 may also be a data storage device (or data repository) for storing data. Computer system 1501 may be operatively coupled to a computer network ("network") 1530 using communication interface 1520. Network 1530 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. Network 1530 may, in some cases, be a telecommunications and / or data network. Network 1530 may include one or more computer servers that enable distributed computing, such as cloud computing. Network 1530 may, in some cases, implement a peer-to-peer network, allowing devices coupled to computer system 1501 to operate as clients or servers, with the help of computer system 1501.

[0147] The CPU 1505 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions may be directed to the CPU 1505, which may then be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 1505 may include fetch, decode, execute, and writeback.

[0148] The CPU 1505 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a digital signal processor (DSP), or a graphics processing unit (GPU).

[0149] Storage device 1515 may store files such as drivers, libraries, and saved programs. Storage device 1515 may store user data, such as user preferences and user programs. Computer system 1501 may optionally include one or more additional data storage devices external to computer system 1501, such as located on a remote server that communicates with computer system 1501 over an intranet or the Internet.

[0150] Computer system 1501 can communicate with one or more remote computer systems via network 1530. For example, computer system 1501 can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a telephone, a smartphone (an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access computer system 1501 via network 1530.

[0151] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of computer system 1501, such as memory 1510 or electronic storage 1515. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by processor 1505. In some cases, the code may be retrieved from storage 1515 and stored in memory 1510 for immediate access by processor 1505. In some situations, electronic storage 1515 may be omitted, and machine-executable instructions are stored in memory 1510.

[0152] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or it may be compiled at run-time. The code may be provided in a programming language that can be selected to allow the code to be executed in a pre-compiled or as-compiled manner.

[0153] Aspects of the systems and methods provided herein, such as computer system 1501, may be embodied in programming. Various aspects of the technology can be considered "products" or "articles of manufacture," typically in the form of machine (or processor) executable code and / or associated data, which are held or embodied in some type of machine-readable medium. The machine-executable code may be stored in electronic storage, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage"-type media may include any or all of the tangible memory of a computer, processor, etc., or associated modules, such as various semiconductor memory, tape drives, disk drives, etc., that may provide non-transitory storage for software programming at any time. All or portions of the software may be communicated via the Internet or various other telecommunications networks. Such communication may, for example, enable loading of the software from one computer or processor to another, such as from an administrative server or host computer to an application server computer platform. Thus, other types of media that may hold software elements include the light waves, radio waves, and electromagnetic waves used across physical interfaces between local devices over wired and optical landline networks, as well as various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media that carry software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0154] Thus, machine-readable media, such as computer-executable code, may take many forms, including, but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices of any computer, such as those that may be used to implement the databases shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include copper wire and fiber optics, including coaxial cables, i.e., the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible diskette, a hard disk, magnetic tape, any other magnetic medium, a CD-ROM, a DVD, or a DVD-ROM, any other optical medium, punched cards, paper tape, any other physical storage medium with a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link carrying such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0155] The computer system 1501 may include or communicate with an electronic display 1535 with a user interface (UI) 1540 to present, for example, (i) the progress of the reaction mixture, (ii) the progress of the sequencing, and (iii) the sequencing information obtained from the sequencing. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0156] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 1505. The algorithms may, for example, determine a sequence readout of one or more target sites during nanopore sequencing. [Example]

[0157] The following examples are presented to further illustrate some embodiments of the present disclosure and are not intended to limit the scope of the disclosure. By their illustrative nature, it will be understood that other procedures, methodologies, or techniques may alternatively be used.

[0158] Example 1: Exemplary Biomolecular Analysis System The biomolecule analysis system disclosed herein enables high-throughput size distribution analysis of biomolecules at single base / amino acid resolution. That is, the system enables the acquisition of high-resolution size distribution maps such as those illustrated in Figure 12. Accurate measurement of biomolecule size (e.g., DNA length in kilobases) is useful for determining the effectiveness of assays such as polymerase chain reaction (PCR).

[0159] FIG. 13 illustrates a biomolecule moving from the upper chamber to the lower chamber of a nanopore unit cell. The nanopore unit cell may include an upper cavity and a lower cavity. The upper cavity may include an upper chamber 1313 encapsulated with a first dielectric. The lower cavity may include a substrate 1322 and a lower chamber 1323 encapsulated with a second dielectric. The nanopore unit cell may include a dielectric membrane 1301 supported by the substrate 1322 and configured to separate the upper and lower cavities. The dielectric membrane 1301 may include a nanopore region 1302. The nanopore unit cell may further include two electrodes, e.g., a positive electrode 1312 and a negative electrode 1311, associated adjacent to the nanopore region 1302. The two electrodes 1311 and 1312 may have sharp tips. In some cases, the nanopore unit cell may comprise an upper electrode 1316 and a lower electrode 1321, and a dielectric separator 1314 separating electrodes 1311 and 1312 from electrode 1316. In some cases, the nanopore unit cell may further comprise an upper lid 1315 and a lower lid 1325. The upper and lower chambers may be enclosed by the upper and lower lids to create a seal that allows fluid to flow in and out of the upper and lower chambers.

[0160] Biomolecule movement 1330 may be driven by an electric field applied between electrodes 1316 and 1321. Biomolecule movement may result in a change in the ionic current between electrodes 1316 and 1321 and a change in the tunneling current between electrodes 1311 and 1312.

[0161] FIG. 14 shows an exemplary measurement of current versus time within a nanopore. The baseline current (L1) is the current level when there is no ion flow within the nanopore, the ion / tunneling current baseline (L2) is the current when an ion blocks the nanopore or tunneling sensor, and the molecular translocation current (L3) is the current measured by the ion / tunneling sensor when a biomolecule translocates through the nanopore. When there is an ion blockage at the nanopore or tunneling sensor, the current drops from baseline L1 to L2. When the biomolecule translocates through the nanopore, the current drops to L3. The length of the biomolecule may be determined by the magnitude of the current drop and the duration of the drop.

[0162] Example 2: Exemplary biomolecular analysis using nanopore unit cells 13, lower chamber 1323 contains ionic liquid 1. A sample containing biomolecules in ionic liquid 2 is introduced into upper chamber 1313. An electric field applied between electrodes 1316 and 1321 can translocate the biomolecules from the upper chamber (cis) to the lower chamber (trans) through the nanopore.

[0163] In the absence of biomolecules, the flow of ions through the nanopore perturbs the ionic current measured between electrodes 1316 and 1321. These perturbations are due to blockage of the ionic current flow. When a biomolecule passes through the nanopore, the blockage of the ionic current is much greater, resulting in a much smaller ionic current. The ionic current may be measured using electrodes immersed in the liquid in the upper and lower chambers. The electrodes are embedded in the nanopore unit cells, thereby allowing for independent control of each individual nanopore unit cell. Furthermore, this allows for the parallel analysis of a large number of biomolecules with a large number (e.g., millions) of nanopores.

[0164] The electrodes 1311 and 1312 used to create the nanopore using corona or arc discharge can be used as a tunneling current sensor. The electrodes 1311 and 1312 can be passivated with an insulating oxide layer. When a high electric field is applied between the electrodes 1311 and 1312, a small amount of current flows between the two electrodes due to quantum mechanical tunneling. As a biomolecule passes through the nanopore, it can affect the charge density in the gap between the electrodes 1311 and 1312. This can cause a change in the tunneling current between the electrodes, thus providing important information about the biomolecule passing through the nanopore.

[0165] Ion currents and tunneling currents and their changes may be used to detect, analyze, and manipulate biomolecules within the nanopore chip.

[0166] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may be made without departing from the present disclosure. It will be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

[Claim 1] A nanopore device for processing fluid samples, wherein the nanopore device is substrate, Upper cavity containing the first dielectric, Lower cavity containing a second dielectric, A first dielectric film supported by the substrate, separating the upper cavity and the lower cavity, A first set of electrodes and a second set of electrodes located in close proximity to the first dielectric film. Equipped with, A nanopore device wherein the first set of electrodes and the second set of electrodes are configured to apply a first electric field to generate a corona discharge, thereby forming one or more nanopores in the first dielectric film.

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