Systems and methods for analyzing biological samples

Abstract

The present disclosure provides methods and systems for analyzing an analyte. In one aspect, the present disclosure provides a method, (a) contacting at least a portion of an analyte with a nanopore system, wherein the nanopore system comprises (i) a fluidic chamber, (ii) a nanopore disposed within a membrane, wherein the membrane separates the fluidic chamber to a first side and a second side, and (iii) a sensor comprising a first electrode and a second electrode; (b) detecting one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) associated with the at least the portion of the analyte; and (c) using the one or more signals to identify one or more characteristics of the at least the portion of the analyte with an accuracy of at least about 90%.

Description

[0001] SYSTEMS AND METHODS FOR ANALYZING BIOLOGICAL SAMPLES

[0002] CROSS REFERENCE

[0003]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 712,954, filed October 28, 2024, which is entirely incorporated herein by reference.

[0004] BACKGROUND

[0005]

[0002] Nucleic acid sequencing is the process of determining the sequence of nucleotides in a nucleic acid sample. Specific nucleic acid sequence information can be used in the discovery of genetic diseases, diagnosis of infectious diseases, and development and monitoring of treatment. A variety of nucleic acid sequencing methods have been investigated, for example, electrophoresis, sequencing by hybridization, mass spectrometry-based method, sequencing by ligation, and sequencing by synthesis (SBS).

[0006] SUMMARY

[0007]

[0003] The present disclosure provides methods, systems, devices, compositions, and kits for analyzing an analyte in a sample. In some cases, an analyte can comprise a small molecule, a nucleotide, a nucleic acid molecule, a polynucleotide, a protein, a polypeptide, or a peptide.

[0004] In an aspect, the present disclosure provides a method, comprising: (a) contacting at least a portion of an analyte with a nanopore system, wherein the nanopore system comprises (i) a fluidic chamber, (ii) a nanopore disposed within a membrane, wherein the membrane separates the fluidic chamber to a first side and a second side, and (iii) a sensor comprising a first electrode and a second electrode; (b) detecting one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) associated with the at least the portion of the analyte; and (c) using the one or more signals to identify one or more characteristics of the at least the portion of the analyte with an accuracy of at least about 90%.

[0008]

[0005] In an aspect, the present disclosure provides a method, comprising: (a) contacting at least a portion of an analyte with a nanopore system, the nanopore system comprising (i) a nanopore disposed within a membrane, (ii) a first electrode disposed adjacent to a first side of the nanopore, and (iii) a second electrode disposed adjacent to a second and different side of the nanopore; (b) applying a time-varying asymmetrical voltage waveform between the first electrode and the second electrode of the nanopore system; and (c) directing the nanopore system to detect one or more electrical signals associated with the at least the portion of the analyte.

[0006] In an aspect, the present disclosure provides a method for analyzing a plurality of samples, comprising: (a) contacting a plurality of samples with a nanopore system comprising at least 500,000 fluidic chambers, wherein the at least 500,000 fluidic chambers are capable of independently analyzing a sample; (b) detecting one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) in the at least 500,000 fluidic chambers; and (c) using the one or more signals to identify one or more characteristics of the plurality of samples.

[0009]

[0007] In an aspect, the present disclosure provides a method, comprising: (a) contacting at least a portion of an analyte with a nanopore system, wherein the nanopore system comprises (i) a nanopore disposed within a membrane, (ii) one or more electrodes in proximity to the nanopore, and (iii) one or more signal generators in electrical communication with the one or more electrodes; (b) applying a potential to the one or more electrodes, thereby detecting one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) associated with the at least the portion of the analyte, wherein the one or more signal generators stabilize the potential; and (c) using the one or more signals to identify one or more characteristics of the at least the portion of the analyte.

[0010]

[0008] In an aspect, the present disclosure provides a method, comprising: (a) providing a single stranded nucleic acid molecule; (b) circularizing the single stranded nucleic acid molecule, in a presence of an oligonucleotide, wherein at least a portion of the oligonucleotide comprises sequence complementarity to at least a portion of an end of the single stranded nucleic acid molecule, and at least an additional portion of the oligonucleotide comprises sequence complementarity to at least an additional portion of an additional end of the single stranded nucleic acid molecule, wherein the circularizing comprises hybridizing the at least the portion of the oligonucleotide with the at least the portion of the end of the single stranded nucleic acid molecule and hybridizing the additional portion of the oligonucleotide with the at least the additional portion of the additional end of the single stranded nucleic acid molecule, thereby generating a complex comprising a circularized single stranded nucleic acid molecule hybridized with the oligonucleotide, wherein the circularized single stranded nucleic acid molecule comprises a nick; and (c) ligating an end of the circularized single stranded nucleic acid molecule and an additional end of the circularized single stranded nucleic acid molecule to generate a closed circularized single stranded nucleic acid molecule.

[0011]

[0009] In an aspect, the present disclosure provides a system comprising: a computer processor and a computer memory coupled thereto, wherein the computer memory comprises a machine executable code that, upon execution by the one or more computer processors, implements any of the methods presented herein.

[0010] In an aspect, the present disclosure provides a nanopore system, comprising: a sensor comprising (i) a fluidic chamber; (ii) a nanopore disposed within a membrane, wherein the membrane separates the fluidic chamber to a first side and a second side; and (iii) a sensor comprising a sensing electrode in proximity to the nanopore; a processor operatively coupled to the sensor, wherein the processor is configured to direct the sensor to (a) detect one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) associated with at least a portion of an analyte, and (b) use the one or more signals to identify one or more characteristics of the at least the portion of the analyte with an accuracy of at least about 90%.

[0012] [Oi l] In an aspect, the present disclosure provides a nanopore system, comprising: a sensor comprising (i) a nanopore disposed within a membrane; (ii) a first electrode disposed adjacent to a first side of the nanopore; and (iii) a second electrode disposed adjacent to a second and different side of the nanopore; a processor operatively coupled to the sensor, wherein the processor is configured to direct the sensor to: (a) apply a time-varying asymmetrical voltage waveform between the first electrode and the second electrode; and (b) detect one or more electrical signals associated with the at least the portion of the analyte.

[0013]

[0012] In an aspect, the present disclosure provides a nanopore system, comprising: a flow cell chip comprising at least 500,000 fluidic chambers; and a plurality of sensors configured to detect one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof), wherein each fluidic chamber of the at least 500,000 fluidic chambers is coupled to a sensing electrode, wherein the each fluidic chamber comprises a nanopore embedded in a membrane.

[0014]

[0013] In an aspect, the present disclosure provides a nanopore system, comprising: a nanopore disposed within a membrane; one or more electrodes in proximity to the nanopore; one or more signal generators in electrical communication with the one or more electrodes; wherein the one or more signal generators are configured to (a) apply one or more potentials to the one or more electrodes, and (b) stabilize the one or more potentials; and one or more controllers operatively coupled to the one or more electrodes, wherein the one or more controllers are individually or collectively configured to (1) detect one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) associated with at least a portion of an analyte, and (2) use the one or more signals to identify one or more characteristics of the at least the portion of the analyte.

[0015]

[0014] In an aspect, the present disclosure provides a nanopore system, comprising: a sensor comprising: (i) a nanopore disposed within a membrane; (ii) a first electrode disposed adjacent to a first side of the nanopore; (iii) a second electrode disposed adjacent to a second and different side of the nanopore; (iv) a capacitor electronically coupled to the second electrode; and (v) an electronic switch for generating a plurality of sequential temporary couplings between the capacitor and a voltage source; a processor operatively coupled to the sensor, wherein the processor is configured to direct the sensor to detect a plurality of electrical signals associated with the at least a portion of an analyte between the plurality of sequential temporary couplings.

[0015] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0016]

[0016] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0017]

[0017] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.

[0018] Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0019] INCORPORATION BY REFERENCE

[0020]

[0018] 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 publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

[0019] 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 present disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0023]

[0020] FIG. 1 shows an exemplary nanopore cell 100 of a nanopore system, in accordance with some embodiments;

[0021] FIG. 2 shows a top view 210 of an exemplary nanopore cell 200 and a side view 220 of the exemplary nanopore cell 200, in accordance with some embodiments;

[0024]

[0022] FIG. 3 shows an exemplary nanopore cell 300, in accordance with some embodiments;

[0023] FIGS. 4A and 4B show an exemplary chip, and FIG. 4C shows an exploded view of the nanopore block 401, in accordance with some embodiments;

[0025]

[0024] FIG. 5A shows a perspective view of a chip 500, FIG. 5B shows a top view of the chip 500, FIG. 5C shows a flow direction in the chip 500, FIG. 5D shows an exploded two- dimensional view of a flow cell, FIG. 5E shows a three-dimensional view of the flow cell, and FIG. 5F shows the flow directions of the fluid in the channels of the flow cell in accordance with some embodiments;

[0026]

[0025] FIG. 6A shows a top view of an exemplary cartridge, FIG. 6B shows an exploded view of the cartridge components, FIG. 6C shows an image of an exemplary assembled cartridge, FIG. 6D shows a perspective view of the assembled cartridge, FIG. 6E shows side views of the assembled cartridge, FIG. 6F shows an exemplary pin and FIG. 6G shows different configurations of the exemplary pin, in accordance with some embodiments;

[0027]

[0026] FIG. 7A shows a schematic of interior components of an exemplary nanopore device, FIG. 7B shows an image of the loading module in a front-side view, FIG. 7C shows an image of the loading module in a top view, FIG. 7D shows an image of an exemplary manifold in a front view, FIG. 7E shows an image of an exemplary manifold in a top view, FIG. 7F shows images of a water reservoir in a side view and top view, FIG. 7G shows images of a KC1 reservoir in side view and top view, FIG. 7H shows images of a waste reservoir in side view and top view, FIG. 71 shows an image of an exemplary connecting mechanism between a reagent reservoir and a manifold, FIG. 7J shows a schematic of an exemplary chip, and FIG. 7K shows a schematic of a cartridge, in accordance with some embodiments;

[0028]

[0027] FIG. 8 shows an exemplary schematic for detection of an analyte, in accordance with some embodiments;

[0029]

[0028] FIG. 9 shows a computer system 901, in accordance with some embodiments;

[0030]

[0029] FIG. 10A shows an exemplary circuit model of the nanopore cell when no membrane or nanopore are assembled, FIG. 10B shows a heat map in wet test, in accordance with some embodiments, FIG. 10C shows an exemplary simplified circuit model of the nanopore cell when membrane is assembled (BLF), FIG. 10D shows a heat map of BLF, FIG. 10E shows an exemplary simplified circuit model of the nanopore cell when membrane and nanopore are assembled, FIG. 10F shows a heat map after nanopore insertion, FIG. 10G shows an illustration of CMEM2 and RCv7 value regions for wet, membrane, and nanopore, FIG. 10H shows an exemplary circuit of the sensor for detecting the one or more signals, FIG. 101 shows an exemplary plot of voltage signals during a measurement cycle, FIG. 10J shows raw voltage signal, FIG. 10K shows the frequency spectrum of the signal, and FIG. 10L shows the pre- processed signal after removing the high frequency signals, in accordance with some embodiments;

[0031]

[0030] FIG. 11A shows exemplary time-resolved impedance measurement traces for tag A, FIG. 11B shows time-resolved impedance measurement traces for Tag B, FIGS. 11C and 11D show detection of 2 level signal with one applied voltage at different read time values, FIG. HE shows raw voltage signal during a sample / analyte detection (scatter plot of raw data), FIG. HF shows the raw voltage signal of a baseline (e.g., no sample), FIG. 11G shows the residual signal after subtracting the baseline from the sample signal, FIG. 11H shows the pre-processed data at a selected tau value, and FIG. HI shows exemplary voltage signal curves for open nanopore and various tags, in accordance with some embodiments;

[0032]

[0031] FIG. 12A shows an exemplary circuit model of the sensor for detecting one or more signals, FIG. 12B shows exemplary simulated voltage levels of the circuit 1200, FIG. 12C shows expanded view of FIG. 12B, FIG. 12D shows a comparison of a bipolar CE scheme and a unipolar CE scheme, FIG. 12E shows an exemplary circuit model of the sensor for detecting one or more signals, FIG. 12F shows exemplary simulated voltage levels of the unipolar CE scheme, FIG. 12G shows expanded view of the simulated voltage levels of the unipolar CE scheme, FIG. 12H shows exemplary simulated voltage levels of the circuit 1230, FIG. 121 shows exemplary simulated voltage levels of the unipolar CE scheme with CE compensation, FIG. 12J shows exemplary simulated voltage levels of the unipolar CE scheme with CE compensation and shorter dark time, FIG. 12K shows exemplary simulated voltage levels of the unipolar CE scheme without CE compensation but with shorter dark time, FIG. 12L and 12M illustrate exemplary data showing measured signal for pore insertion, signal noise check, and sequencing, FIG. 12N and 120 illustrate exemplary data showing measured signal for pore insertion, signal noise check, and sequencing for different nanopore cells in the chip operated with the unipolar CE scheme, in accordance with some embodiments;

[0033]

[0032] FIG. 13A shows an exemplary workflow for analyzing a nucleic acid molecule, and FIG. 13B shows an exemplary process for preparing a single stranded circular nucleic acid molecule in accordance with some embodiments;

[0034]

[0033] FIG. 14 shows single stranded circular DNA (sscDNA) preparation process, in accordance with some embodiments;

[0035]

[0034] FIG. 15A shows an image of 1% agarose gel electrophoresis after fragmentation, FIG. 15B shows an image of 1% agarose gel electrophoresis after size selection, FIG. 15C shows the 1% agarose gel electrophoresis of the amplified DNA aliquots, and FIG. 15D shows the gel image after digestion, in accordance with some embodiments;

[0036]

[0035] FIG. 16A shows an exemplary IP address, FIG. 16B shows an exemplary window to start a new run, FIG. 16C shows an exemplary window to create a new run, FIG. 16D shows an image of cartridge insertion orientation, FIG. 16E shows an exemplary hardware connection screen, FIG. 16F shows an exemplary screen for creating a wet test, FIG. 16G shows an exemplary lipid formation screen, and FIG. 16H shows an exemplary nanopore insertion screen, in accordance with some embodiments;

[0037]

[0036] FIG. 17A shows an exemplary script for calibration the force and FIG. 17B shows an example of the .json file, in accordance with some embodiments;

[0038]

[0037] FIG. 18A shows an image of the chip 1812 covered with the flow cell 1813, FIG. 18B shows an image of the assembled plates, FIG. 18C shows an exemplary script, FIG. 18D shows an exemplary script for launching, FIG. 18E shows an exemplary screen for starting a new run, FIG. 18F shows an exemplary screen for inputting run information, FIG. 18G shows an exemplary screen for creating a run, FIG. 18H shows an exemplary window for inputting dry test parameters, FIG. 181 shows exemplary heat maps, FIG. 18J shows exemplary test result, FIG. 18K shows exemplary screen for bilayer formation, FIG. 18L shows an exemplary internal prefill screen, FIG. 18M shows exemplary heat map of the bilayer, FIG. 18N shows an exemplary sample port prime screen, FIG. 180 shows an exemplary complex insertion screen, FIG. 18P shows an exemplary heat map after the nanopore insertion, FIG. 18Q shows an exemplary screen for sequencing cycle, FIG. 18R shows an exemplary screen for CR reading configuration table, and FIG. 18S shows an exemplary screen for visualization filter configuration table, in accordance with some embodiments;

[0039]

[0038] FIG. 19A shows an exemplary screen for creating step change command, FIG. 19B shows an exemplary step change graph, and FIG. 19C shows an exemplary screen for generating STL plots, in accordance with some embodiments;

[0040]

[0039] FIG. 20 A shows an agarose gel image of PCR product, FIG. 20B shows an agarose gel image of sscDNA, and FIG. 20C shows an agarose gel image of RCA product, in accordance with some embodiments;

[0041]

[0040] FIG. 21A shows an exemplary schematic of sequencing a nucleic acid (e.g., sscDNA) with a nanopore device, FIG. 21B shows a sequencing workflow, FIG. 21C shows an image of a banner with command, FIG. 21D shows images of loading and loaded cartridge, FIG. 21E shows an image of a banner with command, FIG. 21F shows a schematic of cartridge, FIG. 21G shows an image of a banner with command, FIG. 21H shows a schematic of cartridge, FIG. 211 shows a schematic of cartridge, and FIG. 21 J shows an image of a banner with command, in accordance with some embodiments; and

[0041] FIG. 22A shows a biosensing workflow, FIG. 22B shows an exemplary interface illustrating the bilayer formation cycle status, FIG. 22C show exemplary screen interface illustrating real-time step change plots, and FIG. 22D shows an exemplary interface to set reading configuration, in accordance with some embodiments.

[0042] DETAILED DESCRIPTION

[0043]

[0042] While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be employed.

[0043] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0044]

[0044] Whenever the term “at most,” “up to,” “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0045]

[0045] As used in the 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 sequencing sensor” can include a plurality of sequencing sensors.

[0046]

[0046] The term “about” or “approximately,” as used interchangeably herein, generally refers to within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean 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 can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0047]

[0047] The term “binding unit” can mean a molecule capable of interacting (e.g., binding) one or more target molecules. In some cases, binding can be covalent and / or non-covalent (e.g., hydrogen bonding, hydrophobic interactions, etc.). In some cases, the interaction between the binding unit and the analyte(s) can be reversible or irreversible. In some cases, the binding unit can be configured to couple a tag (or probe) that is coupled to an analyte. In some cases, the analyte can be a biomolecule. In some cases, the analyte can be a cell or one or more components or derivatives thereof of a cell.

[0048]

[0048] The term “cell” can mean a biological cell or cell derivative. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, fems, clubmosses, hornworts, liverworts, mosses), an algal cell (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, or an artificial cell).

[0049]

[0049] The terms “nucleotide,” “nucleobase,” and “base,” as used interchangeably herein, can mean a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and deoxyribonucleoside triphosphates (dNTPs) such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. In some cases, the nucleotide comprises dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide can be unlabeled or detectably labeled. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides can include but are not limited fluorescein, 5- carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l- sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein- 15 -d ATP, Fluorescein- 12-dUTP, Tetramethyl- rodamine-6-dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein- 15 -2 '-d ATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY- TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein- 12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5- dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14-dATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin- 11-dUTP, biotin- 16-dUTP, biotin-20-dUTP).

[0050]

[0050] Naturally-occurring nucleotides guanine, cytosine, adenine, thymine, and uracil can be abbreviated as G, C, A, T, and U, respectively. A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e., A or G, or a variant thereof) or a pyrimidine (i.e., C, T or U, or a variant thereof). A subunit can enable individual nucleic acid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or uracil-counterparts thereof) to be resolved.

[0051]

[0051] The terms “polynucleotide,” “oligonucleotide,” “oligomer,” and “nucleic acid,” as used interchangeably herein, can mean a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in 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 fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three dimensional structure, and can perform any function. A polynucleotide can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some nonlimiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g. rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (miRNA), ribozymes, complementary DNA (cDNA, such as double-strand 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., fluorescence in situ hybridization (FISH) probes), and primers. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

[0052]

[0052] The term “gene” can mean a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term “gene” as used herein with reference to genomic DNA can include intervening, non-coding regions as well as regulatory regions and can include 5' and 3' ends. In some uses, the term can encompass the transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'- UTR), exons and introns. In some genes, the transcribed region may contain “open reading frames” that encode polypeptides. In some cases, a “gene” can comprise only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. The genes may not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. The term “gene” can not only include the transcribed sequences, but also non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can be an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can be an “exogenous gene” or a nonnative gene. A non-native gene can be a gene not normally found in the host organism but which is introduced into the host organism by gene transfer (e.g., transgene). A non-native gene can be a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and / or deletions (e.g., non-native sequence).

[0053]

[0053] The term “mutation” can mean a change in the sequence of nucleotides of a normally conserved nucleic acid sequence or in a sequence of amino acids of a normally conserved amino acid sequence resulting in the formation of a mutant as differentiated from the normal (unaltered) or wild type sequence. A position (e.g., relative to a gene, a nucleic acid, a polynucleotide, a protein, or a polypeptide) and sequence of the mutation can be undetermined prior to sequencing. Alternatively, a position (e.g., relative to a gene, a nucleic acid, a polynucleotide, a protein, or a polypeptide) and sequence of the mutation can be determined prior to sequencing, in which case the sequencing can be performed to detect a presence or absence of the mutation in the sample polynucleotide. A mutation can comprise a base-pair substitution (e.g., single nucleotide substitution) and a frame-shift mutation. The frame-shift mutation can require insertion or deletion of one to several nucleotide pairs. A mutation can comprise an amino acid substitution (e.g., single amino acid substitution) and a frame-shift mutation. The frame-shift mutation can require insertion or deletion of one to several amino acids.

[0054]

[0054] The term “probe” can mean a nucleotide or polynucleotide that is tagged with a maker (e.g., a fluorescent marker) useful for detecting or identifying its corresponding target nucleotide or polynucleotide in a hybridization reaction by hybridization with a corresponding target sequence. The terms “nucleotide probe, “nucleotide tag,” and “tagged nucleotide,” as used interchangeable herein, can mean a probe having a single nucleotide. The terms “polynucleotide probe, “polynucleotide tag,” and “tagged polynucleotide,” as used interchangeable herein, can mean a probe having polynucleotide. A polynucleotide probe can be tagged with at least one marker (e.g., one marker per each nucleotide of the polynucleotide probe). A probe can be hybridizable to one or more target nucleotides or polynucleotides. A polynucleotide probe can be entirely complementary to one or more target polynucleotides in a sample, or contain one or more nucleotides that are not complementary (i.e., a mismatch) to one or more nucleotides of the one or more target polynucleotides in the sample.

[0055]

[0055] The terms “complement,” “complements,” “complementary,” and “complementarity,” as used interchangeably herein, can mean a sequence that is complementary to and hybridizable to at least a portion of the given sequence. Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson- Crick pairing. Substantial or sufficient complementary can mean that a sequence in one strand is not completely and / or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the melting temperature (Tm) of hybridized strands, or by empirical determination of Tmby using routine methods.

[0056]

[0056] A sequence hybridized with at least a portion of a given nucleic acid may be referred to as the “complement” or “reverse-complement” of the given molecule if its sequence of bases over a given region is capable of complementarily binding those of its binding partner, such that, for example, A-T, A-U, G-C, and G-U base pairs are formed. In general, a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, such that hybridization to the second sequence or set of second sequences is preferred (e.g., thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art) to hybridization with non-target sequences during a hybridization reaction. In some cases, hybridizable sequences may share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, including at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence complementarity. The respective lengths can comprise a region of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more nucleotides. Sequence identity, such as for the purpose of assessing percent complementarity, can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm, the BLAST algorithm, or the Smith-Waterman algorithm.

[0057] Optimal alignment can be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[0057] The term “hybridization” can mean a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner according to base complementarity. The complex can comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of PCR, or the enzymatic cleavage of a polynucleotide by an endonuclease. A second sequence that is complementary to a first sequence can be referred to as the “complement” of the first sequence. The term “hybridizable,” as applied to a polynucleotide, generally refers to the ability of the polynucleotide to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues in a hybridization reaction.

[0058]

[0058] The term “stringent condition” can mean one or more hybridization conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with a target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions can be sequence-dependent, and can vary depending on a number of factors. In some cases, the longer the sequence, the higher the temperature at which the sequence can specifically hybridize to its target sequence.

[0059]

[0059] The term “label,” or “tag,” as used interchangeably herein, can mean a directly or indirectly detectable molecule that is conjugated directly or indirectly to a target compound or composition to be detected (e.g., a nucleotide molecule, a small molecule, a protein, a peptide, or a polypeptide). The tag can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate compound or composition which is detectable. In some cases, presence or absence of the tag can be detectable by measuring an electrochemical property (e.g., capacitance, impedance, etc.) of an electrochemical cell (e.g., a nanopore sensor) upon addition or removal of the tag, respectively. The tag can be suitable for small scale detection or more suitable for high-throughput screening. As such, non-limiting examples of the tag can include radioisotopes, fluorochromes, chemiluminescent compounds, bioluminescent compounds, dyes, polynucleotides, polypeptides (e.g., enzymes, fluorescent proteins, etc.), and non-polynucleotide / non-polypeptide polymers. The tag can be simply detected. Alternatively or in addition to, the tag can be quantified.

[0060]

[0060] The term “concatemer” can mean a long continuous DNA molecule that may contain multiple copies of the same DNA sequence linked in series formed by rolling circle amplification of a circular nucleic acid molecule. In some cases, concatemeric single strand may comprise at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, or more copies (repeat units) of a single sequence. In some cases, the single sequence may have a length of at least 10 bases, at least 20 bases, at least 50 bases, at least 100 bases, at least 500 bases, at least 1 kilobases (kb), at least 10 kb, at least 50 kb, at least 100 kb, or more. In some cases, the single sequence in each of copies (repeat units) may differ by no more than 5%, no more than 10%, no more than 15%, or no more than 20%.

[0061]

[0061] The term “polymerase” can mean an enzyme (e.g., natural or synthetic) capable of catalyzing a polymerization reaction. Examples of polymerases can include a nucleic acid polymerase (e.g., a DNA polymerase or an RNA polymerase), a transcriptase, and a ligase. A polymerase can be a polymerization enzyme. The term “DNA polymerase” can mean an enzyme capable of catalyzing a polymerization reaction of DNA.

[0062]

[0062] The term “sequencing” can mean a procedure for determining the order in which nucleotides or amino acids occur in a nucleotide sequence or an amino acid sequence. Methods of sequencing can comprise high-throughput sequencing, such as, for example, next-generation sequencing (NGS). Sequencing can be whole-genome sequencing or targeted sequencing. Sequencing can be single molecule sequencing or massively parallel sequencing. Nextgeneration sequencing methods can be useful in obtaining millions of sequences in a single run. In some cases, sequencing can be performed using one or more nanopore sequencing methods (e.g., sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-cleavage).

[0063]

[0063] The term “sequence identity” can mean an exact nucleotide-to-nucleotide or amino acid- to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Sequence identity can be approximately 50% to 100% and integer values therebetween. This disclosure may encompass sequences with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein.

[0064]

[0064] The term “nanopore” can mean a pore, channel, or passage formed or otherwise provided in a membrane. The membrane can be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed of a polymeric material such as a protein nanopore. The membrane can be a solid-state membrane (e.g., silicon substrate). The nanopore can be disposed adjacent or in proximity to a sensing circuit or an electrode coupled to a sensing circuit, such as, for example, a complementary metal-oxide semiconductor (CMOS) or field effect transistor (FET) circuit. The nanopore can be part of the sensing circuit. A nanopore can have a characteristic width or diameter, for example, on the order of about 0.1 nanometer (nm) to 1000 nm. A nanopore can be a biological nanopore, solid state nanopore, hybrid biological solid state nanopore, a variation thereof, or a combination thereof. Examples of the biological nanopore include, but are not limited to, OmpG from E. coli, sp., Salmonella sp., Shigella sp., and Pseudomonas sp., and alpha hemolysin (a-hemolysin) from S. aureus sp., MspA from M. smegmatis sp, a functional variant thereof, or a combination thereof. Examples of the solid state nanopore include, but are not limited to, silicon nitride, silicon oxide, graphene, molybdenum sulfide, a functional variant thereof, or a combination thereof. The solid state nanopore can be fabricated by high-energy beam manufacturing, imprinting (e.g., nanoimprinting), laser ablation, chemical etching, plasma etching (e.g., oxygen plasma etching), etc. In some cases, a nanopore can be used for detecting a molecule or a group of molecules (e.g., polynucleotides, polypeptides, small molecules). In some cases, the detecting can comprise detecting a presence of the molecule or group of molecules. In some cases, the detecting can comprise determining a concentration of the molecule or group of molecules. In some cases, a nanopore can be used to sequence, e.g., obtain a sequence information, of a molecule (e.g., a polynucleotide or a polypeptide). In some cases, sequencing can comprise forward sequencing and / or reverse sequencing.

[0065]

[0065] The terms “sequence variant” and “sequencing variant,” as used interchangeably herein, can mean any variation in sequence relative to one or more reference sequences. Typically, a sequence variant occurs with a lower frequency than a reference sequence for a given population of individuals for whom the reference sequence is provided. For example, a particular bacterial genus can have a consensus reference sequence for the 16S rRNA gene, but individual species within that genus can have one or more sequence variants within the gene or a portion of a gene that are useful in identifying that species in a population of bacteria. As a further example, sequences for multiple individuals of the same species or multiple sequencing reads for the same individual can produce a consensus sequence when optimally aligned, and sequence variants with respect to that consensus can be used to identify mutants in the population indicative of dangerous contamination. In general, a “consensus sequence” refers to a nucleotide sequence that reflects the most common choice of base at each position in the sequence where the series of related nucleic acids has been subjected to intensive mathematical and / or sequence analysis, such as optimal sequence alignment according to any of a variety of sequence alignment algorithms. A reference sequence can be a single reference sequence, such as a predetermined genomic sequence of a single individual. A reference sequence can be a consensus sequence formed by aligning multiple sequences, such as predetermined genomic sequences of multiple individuals serving as a reference population, or multiple sequencing reads of polynucleotides from the same individual. A reference sequence can be a consensus sequence formed by optimally aligning the sequences from a sample under analysis, such that a sequence variant represents a variation relative to corresponding sequences in the same sample. A sequence variant can occur with a low frequency in the population (also referred to as a “rare” sequence variant). For example, a sequence variant can occur with a frequency of or less than 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.1%, 0.075%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.001%, or lower. A sequence variant can occur with a frequency of or less than 0.1%.

[0066] A sequence variant can be any variation with respect to a reference sequence. A sequence variation can consist of a change in, insertion of, or deletion of a single nucleotide, or of a plurality of nucleotides such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. Where a sequence variant comprises two or more nucleotide differences, the nucleotides that are different can be contiguous with one another or discontinuous. Examples of types of sequence variants include single nucleotide polymorphisms (SNP), deletion / insertion polymorphisms (DIP), copy number variants (CNV), short tandem repeats (STR), simple sequence repeats (SSR), variable number of tandem repeats (VNTR), amplified fragment length polymorphisms (AFLP), retrotransposon-based insertion polymorphisms, sequence specific amplified polymorphism, and differences in epigenetic marks that can be detected as sequence variants (e.g., methylation differences).

[0066]

[0067] The term “nanopore sequencing complex” can mean a nanopore linked or coupled to an enzyme, e.g., a polymerase, which in turn is associated with a polymer, e.g., a polynucleotide template. The nanopore sequencing complex can be positioned in a membrane, e.g., a lipid bilayer, where it functions to identify polymer components, e.g., nucleotides or amino acids.

[0068] The term “nanopore sequencing” or “nanopore-based sequencing” can mean a method that determines the sequence of a polynucleotide or a polypeptide with the aid of a nanopore. In some cases, the sequence of the polynucleotide or polypeptide can be determined in a templatedependent manner.

[0067]

[0069] The term “barcode” can mean a predetermined nucleic acid sequence that allows some feature of a polynucleotide with which the barcode is associated to (e.g., a polynucleotide comprising at least a portion of the barcode or a polynucleotide having complementarity to at least a portion of the barcode) be identified. In some examples, the feature of the polynucleotide to be identified can be the sample from which the polynucleotide is derived. A barcode can be at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides in length. A barcode can be at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides in length. A barcode associated with polynucleotides from a first sample can be different (e.g., different sequences and / or different lengths) than the barcode associated with polynucleotides from a second sample that is different than the first sample. In such a case, identification of the barcode in the respective polynucleotides can help identify the sample source of one or more of the polynucleotides. Thus, different samples with different barcodes can be analyzed (e.g., sequenced) together (e.g., in the batch), and separated during analysis based at least in part on the barcode. In some examples, a barcode can be identified accurately even after mutation, insertion, or deletion of one or more nucleotides in the barcode sequence (e.g., the mutation, insertion, or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides). A plurality of polynucleotides from the same sample can have the same barcode. Alternatively, the plurality of polynucleotides from the same sample can have different barcodes. A first barcode can differ from a second barcode by at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions. A plurality of barcodes can be represented in a pool of samples, each sample comprising polynucleotides comprising one or more barcodes that differ from the barcodes contained in the polynucleotides derived from the other samples in the pool. Samples of polynucleotides comprising one or more barcodes can be pooled based on the barcode sequences to which they are joined, such that all four of the nucleotide bases A, G, C, and T are approximately evenly represented at one or more positions along each barcode in the pool (such as at 1, 2, 3, 4, 5, 6, 7, 8, or more positions, or all positions of the barcode). In some examples, the methods of the present disclosure can comprise identifying the sample from which a target polynucleotide is derived based on a barcode sequence to which the target polynucleotide is joined. The barcode can comprise a nucleic acid sequence that when joined to a target polynucleotide can serve as an identifier of the sample from which the target polynucleotide was derived. In an example, an oligonucleotide primer (e.g., an amplification primer) can comprise one or more barcodes. In another example, a nucleic acid molecule can be coupled (e.g., ligated) to an adaptor nucleic acid (e.g., for circularization), and the adaptor nucleic acid can comprise one or more barcodes.

[0068]

[0070] The term “real-time” or “real time,” as used interchangeably herein, can mean an event (e.g., an operation, a process, a measurement, a detection, etc.) that is performed almost immediately after or within a short period of time after another event (e.g., addition of a nucleobase, generation of a growing strand, etc.), such as within at least 0.0001 millisecond (ms), at least 0.0005 ms, at least 0.001 ms, at least 0.005 ms, at least 0.01 ms, at least 0.05 ms, at least 0.1 ms, at least 0.5 ms, at least 1 ms, at least 5 ms, at least 0.01 seconds, at least 0.05 seconds, at least 0.1 seconds, at least 0.5 seconds, at least 1 second, or more. In some cases, a real time event may be performed almost immediately after or within a short period of time after another event, such as within at most 1 second, at most 0.5 seconds, at most 0.1 seconds, at most 0.05 seconds, at most 0.01 seconds, at most 5 ms, at most 1 ms, at most 0.5 ms, at most 0.1 ms, at most 0.05 ms, at most 0.01 ms, at most 0.005 ms, at most 0.001 ms, at most 0.0005 ms, at most 0.0001 ms, or less.

[0069]

[0071] The term “sample” can mean any sample that can include one or more constituents (e.g., nucleic acid molecules) for processing or analysis. The sample can be a biological sample. The sample can be a cellular or tissue sample. The sample can be a cell-free sample, such as blood (e.g., whole blood), plasma, serum, sweat, saliva, or urine. The sample can be obtained in vivo or cultured in vitro.

[0070]

[0072] The term “subject” can mean an individual or entity from which a sample is derived, such as, for example, a vertebrate (e.g., a mammal, such as a human) or an invertebrate. A mammal can be a murine, simian, human, farm animal (e.g., cow, goat, pig, or chicken), or a pet (e.g., cat or dog). The subject can be a plant. The subject can be a patient. The subject can be asymptomatic with respect to a disease (e.g., cancer). Alternatively, the subject can be symptomatic with respect to the disease.

[0071]

[0073] The term “substituted” can mean a functional group as described herein such as an alkyl, or a hydrocarbyl, in which at least one bond to a hydrogen atom contained therein is replaced by a bond to non- hydrogen or non-carbon atom, provided that normal valencies are maintained and that the substitution(s) result(s) in a stable compound. Substituted groups can include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Non-limiting examples of substituents include the functional groups described herein, and for example, N, e.g., so as to form -CN.

[0072] Overview

[0073]

[0074] The methods, systems, devices, and / or kits disclosed herein may be used to may be used to detect or analyze a molecule or an analyte (e.g., a nucleic acid molecule, a protein, or a small molecule) with low cost, high speed, and high accuracy. The analyte may be processed or transformed to a library of molecules (e.g., library molecules) prior to the detection or analysis. In some cases, the detection or analysis may comprise identifying an analyte. In some cases, the detection or analysis may comprise quantifying an analyte. In some cases, the detection or analysis may comprise obtaining a sequence information of an analyte (e.g., a nucleic acid molecule, a polynucleotide, a protein, a polypeptide, or a peptide).

[0074] Nanopore Systems and Methods Thereof

[0075]

[0075] In an aspect, the present disclosure provides a nanopore system for analyzing an analyte. In some cases, the nanopore system may comprise a fluidic chamber. In some cases, the nanopore system may comprise an analytical cell (or a nanopore cell). In some cases, the nanopore system may comprise a plurality of nanopore cells. In some cases, the nanopore system may comprise a plurality of fluidic chambers. In some cases, a nanopore cell may comprise a fluidic chamber.

[0076]

[0076] In some cases, the fluidic chamber may comprise a membrane. In some cases, the membrane may comprise a lipid bilayer. In some cases, the membrane may comprise one or more types of lipids. In some cases, the membrane may comprise one or more fatty acids, phospholipids, triglycerides, or glycolipids, or any combination thereof. In some cases, the membrane may comprise one or more phospholipids. In some cases, the membrane may comprise phosphocholine (e.g., l,2-di-O-phytanyl-sn-glycero-3 -phosphocholine (DoPhPC) or l,2-di-phytanoyl-sn-glycero-3 -phosphocholine (DPhPC)), phosphatidylcholine (e.g., palmitoyl- oleoyl-phosphatidylcholine, dipalmitoylphosphatidylcholine), phosphatidylglycerol (1,2-di-O- phytanyl-sn-glycerol), a phosphatidylethanolamine (e.g., l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-lactosyl, 1 ,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine-N- [methoxy(polyethylene glycol)-350] (DPPE-PEG350), DPPE-PEG550, DPPE-PEG750, DPPE- PEG1000, DPPE-PEG1500, or DPPE-PEG2000), lysophosphatidylcholine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, sphingomyelin, or di-oleoyl-phosphatidyl-methylester, or combinations thereof. In some cases, the membrane may comprise 1,2-di-O-phytanyl-sn- glycero-3 -phosphocholine (DoPhPC). In some cases, the membrane may comprise 1,2-di- phytanoyl-sn-glycero-3-phosphocholine (DPhPC). In some cases, the membrane may have a thickness (e.g., from the top surface to the bottom surface) of at least about 3 nanometers (nm), at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, or at least about 8 nm. In some cases, the thickness may be at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, or at most about 4 nm. In some cases, the thickness may be from about 3 nm to about 4 nm, from about 3 nm to about 5 nm, from about 3 nm to about 6 nm, from about 3 nm to about 7 nm, from about 3 nm to about 8 nm, from about 3 nm to about 9 nm, from about 3 nm to about 10 nm, from about 4 nm to about 5 nm, from about 4 nm to about 6 nm, from about 4 nm to about 7 nm, from about 4 nm to about 8 nm, from about 4 nm to about 9 nm, from about 4 nm to about 10 nm, from about 5 nm to about 6 nm, from about 5 nm to about 7 nm, from about 5 nm to about 8 nm, from about 5 nm to about 9 nm, from about 5 nm to about 10 nm, from about 6 nm to about 7 nm, from about 6 nm to about 8 nm, from about 6 nm to about 9 nm, from about 6 nm to about 10 nm, from about 7 nm to about 8 nm, from about 7 nm to about 9 nm, from about 7 nm to about 10 nm, from about 8 nm to about 9 nm, from about 8 nm to about 10 nm, or from about 9 nm to about 10 nm. In some cases, the membrane may separate the fluidic chamber to a first side and a second side. In some cases, the first side of the fluidic chamber may be a cis chamber, and the second side of the fluidic chamber may be a trans chamber. In some cases, the first side of the fluidic chamber may be a trans chamber, and the second side of the fluidic chamber may be a cis chamber. In some cases, the nanopore cell may comprise a nanopore disposed within the membrane. In some cases, the nanopore cell may comprise a sensor. In some cases, the sensor may comprise one or more electrodes. In some cases, the sensor may comprise a working electrode (or sensing electrode) and / or a counter electrode. In some cases, the working electrode may be disposed in the cis chamber and the counter electrode may be disposed in the trans chamber. In some cases, the working electrode may be disposed in the trans chamber and the counter electrode may be disposed in the cis chamber. In some cases, the sensor may comprise a reference electrode.

[0077] In some cases, the nanopore may comprise a solid state nanopore. In some cases, the nanopore may comprise a biological nanopore. In some cases, the nanopore may comprise a protein nanopore. In some cases, the nanopore may comprise one or more proteins or polypeptides. In some cases, the one or more proteins or polypeptides may comprise alpha hemolysin (a-hemolysin), mycobacterium smegmatis porin A (MspA), aerolysin, or vacuolating cytotoxin A (VacA), or any combination thereof. In some cases, the nanopore may comprise one or more proteins or polypeptides that are wild type proteins or polypeptides. In some cases, the nanopore may comprise one or more linkers coupled to at least a portion of the nanopore. In some cases, the one or more linkers may comprise one or more peptides or polypeptides. In some cases, the one or more linkers may be configured to couple at least a portion of the analyte. In some cases, the one or more linkers may be configured to couple a polymerase. In some cases, the one or more linkers may comprise a spytag peptide configured to couple a polymerase. In some cases, the nanopore may comprise one or more monomers. The nanopore may comprise one or more modified monomers. A modified monomer may be a monomer coupled to a linker. The one or more monomers may comprise at least one mutant monomer or at least one wild type monomer. For example, the nanopore may comprise (1) six wild type proteins or polypeptides and / or (2) one wild type protein coupled to a linker or one polypeptide coupled to a linker. As another example, the nanopore may comprise (1) six wild type a-hemolysin proteins or polypeptides and (2) one a-hemolysin protein coupled to a spytag or one a-hemolysin polypeptide coupled to a spytag. In some cases, the a-hemolysin protein may comprise a sequence of:

[0077] MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAG QYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTG DDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNP VYGNQLFMKTRNGSMKAAENFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVR DDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN (SEQ ID NO: 1).

[0078]

[0078] In some cases, the a-hemolysin protein may comprise at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1. In some cases, the a-hemolysin protein may be coupled to a linker (e.g., GGSSGGSSGG). In some cases, the a-hemolysin protein may be coupled to a TEV protease (e.g., ENLYFQG). In some cases, the a-hemolysin protein may be coupled to a StrepTag (e.g., WSHPQFEK). In some cases, the a-hemolysin protein complex (e.g., a-hemolysin protein and the linker or tags) may comprise a sequence of:

[0079] MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAG QYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTG DDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNP VYGNQLFMKTRNGSMKAAENFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVR DDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNGGSSGGSSGGEALFFQG|WSHPQ FEK| (SEQ ID NO: 2, underlined is a linker sequence, italicized is a TEV protease sequence, and boxed is a StrepTag sequence).

[0080]

[0079] In some cases, the a-hemolysin protein may be coupled to a linker (e.g., GGSSGGSSGG, SEQ ID NO: 12). In some cases, the a-hemolysin protein may be coupled to a SpyTag (AHIVMVDAYKPTK, SEQ ID NO: 3). In some cases, the a-hemolysin protein may be coupled to a his-tag (e.g., HHHHHH) linked to the SpyTag through an additional linker (e.g., KG). In some cases, the a-hemolysin protein complex (e.g., a-hemolysin protein and the linker or tags) may have a sequence of:

[0081] MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAG QYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTG DDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNP VYGNQLFMKTRNGSMKAAENFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVR DDYOLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNGGSSGGSSGG|AHIVMVDAYKP TK| FGHHHHHH (SEQ ID NO: 4, underlined is a linker sequence, italicized is an additional linker sequence, boxed is a SpyTag sequence, and bolded is a his-tag sequence).

[0082]

[0080] FIG. 1 shows an exemplary nanopore cell 100 of a nanopore system. The nanopore cell 100 may comprise a fluidic chamber that may be separated by a membrane 102 to a first side 111 (or a cis chamber) and a second side 112 (or a trans chamber). 103 shows a surface of a chip that the fluidic chamber is built upon. A nanopore 101 may be embedded in the membrane 102. The nanopore 101 may comprise a channel 104 through the nanopore.

[0083]

[0081] FIG. 2 shows a top view 210 of an exemplary nanopore cell 200 and a side view 220 of the exemplary nanopore cell 200. The nanopore cell 200 may comprise a membrane 202 which may separate the fluidic chamber to a cis chamber 211 and a trans chamber 212. The trans chamber 212 may have a wall 213. A nanopore 201 may be embedded in the membrane 202. The nanopore 201 may comprise a channel 204 through the membrane. In the top view 210, 205 shows the outer perimeter of the nanopore, 214 shows the outer perimeter of the cis chamber, 215 shows the outer perimeter of the trans chamber, and 213 shows the annulus area of the wall 213. In the side view 220, 223 shows a height of the trans chamber, 222 shows a diameter of the trans chamber, and 221 shows a diameter of the cis chamber.

[0082] In some cases, the channel of the nanopore (e.g., 104 or 204) may have a diameter from about 0.5 nm to about 1 nm, from about 0.5 nm to about 1.5 nm, from about 0.5 nm to about 2 nm, from about 1 nm to about 1.5 nm, from about 1 nm to about 2 nm, or from about 1.5 nm to about 2 nm. In some cases, the diameter of the channel of the nanopore may be at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, or at least about 2 nm. In some cases, the diameter of the channel of the nanopore may be at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or at most about 0.5 nm. In some cases, the nanopore may have diameter at the biggest cross section (e.g., 205) from about 2 nm to about 5 nm, from about 2 nm to about 10 nm, from about 2 nm to about 12 nm, from about 5 nm to about 10 nm, from about 5 nm to about 12 nm, or from about 10 nm to about 12 nm. In some cases, the diameter at the biggest cross section of the nanopore may be at least about 2 nm, at least about 5 nm, at least about 10 nm, or at least about 12 nm. In some cases, the diameter at the biggest cross section of the nanopore may be at most about 12 nm, at most about 10 nm, at most about 5 nm, or at most about 2 nm. In some cases, a height of the nanopore (e.g., from the highest point to the lowest point of the nanopore) may be from about 2 nm to about 5 nm, from about 2 nm to about 10 nm, from about 2 nm to about 12 nm, from about 5 nm to about 10 nm, from about 5 nm to about 12 nm, or from about 10 nm to about 12 nm. In some cases, the height of the nanopore may be at least about 2 nm, at least about 5 nm, at least about 10 nm, or at least about 12 nm. In some cases, the height of the nanopore may be at most about 12 nm, at most about 10 nm, at most about 5 nm, or at most about 2 nm.

[0083] In some cases, the nanopore system may comprise a plurality of fluidic chambers. In some cases, the nanopore system may comprise at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000, at least 500000, at least 600000, at least 700000, at least 800000, at least 900000, at least 1000000, or more nanopore cells. In some cases, the nanopore system may comprise at most 100, at most 500, at most 1000, at most 5000, at most 10000, at most 50000, at most 60000, at most 70000, at most 80000, at most 90000, at most 100000, at most 500000, at most 600000, at most 700000, at most 800000, at most 900000, or at most 1000000 nanopore cells. In some cases, the nanopore system may comprise at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000, at least 500000, at least 600000, at least 700000, at least 800000, at least 900000, at least 1000000, or more fluidic chambers. In some cases, the nanopore system may comprise at most 100, at most 500, at most 1000, at most 5000, at most 10000, at most 50000, at most 60000, at most 70000, at most 80000, at most 90000, at most 100000, at most 500000, at most 600000, at most 700000, at most 800000, at most 900000, or at most 1000000 fluidic chambers. In some cases, a fluidic chamber of the plurality of fluidic chambers may comprise a membrane. In some cases, the fluidic chamber may comprise a nanopore disposed in the membrane. In some cases, the fluidic chamber may comprise one nanopore. In some cases, the fluidic chamber may comprise two or more nanopores. In some cases, each fluidic chamber of the plurality of fluidic chambers may be coupled to a working electrode. In some cases, each fluidic chamber of the plurality of fluidic chambers may be coupled to a counter electrode. In some cases, two or more fluidic chambers of the plurality of fluidic chambers may be coupled to a counter electrode. In some cases, all fluidic chambers of the plurality of fluidic chambers may be coupled to a counter electrode.

[0084]

[0084] In some cases, the nanopore system may comprise a nanopore array. In some cases, the nanopore system may comprise a chip. In some cases, the chip may comprise a nanopore array. In some cases, the chip may comprise at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000, at least 500000, at least 600000, at least 700000, at least 800000, at least 900000, at least 1000000, or more nanopore cells. In some cases, the chip may comprise at most 100, at most 500, at most 1000, at most 5000, at most 10000, at most 50000, at most 60000, at most 70000, at most 80000, at most 90000, at most 100000, at most 500000, at most 600000, at most 700000, at most 800000, at most 900000, or at most 1000000 nanopore cells. In some cases, the chip may comprise at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000, at least 500000, at least 600000, at least 700000, at least 800000, at least 900000, at least 1000000, or more fluidic chambers. In some cases, the chip may comprise at most 100, at most 500, at most 1000, at most 5000, at most 10000, at most 50000, at most 60000, at most 70000, at most 80000, at most 90000, at most 100000, at most 500000, at most 600000, at most 700000, at most 800000, at most 900000, or at most 1000000 fluidic chambers. In some cases, the chip may comprise at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000, at least 500000, at least 600000, at least 700000, at least 800000, at least 900000, at least 1000000, or more nanopores. In some cases, the chip may comprise at most 100, at most 500, at most 1000, at most 5000, at most 10000, at most 50000, at most 60000, at most 70000, at most 80000, at most 90000, at most 100000, at most 500000, at most 600000, at most 700000, at most 800000, at most 900000, or at most 1000000 nanopores. In some cases, the chip may comprise from 10000 to 20000, from 10000 to 30000, from 10000 to 40000, from 10000 to 50000, from 10000 to 60000, from 10000 to 80000, from 10000 to 100000, from 20000 to 30000, from 20000 to 40000, from 20000 to 50000, from 20000 to 60000, from 20000 to 80000, from 20000 to 100000, from 30000 to 40000, from 30000 to 50000, from 30000 to 60000, from 30000 to 80000, from 30000 to 100000, from 40000 to 50000, from 40000 to 60000, from 40000 to 80000, from 40000 to 100000, from 50000 to 60000, from 50000 to 80000, from 50000 to 100000, from 60000 to 80000, or from 60000 to 100000 nanopore cells. In some cases, the chip may comprise from 10000 to 20000, from 10000 to 30000, from 10000 to 40000, from 10000 to 50000, from 10000 to 60000, from 10000 to 80000, from 10000 to 100000, from 20000 to 30000, from 20000 to 40000, from 20000 to 50000, from 20000 to 60000, from 20000 to 80000, from 20000 to 100000, from 30000 to 40000, from 30000 to 50000, from 30000 to 60000, from 30000 to 80000, from 30000 to 100000, from 40000 to 50000, from 40000 to 60000, from 40000 to 80000, from 40000 to 100000, from 50000 to 60000, from 50000 to 80000, from 50000 to 100000, from 60000 to 80000, or from 60000 to 100000 nanopores.

[0085]

[0085] In some cases, the nanopore cells and / or the nanopores may be independently addressable on the chip. An individually addressable nanopore can be individually readable. An individually addressable nanopore can be individually writable. An individually addressable nanopore can be individually readable and individually writable. The system can include one or more computer processors for facilitating sample preparation and various operations of the disclosure, such as polynucleotide sequencing. The processor can be coupled to nanopore system. In some cases, the chip may comprise a substrate. In some cases, the nanopore cells may be formed on the substrate. In some cases, the substrate may comprise silicon. In some cases, the substrate may comprise a silicon complementary metal-oxide semiconductor (Si CMOS). In some cases, the substrate may comprise a dielectric layer. In some cases, the dielectric layer may comprise a dielectric material (e.g., oxides, nitrides, or glass). In some cases, the substrate may comprise a 300 mm wafer. In some cases, the chip may be made by a 65 nm process on the wafer.

[0086]

[0086] FIG. 3 shows an exemplary nanopore cell 300. The nanopore cell 300 may be formed on a substrate disclosed herein. The nanopore cell 300 may comprise a fluidic chamber. The fluidic chamber may be separated by a membrane 302 to a cis chamber 311 and a trans chamber 312. A nanopore 301 may be embedded in the membrane 302. The nanopore cell may comprise a counter electrode (CE) 322 disposed in the cis chamber. The nanopore system may comprise a sensing electrode or working electrode (WE) 321 disposed in the trans chamber. In some cases, the sensing electrode or WE may comprise a metal electrode. In some cases, the sensing electrode or WE may comprise gold, copper, silver, platinum, titanium nitride, or graphite, or combinations thereof. The trans chamber may comprise one or more walls (e.g., 306). In some cases, the walls may comprise a dielectric material. The dielectric material may comprise an oxide, glass, a nitride (e.g., silicon mononitride (SiN)), or a polymer (e.g., polyimide), or combinations thereof. The trans chamber may be coupled to a cell circuit (or circuit) 323. The cell circuit 323 may be disposed at the bottom of the trans chamber. In some cases, the cell circuit may be configured to control electrical stimulation. In some cases, the cell circuit may be configured to collect signals from the sensor. In some cases, the cell circuit may be configured to process the signals. In some cases, the cell circuit may be formed on the substrate. In some cases, the cell circuit may comprise one or more signal generators. In some cases, the cell circuit may comprise one or more amplifiers. In some cases, the cell circuit may comprise one or more transistors. In some cases, the cell circuit may comprise one or more analog-to digital (ADC) converters. In some cases, the cell circuit may comprise one or more integrators. In some cases, the cell circuit may comprise one or more noise filters (e.g., low pass filters). In some cases, the cell circuit may comprise one or more processors. In some cases, the cell circuit may be coupled to one or more processors. In some cases, the cell circuit may comprise one or more memory devices.

[0087]

[0087] In some cases, the chip may comprise a plurality of nanopore blocks. In some cases, a nanopore block of the plurality of nanopore blocks may comprise a plurality of nanopore cells and / or fluidic chambers. In some cases, a nanopore block of the plurality of nanopore blocks may comprise a plurality of nanopores. In some cases, the chip may comprise n times m (n*m) nanopore blocks, where n can be from 1 to 100 and m can be from 1 to 100. In some cases, n can be same as m. in some cases, n can be different from m. In some cases, the chip may comprise at least 2, at least 4, at least 8, at least 9, at least 10, at least 16, at least 20, at least 24, at least 25, at least 36, at least 40, at least 48, at least 49, at least 64, at least 81, at least 100, or more nanopore blocks.

[0088]

[0088] In some cases, a nanopore block of the plurality of nanopore blocks may comprise at least 10, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, or more nanopore cells and / or fluidic chambers. In some cases, a nanopore block of the plurality of nanopore blocks may comprise at least 10, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, at least 100000, or more nanopores.

[0089]

[0089] FIGS. 4A and 4B show an exemplary chip. The chip may comprise a nanopore array. The nanopore array may comprise a plurality of nanopore blocks (e.g., 401). The chip in FIGS. 4A and 4B has 16 nanopore blocks. The chip may comprise any number of nanopore blocks disclosed herein. FIG. 4C shows an exploded view of the nanopore block 401. The nanopore block 401 may comprise a plurality of nanopores (e.g., 402). In some cases, the nanopore block 401 may comprise 65,536 nanopores.

[0090]

[0090] In some cases, the nanopore system may be manufactured to comprise the membrane and the nanopore. In some cases, the nanopore system may be manufactured to comprise the membrane but without a nanopore. In some cases, the nanopore system may be manufactured to comprise empty fluidic chambers. In some cases, the nanopore system may be manufactured without a membrane or nanopore. In some cases, the membrane and nanopore may be formed by a user. In some cases, the membrane and / or nanopore may be formed prior to use. In some cases, the membrane and / or nanopore may be formed on site. In some cases, the membrane may be formed in a fluidic chamber by loading a solution comprising a plurality of lipids (e.g., phospholipids) to the fluidic chamber. In some cases, after loading the plurality of lipids (e.g., phospholipids) to the fluidic chamber, the plurality of lipids (e.g., phospholipids) may assemble to form the lipid (e.g., phospholipid) bilayer. In some cases, the lipid may comprise a phospholipid. In some cases, the phospholipid may comprise diphytanoylphosphatidylcholine. In some cases, the nanopore may be deposited or inserted in the membrane. In some cases, depositing or inserting the nanopore in the membrane may comprise flowing a solution comprising one or more nanopores to the fluidic chamber. In some cases, a nanopore of the one or more nanopores may diffuse into the membrane. In some cases, an electrical field may be applied during the nanopore deposition. In some cases, an electrical field may not be applied during the nanopore deposition.

[0091]

[0091] In some cases, a first fluidic chamber of the plurality of fluidic chambers may be in fluidic communication with a second fluidic chamber of the plurality of fluidic chambers. In some cases, the nanopore system may comprise a flow cell. In some cases, the flow cell may be configured to flow or deliver a fluid to the nanopore system. In some cases, the flow cell may be configured to flow a fluid to the plurality of fluidic chambers of the nanopore system. In some cases, the fluid may comprise a buffer solution. In some cases, the fluid may comprise a plurality of lipids (e.g., phospholipids). In some cases, the fluid comprising the plurality of lipids may be configured to form a membrane in the fluidic chamber. In some cases, the fluid may comprise a plurality of nanopores. In some cases, the fluid may comprise a plurality of nanopore- polymerase complexes. In some cases, the fluid may comprise a plurality of nanopore- polymerase-analyte complexes (or triplexes). In some cases, the fluid may comprise a plurality of nanopore-polymerase-nucleic acid complexes (or triplexes). In some cases, the fluid may comprise an analyte. In some cases, the fluid may comprise one or more analytes. In some cases, the fluid may comprise a plurality of nucleotides. In some cases, the fluid may comprise a wash solution. In some cases, the fluid may comprise a priming solution. In some cases, the fluid may comprise an electrolyte solution. In some cases, the electrolyte solution may comprise one or more electrolytes. In some cases, the electrolyte may comprise one or more cations (e.g., sodium, calcium, magnesium, lithium, strontium, manganese, or potassium cations). In some cases, the electrolyte may comprise one or more anions (e.g., chloride, bromide, glutamate, or acetate anions). In some cases, the electrolyte may comprise potassium chloride. In some cases, the electrolyte solution may comprise a concentration of the one or more electrolytes of at least about 50 mM, at least about 100 mM, at least about 200 mM, at least about 300 mM, at least about 400 mM, or at least about 500 mM. In some cases, the electrolyte solution may comprise a concentration of the one or more electrolytes of at most about 500 mM, at most about 400 mM, at most about 300 mM, at most about 200 mM, at most about 100 mM, or at most about 50 mM. In some cases, the fluid may be distributed to the plurality of fluidic chambers.

[0092]

[0092] FIG. 5A shows a perspective view of a chip on board (COB) 500. The COB 500 may comprise a chip 501 formed on a substrate 502. The chip 501 may comprise a nanopore array. FIG. 5B shows a top view of the chip 501. In some cases, the chip may have a length of at least about 20 mm, at least about 30 mm, at least about 40 mm, at least about 50 mm, or at least about 60 mm. In some cases, the chip may have a length of at most about 60 mm, at most about 50 mm, at most about 40 mm, at most about 30 mm, or at most about 20 mm. In some cases, the chip may have a length of at least about 20 mm, at least about 30 mm, at least about 40 mm, at least about 50 mm, or at least about 60 mm. In some cases, the chip may have a length of at most about 60 mm, at most about 50 mm, at most about 40 mm, at most about 30 mm, or at most about 20 mm. In some cases, the chip may have a height of at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 7 mm, or at least about 10 mm. In some cases, the chip may have a height of at most about 10 mm, at most about 7 mm, at most about 5 mm, or at most about 4 mm. The chip 501 may comprise a plurality of nanopore blocks (e.g., blocks 0-15). A block of the plurality of nanopore blocks may comprise a plurality of nanopore cells. A block of the plurality of nanopore blocks may comprise a plurality of fluidic chambers. FIG. 5C shows a flow direction in the chip 501. The arrows show the flow direction. Using numbered nanopore blocks of FIG. 5B as example, the fluid may be added in block 12, and flow to blocks 8, 4, and 0. The fluid may be flown to the next row to blocks 1, 5, 9, and 13. The fluid may be flown to the next row to blocks 14, 10, 6, and 2. The fluid may be flown to the next row to blocks 3, 7, 11, and 15. The fluid may be flown out of the chip from block 15.

[0093]

[0093] FIG. 5D shows an exploded two-dimensional view of a flow cell. The flow cell 510 may comprise a plurality of flow channels (e.g., 511). The arrows in FIG. 5D shows the flow direction in the flow cell. FIG. 5E shows a three-dimensional view of the flow cell. The arrows in FIG. 5E shows the flow direction in the flow cell. In some cases, the flow in the flow cell may be in a single plane. In some cases, the flow in the flow cell may not be in a single plane. In some cases, the flow in a channel of the flow cell may be in a same plane. In some cases, the same plane may be substantially horizontal (e.g., horizontal or having an angle of less than 10° from the horizontal plane). In some cases, the flow from one channel to an adjacent channel may be in a different plane. The fluid may be added to the flow cell in a substantially vertical (e.g., vertical or having an angle of less than 10° from the vertical direction) direction. When the fluid is flown in the channel 521, the flow may be substantially horizontal. The channel 521 and channel 522 may be connected by a bypass channel. The bypass channel may comprise a first portion that is substantially vertical, a second portion that is substantially horizontal, and a third portion that is substantially vertical. At the connection of channel 521 and 522, the flow may flow substantially vertically (e.g., flowing upwards) in the first portion of the bypass channel. The flow may then flow substantially horizontally in the second portion of the bypass channel. The flow may then flow substantially vertically (e.g., flowing downwards) in the third portion of the bypass channel. The flow may then flow to the channel 522. FIG. 5F shows the flow directions of the fluid in the channels of the flow cell.

[0094]

[0094] In some cases, the chip and flow cell may be assembled to a cartridge. FIG. 6A shows a top view of an exemplary cartridge. The nanopore array is not visible in this top view. The cartridge may comprise a plurality of inlets. The cartridge may comprise a plurality of connections. The cartridge may comprise one or more loading ports or inlets (e.g., 621, 622, 623, and 625) for loading one or more fluidic samples (e.g., lipid solution, nanopore solution, tag solution, and / or buffer solution). In some cases, the cartridge may comprise a lipid inlet (e.g., 623) configured to flow a lipid solution (e.g., a solution comprising lipids for membrane formation) in. In some cases, the cartridge may comprise a system inlet (e.g., 625) configured to flow in buffer solutions (e.g., priming buffer, wash buffer, etc.). In some cases, the cartridge may comprise a sample inlet (e.g., 622). In some cases, the sample inlet may be configured to flow in a nanopore solution (e.g., a solution comprising nanopore proteins for nanopore formation in the fluidic chamber). In some cases, the sample inlet may be configured to flow in an analyte solution (e.g., a solution comprising the analyte). In some cases, the cartridge may comprise a tag inlet (e.g., 621). The tag inlet may be configured to flow in a nucleotide solution. The nucleotide solution may comprise a plurality of nucleotides configured to contact with a primer molecule or an analyte molecule in the detection of a nucleic acid molecule. In some cases, the cartridge may comprise a system outlet (e.g., 624). In some cases, the system outlet may be configured to flow out a fluid from the chip. The cartridge may comprise an outlet valve (e.g.,

[0095] 640) to control the fluidic flow exiting the cartridge. In some cases, the cartridge may comprise a plurality of channels (e.g., 626) and one or more valves (e.g., 627, 628, 629) configured to facilitate the flow in the flow cell and chip. In some cases, the one or more valves (e.g., 627, 628, 629) may be configured to control the flow of a fluidic sample from a loading port or inlet to the flow cell. The one or more valves may comprise one or more actuators to control or regulate the opening and closing of the valves. The cartridge may comprise a bypass valve (e.g.,

[0096] 641). A valve (e.g., 627) may be closed when a sample is loaded to an inlet (e.g., 623). The sample may flow through the channel 626. When the valve 627 is open, the sample may flow through the channel 642 to an inlet 643. The sample may flow to the flow cell via the inlet 643 and be distributed to the fluidic chambers of the chip via the flow cell. The valve 627 may be controlled or regulated by an actuator. In some cases, the actuator may comprise a mechanical actuator. The chip may comprise a plurality of nanopore cells. In some cases, each nanopore cell may comprise a working electrode or sensing electrode. In some cases, the chip may comprise a counter electrode. In some cases, the counter electrode may be coupled to each of the nanopore cells. 644 shows the wire of the counter electrode.

[0097]

[0095] FIG. 6B shows an exploded view of the cartridge components. The cartridge 610 may comprise a chip on board (COB) 608 as disclosed herein. The cartridge 610 may comprise a flow cell 607 adjacent to the COB 608. The flow cell may be configured to regulate or control the fluidic flow in the chip. The flow cell may comprise a plurality of channels (e.g., 612) for flowing and distributing fluid samples. The cartridge 610 may comprise a layer plate 606. The layer plate 606 may comprise one or more s-channels (e.g., 611) for connecting fluidic flow in the flow cell. In some cases, a s-channel may connect one channel of the flow cell to the next channel. In some cases, the s-channel may comprise one or more channels that are perpendicular to the channel of the flow cell. The s-channel may comprise one or more additional channels to connect the one or more channels. The layer plate 606 may comprise counter electrode with metallic traces by physical vapor deposition (PVD) (e.g., silver trace). The counter electrode may be deposited on the bottom layer of the layer plate 606. The counter electrode may comprise silver or gold. The cartridge 610 may comprise a lamination pressure sensitive adhesive (PSA) 605 adjacent to the layer plate 606. In some cases, the lamination PSA 605 may be configured to provide adhesion and / or sealing between the layer plate 606 and polycarbonate plate 604. The cartridge 610 may comprise a polycarbonate plate 604 adjacent to the lamination PSA 605. The polycarbonate plate 604 may comprise counter electrode contact path. The polycarbonate plate 604 may be configured for channel and sample containment. The polycarbonate plate 604 may have a thickness from about 0.2 mm to about 1 mm (e.g., 0.5 mm). The polycarbonate plate 604 may have a thickness of at least about 0.2 mm, at least about 0.5 mm, or at least about 1 mm. The polycarbonate plate 604 may have a thickness of at most about 1 mm, at most about 0.5 mm, or at most about 1 mm. The cartridge 610 may comprise an additional lamination PSA 603 adjacent to the polycarbonate plate 604. The additional lamination PSA 603 may be configured to provide adhesion and / or sealing for adjacent components (e.g., 602 and 604). The cartridge 610 may comprise an additional layer plate (or fluid delivery plate) 602. The additional layer plate 602 may comprise delivery channels for loading or collecting fluids. The additional layer plate may be made of polycarbonate. The additional layer plate may have a thickness of about 3 mm to about 5 mm (e.g., 4 mm). The additional layer plate may have a thickness of at least about 3 mm, at least about 4 mm, or at least about 5 mm. The additional layer plate may have a thickness of at most about 5 mm, at most about 4 mm, or at most about 3 mm. The cartridge 610 may comprise a plurality of valves and O-rings. In some cases, the plurality of valves may be configured to control the distribution of a fluidic sample from a sample port or inlet to the flow cell and / or fluidic chamber. In some cases, the plurality of valves may comprise or be coupled to one or more actuators to regulate the valve (e.g., open or close the valve). The O-rings may be configured as fluidic port (e.g., inlet and / or outlet) attachments. The O-rings may be made of silicone material. The valves may be made of silicone material. The valves may comprise a lamination PSA 609 to provide sealing to connections or cavities. The lamination PSA may be configured as an adhesive for O-rings. In some cases, prior to assembly of the cartridge, silicone surface primer can be applied to the O- rings, the valves, and / or the lamination PSAs. In some cases, the silicone surface primer may improve quality of seal between the different components. In some cases, the layer plate 606 and the additional layer plate 602 may be computer numerical control (CNC) machined from a stock polycarbonate. Surfaces of the assembly components may be left both clear and flat. In some cases, the additional lamination PSA 603, the polycarbonate plate 604, and / or the additional lamination PSA 605 may be laser cut. After machining, a silver or gold trace (CE) may be deposited (e.g., by PVD) to the layer plate 606. FIG. 6C shows an image of an exemplary assembled cartridge. The cartridge 630 may comprise one or more loading ports or inlets (e.g., 631, 632, and 633) for loading one or more fluidic samples (e.g., lipid solution, nanopore solution, tag solution, and / or buffer solution). The one or more loading ports or inlets may be configured to load a testing solution (e.g., solution comprising the analyte), a bilayer forming solution (e.g., lipid solution), a tag solution (e.g., solution comprising one or more nucleotides coupled to one or more tags), a nanopore solution, a nanopore-polymerase solution, or a nanopore-polymerase-nucleic acid solution. In some cases, the cartridge 630 may comprise a lipid inlet (e.g., 633) configured to flow a lipid solution in. In some cases, the fluid delivery plate may comprise a system inlet (e.g., 637) configured to flow in buffer solutions (e.g., priming buffer, wash buffer, etc.). In some cases, the fluid delivery plate may comprise a sample inlet (e.g., 632). In some cases, the sample inlet may be configured to flow in a nanopore solution. In some cases, the sample inlet may be configured to flow in an analyte solution. In some cases, the cartridge may comprise a tag inlet (e.g., 631). The tag inlet may be configured to flow in a nucleotide solution. The nucleotide solution may comprise a plurality of nucleotides configured to contact with a primer molecule or an analyte molecule in the detection of a nucleic acid molecule. The cartridge 630 may comprise one or more valves (e.g., 634, 635, and 636) for controlling the flow of the fluidic sample from a loading port to the flow cell. The one or more valves may comprise or be coupled to one or more actuators to control or regulate the opening and closing of the valves. The cartridge 630 may comprise a bypass valve 639. In some cases, the bypass valve can be used for solution recirculation to improve mixing or washing with minimum volume of solution used. The cartridge 630 may comprise an outlet 640. The fluid delivery plate 630 may comprise an outlet valve 638 to control the fluidic flow exiting the cartridge. A valve (e.g., 634) may be closed when a sample is loaded to a loading port (e.g., 631), and after the sample is loaded to the loading port, the valve may be controlled to flow the sample to the flow cell. In some cases, the lamination PSA and / or additional lamination PSA may comprise a plastic coated with one or more layers of acrylic pressure sensitive adhesives. In some cases, the lamination PSA and / or additional lamination PSA may further comprise one or more layers of release liners (e.g., polyester release liner). In some cases, the plastic may have a thickness of at least about 20 micrometers (pm), at least about 25 pm, or at least about 30 pm. In some cases, the plastic may have a thickness of at most about 30 pm, at most about 25 pm, or at most about 20 pm. In some cases, the acrylic pressure sensitive adhesive may have a thickness of at least about 20 pm, at least about 25 pm, or at least about 30 pm. In some cases, the acrylic pressure sensitive adhesive may have a thickness of at most about 30 pm, at most about 25 pm, or at most about 20 pm. In some cases, the release liner may have a thickness of at least about 20 pm, at least about 25 pm, or at least about 30 pm. In some cases, the release liner may have a thickness of at most about 30 pm, at most about 25 pm, or at most about 20 pm.

[0098]

[0096] FIG. 6D shows a perspective view of the assembled cartridge. FIG. 6E shows side views of the assembled cartridge. In some cases, the cartridge may have a length of at least about 30 millimeters (mm), at least about 40 mm, at least about 50 mm, at least about 60 mm, at least about 70 mm, or at least about 80 mm. In some cases, the cartridge may have a length of at most about 80 mm, at most about 70 mm, at most about 60 mm, at most about 50 mm, at most about 40 mm, or at most about 30 mm. In some cases, the cartridge may have a width of at least about 30 mm, at least about 40 mm, at least about 50 mm, at least about 60 mm, at least about 70 mm, or at least about 80 mm. In some cases, the cartridge may have a width of at most about 80 mm, at most about 70 mm, at most about 60 mm, at most about 50 mm, at most about 40 mm, or at most about 30 mm. In some cases, the cartridge may have a height of at least about 4 mm, at least about 5 mm, at least about 7 mm, at least about 10 mm, at least about 12 mm, or at least about 15 mm. In some cases, the cartridge may have a height of at most about 15 mm, at most about 12 mm, at most about 10 mm, at most about 7 mm, at most about 5 mm, or at most about 4 mm. In some cases, the cartridge may have a length of about 57 mm. In some cases, the cartridge may have a width of about 40 mm. In some cases, the cartridge may have a height of about 12 mm. In some cases, the cartridge may have a height of about 8.66 mm. In some cases, the top compartment (comprising the additional layer plate 602, the additional lamination PSA 603, and the polycarbonate plate 604 may have a height of about 4.58 mm.

[0099]

[0097] In some cases, the cartridge may comprise a plurality of pins configured to provide electrical connections between the electrode and a signal generator. FIG. 6F shows an exemplary pin. FIG. 6G shows different configurations of the exemplary pin. The pin may have a maximum working height (1) from about 3 mm to about 6.5 mm. The pin may have a normal working height (2) from about 2.9 mm to about 6.2 mm. The pin may have a minimal working height (3) from about 2.7 mm to about 6.0 mm.

[0100]

[0098] After the cartridge assembly, a post-production inspection may be conducted to ensure correct assembly and quality of pieces. In some cases, the post-production inspection may comprise a visual examination of cosmetic features. In some cases, the post-production inspection may be performed on-site. In some cases, the post-production inspection may comprise an inspection of the structure. The structure inspection may comprise determining if the shape of pieces matches the design files to ensure no top-level deviations or machining errors. In some cases, the post-production inspection may comprise an assembly inspection. The assembly inspection may comprise determining if an orientation of each layer matches that in the design files. The assembly inspection may comprise determining if the engraved channel features map out an identical path. In some cases, the post-production inspection may comprise a color inspection to ensure fluidic piece material visually matches the material specification (i.e., clear polycarbonate) and the surfaces of stock material are clear as instructed. In some cases, the post-production inspection may comprise a visual particle contamination inspection to ensure no foreign material or discolored spots are observed on or in the assembly. In some cases, the postproduction inspection may comprise an adhesive overflow inspection to ensure no additional adhesive around the outer bounds of the fluidic piece and no adhesive seeping into features in the assembly. In some cases, the post-production inspection may comprise a surface scratches inspection to ensure no deep scratches on the surface of the assembly. Minor scratches may be investigated to insure they are superficial. In some cases, the post-production inspection may comprise determining if the surface is clean. In some cases, the post-production inspection may comprise assessing the quality of trace visually (i.e., no visual cracks, smudges or discoloration). In some cases, the assembled cartridges may be packaged independently in plastic.

[0101]

[0099] In some cases, the cartridge may be loaded into a nanopore device. FIG. 7A shows a schematic of interior components of an exemplary nanopore device. The nanopore device 700 may comprise one or more reagent reservoirs (e.g., 701). The one or more reagent reservoirs may be configured to store and supply one or more reagent solutions to the cartridge. In some cases, the one or more reagent reservoirs may comprise water, electrolyte solutions, buffer solutions, lipid solutions, nanopore solutions (or nanopore-polymerase solutions or nanopore- polymerase-analyte solutions), or sample solutions (e.g., analyte containing solutions). In some cases, the nanopore device may comprise one or more reagent reservoirs for washing buffer. In some cases, the nanopore device may comprise one or more reagent reservoirs for lipid solvent. In some cases, the nanopore device may comprise one or more reagent reservoirs for salt solutions. In some cases, the nanopore device may comprise five or more reagent reservoirs. In some cases, the nanopore device may comprise seven or more reagent reservoirs. The nanopore device may comprise one or more manifolds (e.g., 702). In some cases, the one or more manifolds may comprise one or more valves for controlling flow and distribution of fluidic samples from the one or more reagent reservoirs to the cartridge. The one or more manifolds may be fluidically connect to the one or more reservoirs. The one or more manifolds may be fluidically connect to and a flow control module. The nanopore device may comprise a flow control module (e.g., 703). The flow control module may comprise one or more flow control pumps (e.g., syringe pumps). The flow control module may comprise one or more motors. The flow control module may be fluidly coupled to one or more reagent reservoirs. The flow control module may be fluidly coupled to one or more manifolds. In some cases, the flow control unit may be fluidly coupled to one or more sample preparation units. In some cases, the one or more sample preparation units may be configured to prepare one or more reagent solutions. The nanopore device may comprise a mother board. The mother board may be configured to control one or more electronic components of the nanopore device. The nanopore device may comprise one or more sensors (e.g., 704). The one or more sensors may connect with the cartridge. The one or more sensors may be configured to detect one or more signals associated with at least a portion of the analyte. The nanopore device may comprise a loading module (e.g., 705). The loading module may be configured to load the cartridge. A cartridge comprising a nanopore chip and flow cell may be loaded to the loading module. The loading module may comprise one or more actuators configured to independently regulate one or more valves in the cartridge. The one or more valves may control the fluidic flow from the loading ports to the flow cell and / or nanopore chip. The nanopore device may comprise one or more waste reservoirs (e.g., 706). The one or more waste reservoirs may be configured to collect one or more waste solutions exiting the cartridge.

[0102]

[0100] In some cases, the nanopore device may be fully automated. In some cases, the nanopore device may be partially automated. In some cases, the nanopore device may be semi-automated.

[0103]

[0101] FIG. 7B shows an image of the loading module in a front-side view. FIG. 7C shows an image of the loading module in a top view. The loading module 710 may comprise one or more actuators (e.g., 711) configured to independently regulate one or more valves in the cartridge. In some cases, the loading module 710 may comprise five actuators to regulate five valves (e.g., 634, 635, 636, 638, and 639 of FIG. 6C) in the cartridge. The loading module may comprise one or more plates configured to hold a cartridge. In some cases, the loading module may comprise a top plate 712 and a bottom plate 713. The top plate and the bottom plate may be configured to hold the cartridge in between the top plate and the bottom plate. The bottom plate may comprise one or more holes (e.g., 717) for one or more nails or screws (e.g., 718) to tighten the top plate and the bottom plate. The bottom plate may comprise one or more spacers (e.g., 714, 715 and 716) configured to fix the distance between the top plate and the bottom plate. With the spacer, the force can be applied evenly with a fixed distance between top and bottom plates. In some cases, the spacer may be made of stainless steel. In some cases, the spacer may be made of plastics. In some cases, the spacer may comprise a plastic coated with one or more layers of acrylic pressure sensitive adhesives. In some cases, the spacer may further comprise one or more layers of release liners (e.g., polyester release liner). In some cases, the plastic may have a thickness of at least about 20 pm, at least about 25 pm, or at least about 30 pm. In some cases, the plastic may have a thickness of at most about 30 pm, at most about 25 pm, or at most about 20 pm. In some cases, the acrylic pressure sensitive adhesive may have a thickness of at least about 20 pm, at least about 25 pm, or at least about 30 pm. In some cases, the acrylic pressure sensitive adhesive may have a thickness of at most about 30 pm, at most about 25 pm, or at most about 20 pm. In some cases, the release liner may have a thickness of at least about 20 pm, at least about 25 pm, or at least about 30 pm. In some cases, the release liner may have a thickness of at most about 30 pm, at most about 25 pm, or at most about 20 pm. In some cases, the spacer may have a length of at least about 20 mm, at least about 25 mm, at least about 30 mm, at least about 40 mm, at least about 50 mm, at least about 60 mm, or at least about 70 mm. In some cases, the spacer may have a length of at most about 70 mm, at most about 60 mm, at most about 50 mm, at most about 40 mm, at most about 30 mm, or at most about 20 mm. In some cases, the spacer may have a width of at least about 2 mm, at least about 5 mm, at least about 7.5 mm, or at least about 10 mm. In some cases, the spacer may have a width of at most about 10 mm, at most about 7.5 mm, at most about 5 mm, or at most about 2 mm. In some cases, the spacer may have a height of at least about 0.5 mm, at least about 1 mm, at least about 1.5 mm, or at least about 2 mm. In some cases, the spacer may have a height of at most about 2 mm, at most about 1.5 mm, at most about 1 mm, or at most about 0.5 mm. In some cases, a spacer (e.g., 714) may have a length of 55 mm, a width of 7.5 mm, and a height of 2 mm. In some cases, a spacer (e.g., 715) may have a length of 25.5 mm, a width of 7.5 mm, and a height of 2 mm.

[0102] FIG. 7D shows an image of an exemplary manifold in a front view. FIG. 7E shows an image of an exemplary manifold in a top view. FIG. 7F shows images of a water reservoir in a side view and top view. FIG. 7G shows images of a KC1 reservoir in a side view and top view. FIG. 7H shows images of a waste reservoir in side view and top view. The nanopore device may comprise one or more loader motors. The nanopore device may comprise one or more syringe pumps. The syringe pump may be configured to control a flow rate of a fluidic sample. The nanopore device may further comprise one or more blower fans. In some cases, the blower fans may be embedded on the bottom panel. In some cases, the blower fans may be configured to blow cooler air into the nanopore system and expel hotter air out of the nanopore system. In some cases, the blower fans may be configured to cool the nanopore system. The nanopore device may further comprise one or more temperature control units. The temperature control units may be configured to control a temperature of the reagents, the chip, and / or the cartridge.

[0104]

[0103] FIG. 71 shows an image of an exemplary connecting mechanism between a reagent reservoir and a manifold. 731 shows an exemplary reagent reservoir. 732 shows an exemplary connecting mechanism. The connecting mechanism may comprise an assembly of quick connect (e.g., no need to open bottle) between the reservoir and the manifold. The connecting mechanism may comprise a metal tube (or needle) to attach to a flex liquid tube. A connecting mechanism may comprise a head and a needle. The tip of the needle may comprise a conical shape to ensure fit of the needle to the manifold.

[0105]

[0104] FIG. 7J shows a schematic of an exemplary chip. The chip 750 may comprise a plurality of bond pads (e.g., 754). In some cases, the chip may comprise 204 bond pads. The pond pad may comprise metal wires. The pond pad may be configured to connect Si chip to printed circuit boards (PCBs). Bond pads on chip are too small to contact mechanically without high contact resistance. The bond pad may have a length of at least about 50 pm, at least about 100 pm, or at least about 150 pm. The bond pad may have a length of at most about 150 pm, at most about 100 pm, or at most about 50 pm. The bond pad may have a width of at least about 50 pm, at least about 100 pm, or at least about 150 pm. The bond pad may have a width of at most about 150 pm, at most about 100 pm, or at most about 50 pm. The bond pad may be spaced from an adjacent bond pad by at least about 50 pm, at least about 100 pm, or at least about 150 pm. The bond pad may be spaced from an adjacent bond pad by at most about 150 pm, at most about 100 pm, or at most about 50 pm. In some cases, the bond pad may have a length of 100 pm, a width of 100 pm, and a distance of 100 pm from the adjacent bond pad. The chip may comprise a plurality of printed circuit boards (PCBs). The PCB may be connected to a fluidic chamber. The PCB may be connected to a working electrode. The PCB may have a width of at least about 0.05 mm, at least about 0.1 mm, at least about 0.15 mm, or at least about 0.2 mm. The PCB may have a width of at most about 0.2 mm, at most about 0.15 mm, at most about 0.1 mm, or at most about 0.05 mm. The PCB may have a length of at least about 5 mm, at least about 10 mm, at least about 15 mm, at least about 20 mm, or at least about 40 mm. The PCB may have a length of at most about 40 mm, at most about 20 mm, at most about 15 mm, at most about 10 mm, or at most about 5 mm. The PCB may have a space (e.g., distance between adjacent PCBs) of at least about 0.01 mm, at least about 0.02 mm, at least about 0.05 mm, or at least about 0.1 mm. The PCB may have a space of at most about 0.1 mm, at most about 0.05 mm, at most about 0.02 mm, or at most about 0.01 mm. The PCB may have a solder mask opening of at least about 0.1 mm, at least about 0.2 mm, at least about 0.5 mm, or at least about 1 mm. The PCB may have a solder mask opening of at most about 1 mm, at most about 0.5 mm, at most about 0.2 mm, or at most about 0.1 mm. In some cases, the PCB may have a width of 0.005”, a space of 0.003”, and a solder mask opening of 0.02’ ’ . Solder mask opening on PCB may be configured to connect the chip to PCB. Metal pattern on PCB may be covered by a dielectric material and then holes may be opened in the dielectric material to provide electrical contact. In some cases, at least a portion of the chip may be covered by an epoxy (e.g., 751). In some cases, wires connecting bond pads on chip to PCB may be fragile. In some cases, the epoxy deposited over wires may be configured to protect the wires. The epoxy may cover chip edge and PCB. The epoxy may have an inner keep out distance 755 (e.g., inner perimeter of the epoxy from the bond pad). The inner keep out distance may be from about 0.1 mm to about 0.6 mm (e.g., 0.3 mm). The epoxy may have an outer keep out distance 753 (e.g., outer perimeter of the epoxy from the bond pad). The outer keep out distance may be from about 0.1 mm to about 1 mm (e.g., 0.7 mm). The epoxy may have a height of at least about 0.5 mm, at least about 1 mm, at least about 1.5 mm, or at least about 2 mm. The epoxy may have a height of at most about 2 mm, at most about 1.5 mm, at most about 1 mm, or at most about 0.5 mm. In some cases, the epoxy may have a height of about 1.2 mm.

[0106]

[0105] FIG. 7K shows a schematic of a cartridge. The chip may be formed on a COB substrate, hl shows a height of flow cell above chip. h2 shows a height of silicon chip. h3 shows a thickness of chip adhesive to PCB. h4 shows a total height of flow cell. h5 shows a recess into flow cell to accommodate epoxy bead. h6 shows a width of the recess.

[0107]

[0106] In some cases, the nanopore system may comprise a sensor. In some cases, the sensor may be configured to detect one or more signals. In some cases, the one or more signals may comprise one or more voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof. In some cases, the one or more signals may be indicative of an impedance or change thereof in the sensor when at least a portion of an analyte is bound by or in proximity to at least a portion of the nanopore system. In some cases, the one or more signals may be indicative of a resistance or change thereof in the sensor when at least a portion of an analyte is bound by or in proximity to at least a portion of the nanopore system. In some cases, the one or more signals may be indicative of a capacitance or change thereof in the sensor when at least a portion of an analyte is bound by or in proximity to at least a portion of the nanopore system. In some cases, the one or more signals may be indicative of a conductance or change thereof in the sensor when at least a portion of an analyte is bound by or in proximity to at least a portion of the nanopore system. In some cases, a conductance and / or resistance of the nanopore may be measured via a current passing through the nanopore.

[0108]

[0107] In some cases, the one or more signals can be usable or used to analyze or identify the at least the portion of the analyte. In some cases, the impedance or change thereof can be usable or used to analyze or identify the at least the portion of the analyte. In some cases, the resistance or change thereof can be usable or used to analyze or identify the at least the portion of the analyte. In some cases, the capacitance or change thereof can be usable or used to analyze or identify the at least the portion of the analyte. In some cases, the conductance or change thereof can be usable or used to analyze or identify the at least the portion of the analyte.

[0109]

[0108] In some cases, the nanopore system can comprise at least one of the sensors disclosed herein. In some cases, the nanopore system can comprise at least 1, at least 2, at least 3, at least

[0110] 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000 or more sensors. The system can comprise at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or less sensor(s).

[0111]

[0109] In some cases, the one or more signals can be a single measurement. In some cases, the one or more signals can be multiple measurements or a plurality of measurements. In some cases, the multiple measurements may comprise at least 1, at least 2, at least 3, at least 4, at least

[0112] 5, at least 6, at least 7, at least 8, at least 9, or at least 10 measurements. In some cases, the multiple measurements may comprise at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, or at most 2 measurements. In some cases, the one or more signals can be a median or average of a plurality of measurements.

[0113]

[0110] In some cases, when detecting the one or more signals in the sensor, at least a portion of the analyte can be coupled to a binding moiety of the nanopore system. In some cases, the binding moiety can be configured to couple at least a portion of the analyte. In some cases, the nanopore system disclosed herein can comprise at least one binding moiety. In some cases, the nanopore system can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, or more binding moi eties. In some cases, the nanopore system can comprise at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or less binding moieties. In some cases, a binding moiety can comprise a small molecule, a nucleotide, a polynucleotide, a peptide, a polypeptide, a polymer, or a protein, or a combination thereof. In some cases, the binding moiety can comprise an antibody or a fragment thereof. In some cases, the binding moiety can comprise an antigen or a fragment thereof. In some cases, the binding moiety may be specific to at least a portion of an analyte. In some cases, the binding moiety may comprise a polymerase. In some cases, the binding moiety can be coupled to at least a portion of the membrane of the nanopore system. In some cases, the binding moiety can be coupled to at least a portion of the nanopore of the nanopore system. In some cases, the binding moiety can be coupled to at least a portion of a working electrode (or sensing electrode) of the nanopore system. In some cases, the binding moiety can be coupled to at least a portion of the membrane, the nanopore, or the working electrode via a non-covalent binding. In some cases, the non- covalent binding may comprise an ionic interaction, a hydrogen bonding, and / or a Van der Waals interaction. In some cases, the binding moiety can be coupled to at least a portion of the membrane, the nanopore, or the working electrode via a covalent binding. In some cases, the covalent binding can comprise an azide, alkyl, alkene, biotin, carboxyl, amine, tosyl, epoxy, sulfhydryl, ester, hydrazone, or heterobifunctional linkers, or combinations thereof.

[0114] [I l l] In some cases, the working electrode and the counter electrode may be in electrical communication with one another. In some cases, the working electrode and / or the counter electrode may comprise a dielectric material. In some cases, the dielectric material may increase the accuracy of the detection and / or reduce the noise in the detection. In some cases, the dielectric material may increase the lifetime of the working electrode and / or the counter electrode. In some cases, the dielectric material may cover a first portion of a surface of the working electrode and / or the counter electrode. In some cases, the working electrode and / or the counter electrode may comprise a conducting material coupled to the working electrode and / or the counter electrode. In some cases, the conducting material may cover a second portion of the surface of the working electrode and / or the counter electrode. In some cases, the conducting material may be configured to transfer an electric signal and / or conduct an electron or ions between (i) the working electrode and / or the counter electrode and (ii) an analyte, a solution, or an additional component of the nanopore system (e.g., a binding unit). In some cases, the working electrode and / or the counter electrode may comprise a binding unit coupled to the conducting material. In some cases, the binding unit may be configured to couple at least a portion of the analyte. In some cases, the conducting material can be a bond (e.g., a chemical bond) or can comprise a linking unit (e.g., a nanorod, a peptide, a small molecule, etc.) of any desired dimension (e.g., length, cross-sectional diameter or area, volume, etc.). In some cases, the binding unit can be directly coupled to the working and / or the counter electrode. In some cases, the binding unit can be coupled to at least a portion of the dielectric material that is coupled to the working electrode and / or the counter electrode.

[0115]

[0112] The first portion of the working electrode and / or counter electrode that is covered by the dielectric material can be at least 50 percent (%) of the surface of the working electrode and / or counter electrode. In some cases, the first portion of the working electrode and / or counter electrode can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more of the surface of the working electrode and / or counter electrode. In some cases, the first portion of the working electrode and / or counter electrode can be at most 100%, at most 95%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, or less of the surface of the working electrode and / or counter electrode.

[0116]

[0113] The second portion of the working electrode and / or counter electrode can be at most 50% of the surface of the working electrode and / or counter electrode. In some cases, the second portion of the working electrode and / or counter electrode can be at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or less of the surface of the working electrode and / or counter electrode. In some cases, the second portion of the working electrode and / or counter electrode can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more of the surface of the working electrode and / or counter electrode.

[0117]

[0114] An average cross-sectional dimension (or a surface area that is coupled to the dielectric material and the conducting material) of the working electrode can be no more than 100-fold greater than an average size of the analyte. In some cases, the average cross-sectional dimension of the working electrode can be at most 100-fold, at most 90-fold, at most 80-fold, at most 70- fold, at most 60-fold, at most 50-fold, at most 40-fold, at most 30-fold, at most 25-fold, at most 20-fold, at most 15-fold, at most 10-fold, at most 9-fold, at most 8-fold, at most 7-fold, at most

[0118] 6-fold, at most 5-fold, at most 4-fold, at most 3-fold, at most 2-fold, at most 1-fold, at most 0.5- fold, or at most 0.1 -fold greater than the average size of the analyte. In some cases, the average cross-sectional dimension of the working electrode can be at least 0.1-fold, at least 0.5-fold, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least

[0119] 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25- fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold greater than the average size of the analyte. In some cases, the average cross-sectional dimension of the working electrode can be no more than at least 0.1 nanometers (nm), at least 0.5 nm, at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 10 nm, at least 50 nm, at least 100 nm, at least 500 nm, at least 1,000 nm, at least 5,000 nm, at least 10,000 nm, or more than the average size of the analyte. In some cases, the average cross-sectional dimension of the working electrode can be at most 10,000 nm, at most 5,000 nm, at most 1,000 nm, at most 500 nm, at most 100 nm, at most 50 nm, at most 10 nm, at most 5 nm, at most 4 nm, at most 3 nm, at most 2 nm, at most 1 nm, at most 0.5 nm, at most 0.1 nm, or less than the average size of the analyte.

[0120]

[0115] In some cases, the average cross-sectional dimension of the working electrode can be smaller than the average size of the analyte. In some cases, the average cross-sectional dimension of the working electrode can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% smaller than the average size of the analyte. In some cases, the average cross-sectional dimension of the working electrode can be at most 50%, at most 40%, at most 30%, at most 20%, at most 15%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1% smaller than the average size of the analyte.

[0121]

[0116] The area of the second portion of the surface of the working electrode may be no more than 500 Angstrom squared (A2), no more than 100 A2, no more than 50 A2, no more than 10 A2, no more than 9 A2, no more than 8 A2, no more than 7 A2, no more than 6 A2, no more than 5 A2, no more than 4 A2, no more than 3 A2, no more than 2 A2, no more than 1 A2, or less. In some cases, a cross-sectional dimension or the diameter of the second portion of the surface of the working electrode can be approximately equal to a diameter (e.g., twice that of the Van der Waals radius) of an atom of the conducting material.

[0122]

[0117] In some cases, the dielectric material can be a solid layer (e.g., a solid metal or semiconducting material) or a self-assembled monolayer (SAM). In some cases, the conducting material can be a single molecule (e.g., a single conducting polymeric chain). In some cases, one or more features of atomic force microscopy (AFM) (e.g., a piezoelectric cantilever probe of the AFM) may be used to couple the single molecule to a specific (or random) position within the surface of the working electrode. In some cases, the conducting material can be a plurality of molecules (e.g., a plurality of identical and / or different conducting polymeric chains). In some cases, the conducting material can comprise at least 1, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, at least 100,000, or more molecules. In some cases, the conducting material can comprise at most 100,000, at most 50,000, at most 10,000, at most 5,000, at most 1,000, at most 500, at most 100, at most 50, at most 10, at most 5, or at most 1 molecule.

[0123]

[0118] In some cases, a substrate can be bound to the binding unit (e.g., a nuclease or a variant thereof) of the sensor of the present disclosure, and the binding unit or an additional element (e.g., an additional enzyme) can be configured to cleave at least a portion of the substrate. The sensor can be configured to (i) detect a difference in the one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) in the sensor following such cleavage, and / or (ii) identify what has been cleaved off based on analyzing the one or more signals.

[0124]

[0119] In some cases, the binding unit can be an enzyme, an antibody, an aptamer, a non- biological material (e.g., a synthetic polymer), or a functional fragment thereof, or a functional variant thereof, or a combination thereof. In some cases, an enzyme can comprise a polymerase, a nuclease (e.g., double-stranded nuclease), nickase, transcriptional activator, transcriptional repressor, nucleic acid methylation enzyme, nucleic acid demethylation enzyme, and / or recombinase. In some cases, the antibody can be a whole antibody or antigen-binding fragment thereof, such as an scFv, a Fab fragment, a VHH domain, or a VH domain of a heavy-chain only antibody. In some cases, the antibody can be mono-specific or multi-specific (e.g., bi-specific, tri-specific, etc.). In some cases, the antibody can be mono-valent or multi-valent (e.g., bi-valent, tri -valent, etc.).

[0125]

[0120] In some cases, the binding unit can be directly coupled to the conducting material (e.g., covalently or non-covalently attached to the conducting material). In some cases, the binding unit can be indirectly coupled to the working electrode (e.g., via a linker that binds the conducting material on one side and the binding unit on the other side). In such cases, the linker can also be a conducting linker as to minimize interference of the sensing capabilities of the sensor. Alternatively, the linker may not be conducting.

[0126]

[0121] A thickness of the dielectric material that is coupled to at least a portion of a surface of a working electrode can be about at least about 0.1 nm, at least about 0.5 nm, at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm, at least about 500 nm, at least about 600 nm, at least about 700 nm, at least about 800 nm, at least about 900 nm, at least about 1 pm, at least about 2 pm, at least about 3 pm, at least about 4 pm, at least about 5 pm, at least about 6 pm, at least about 7 pm, at least about 8 pm, at least about 9 pm, at least about 10 pm, at least about 20 pm, at least about 30 pm, at least about 40 pm, at least about 50 pm, at least about 60 pm, at least about 70 pm, at least about 80 pm, at least about 90 pm, at least about 100 pm, at least about 200 pm, at least about 300 pm, at least about 400 pm, at least about 500 pm, at least about 600 pm, at least about 700 pm, at least about 800 pm, at least about 900 pm, at least about 1,000 pm, or more. A thickness of the dielectric material that is coupled to at least a portion of a surface of a working electrode can be at most about 1000 pm, at most about 900 pm, at most about 800 pm, at most about 700 pm, at most about 600 pm, at most about 500 pm, at most about 400 pm, at most about 300 pm, at most about 200 pm, at most about 100 pm, at most about 90 pm, at most about 80 pm, at most about 70 pm, at most about 60 pm, at most about 50 pm, at most about 40 pm, at most about 30 pm, at most about 20 pm, at most about 10 pm, at most about 9 pm, at most about 8 pm, at most about 7 pm, at most about 6 pm, at most about 5 pm, at most about 4 pm, at most about 3 pm, at most about 2 pm, at most about 1 pm, at most about 900 nm, at most about 800 nm, at most about 700 nm, at most about 600 nm, at most about 500 nm, at most about 400 nm, at most about 300 nm, at most about 200 nm, at most about 100 nm, at most about 90 nm, at most about 80 nm, at most about 70 nm, at most about 60 nm, at most about 50 nm, at most about 40 nm, at most about 30 nm, at most about 20 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4 nm, at most about 3 nm, at most about 2 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.1 nm, or less.

[0127]

[0122] In some cases, the sensor can be configured to detect one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof), e.g., between the working electrode and the counter electrode or a reference electrode, when at least a portion of an analyte (and / or a tag coupled to the analyte) is bound (e.g., directly, or indirectly via the binding unit and / or the conducting material) to at least a portion of the sensor. In some cases, the sensor can be configured to detect one or more signals indicative of the impedance or impedance change, e.g., between the working electrode and the counter electrode or the reference electrode, when at least a portion of an analyte (and / or a tag coupled to the analyte) is not bound but in proximity to at least a portion of the sensor.

[0123] The sensor of the present disclosure can be configured to detect one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof), e.g., between the working electrode and the counter electrode or the reference electrode, when a distance between (i) at least a portion of an analyte (and / or a tag coupled to the analyte) and (ii) the working electrode is at least about 0.1 nm, at least about 0.5 nm, at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm, at least about 500 nm, at least about 600 nm, at least about 700 nm, at least about 800 nm, at least about 900 nm, at least about 1 pm, at least about 2 pm, at least about 3 pm, at least about 4 pm, at least about 5 pm, at least about 6 pm, at least about 7 pm, at least about 8 pm, at least about 9 pm, at least about 10 pm, at least about 20 pm, at least about 30 pm, at least about 40 pm, at least about 50 pm, at least about 60 pm, at least about 70 pm, at least about 80 pm, at least about 90 pm, at least about 100 pm, at least about 200 pm, at least about 300 pm, at least about 400 pm, at least about 500 pm, at least about 600 pm, at least about 700 pm, at least about 800 pm, at least about 900 pm, at least about 1,000 pm, or more. The sensor as disclosed herein can be configured to detect one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof), e.g., between the working electrode and the counter electrode or the reference electrode, when a distance between (i) at least a portion of an analyte (and / or a tag coupled to the analyte) and (ii) the working electrode is at most about 1,000 pm, at most about 900 pm, at most about 800 pm, at most about 700 pm, at most about 600 pm, at most about 500 pm, at most about 400 pm, at most about 300 pm, at most about 200 pm, at most about 100 pm, at most about 90 pm, at most about 80 pm, at most about 70 pm, at most about 60 pm, at most about 50 pm, at most about 40 pm, at most about 30 pm, at most about 20 pm, at most about 10 pm, at most about 9 pm, at most about 8 pm, at most about 7 pm, at most about 6 pm, at most about 5 pm, at most about 4 pm, at most about 3 pm, at most about 2 pm, at most about 1 pm, at most about 900 nm, at most about 800 nm, at most about 700 nm, at most about 600 nm, at most about 500 nm, at most about 400 nm, at most about 300 nm, at most about 200 nm, at most about 100 nm, at most about 90 nm, at most about 80 nm, at most about 70 nm, at most about 60 nm, at most about 50 nm, at most about 40 nm, at most about 30 nm, at most about 20 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4 nm, at most about 3 nm, at most about 2 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.1 nm, or less.

[0124] The sensor of the present disclosure can be configured to detect one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof), e.g., between the working electrode and the counter electrode or the reference electrode, when at least a portion of an analyte or at least a portion of a nucleotide (and / or a tag coupled to the nucleotide) is within a predetermined space that is near or adjacent to the working electrode. The predetermined space can be characterized by having a volume of at least about 0.1 square nanometers (nm2), at least about 0.5 nm2, at least about 1 nm, at least about 2 nm2, at least about 3 nm2, at least about 4 nm2, at least about 5 nm2, at least about 6 nm2, at least about 7 nm2, at least about 8 nm2, at least about 9 nm2, at least about 10 nm2, at least about 20 nm2, at least about 30 nm2, at least about 40 nm2, at least about 50 nm2, at least about 60 nm2, at least about 70 nm2, at least about 80 nm2, at least about 90 nm2, at least about 100 nm2, at least about 200 nm2, at least about 300 nm2, at least about 400 nm2, at least about 500 nm2, at least about 600 nm2, at least about 700 nm2, at least about 800 nm2, at least about 900 nm2, at least about 1 square micrometer (pm2), at least about 2 pm2, at least about 3 pm2, at least about 4 pm2, at least about 5 pm2, at least about 6 pm2, at least about 7 pm2, at least about 8 pm2, at least about 9 pm2, at least about 10 pm2, at least about 20 pm2, at least about 30 pm2, at least about 40 pm2, at least about 50 pm2, at least about 60 pm2, at least about 70 pm2, at least about 80 pm2, at least about 90 pm2, at least about 100 pm2, at least about 200 pm2, at least about 300 pm2, at least about 400 pm2, at least about 500 pm2, at least about 600 pm2, 7 at least about 00 pm2, at least about 800 pm2, at least about 900 pm2, at least about 1,000 pm2, or more. The predetermined space can be characterized by having a volume of at most about 1,000 pm2, at most about 900 pm2, at most about 800 pm2, at most about 700 pm2, at most about 600 pm2, at most about 500 pm2, at most about 400 pm2, at most about 300 pm2, at most about 200 pm2, at most about 100 pm2, at most about 90 pm2, at most about 80 pm2, at most about 70 pm2, at most about 60 pm2, at most about 50 pm2, at most about 40 pm2, at most about 30 pm2, at most about 20 pm2, at most about 10 pm2, at most about 9 pm2, at most about 8 pm2, at most about 7 pm2, at most about 6 pm2, at most about 5 pm2, at most about 4 pm2, at most about 3 pm2, at most about 2 pm2, at most about 1 pm2, at most about 900 nm2, at most about 800 nm2, at most about 700 nm2, at most about 600 nm2, at most about 500 nm2, at most about 400 nm2, at most about 300 nm2, at most about 200 nm2, at most about 100 nm2, at most about 90 nm2, at most about 80 nm2, at most about 70 nm2, at most about 60 nm2, at most about 50 nm2, at most about 40 nm2, at most about 30 nm2, at most about 20 nm2, at most about 10 nm2, at most about 9 nm2, at most about 8 nm2, at most about 7 nm2, at most about 6 nm2, at most about 5 nm2, at most about 4 nm2, at most about 3 nm2, at most about 2 nm2, at most about 1 nm2, at most about 0.5 nm2, at most about 0.1 nm2, or less.

[0125] The sensor of the present disclosure may not require at least a portion of an analyte (and / or a tag coupled to the analyte) to enter and / or pass through a nanopore to detect one or more signals. For example, the sensor may not comprise or may not be operatively coupled to a nanopore. Alternatively, at least a portion of an analyte (and / or a tag coupled to the analyte) can enter and / or pass through a nanopore in order for the sensor to detect one or more signals.

[0128]

[0126] In some cases, the working electrode and the counter electrode or the reference electrode can provide a first electric field along a first direction. In addition, the nanopore system can further comprise an additional electric field generator (e.g., an additional set of electrodes in a different circuit) configured to apply a second electric field in a second direction that is different (e.g., substantially perpendicular) to the first direction of the first electric field. A difference between the first direction and the second direction can be at least about 1 degree, at least about 2 degrees, at least about 3 degrees, at least about 4 degrees, at least about 5 degrees, at least about 6 degrees, at least about 7 degrees, at least about 8 degrees, at least about 9 degrees, at least about 10 degrees, at least about 15 degrees, at least about 20 degrees, at least about 30 degrees, at least about 40 degrees, at least about 50 degrees, at least about 60 degrees, at least about 70 degrees, at least about 80 degrees, at least about 90 degrees, at least about 100 degrees, at least about 110 degrees, at least about 120 degrees, at least about 130 degrees, at least about 140 degrees, at least about 150 degrees, at least about 160 degrees, at least about 170 degrees, or more. A difference between the first direction and the second direction can be at most about 180 degrees, at most about 170 degrees, at most about 160 degrees, at most about 150 degrees, at most about 140 degrees, at most about 130 degrees, at most about 120 degrees, at most about 110 degrees, at most about 100 degrees, at most about 90 degrees, at most about 80 degrees, at most about 70 degrees, at most about 60 degrees, at most about 50 degrees, at most about 40 degrees, at most about 30 degrees, at most about 20 degrees, at most about 15 degrees, at most about 10 degrees, at most about 9 degrees, at most about 8 degrees, at most about 7 degrees, at most about 6 degrees, at most about 5 degrees, at most about 4 degrees, at most about 3 degrees, at most about 2 degrees, at most about 1 degree, or less. In some cases, the difference between the first direction and the second direction can be about 90 degrees (i.e., the first direction and the second direction can be substantially orthogonal to one another).

[0129]

[0127] The nanopore system can comprise at least 1, at least 2, at least 3, at least 4, at least 5, or more additional electric field generators. The nanopore system can comprise at most 5, at most 4, at most 3, at most 2, or at most 1 additional electric field generator. When comprising a plurality of additional electric field generators, the plurality of additional electric field generators can apply a plurality of electric fields that are along the same or different directions. In some cases, the nanopore system may not comprise any additional electric field generator.

[0128] In some cases, an analyte can comprise one or more redox moieties (e.g., as tags), and the sensor of the present disclosure can be configured to measure redox potential (e.g., reduction potential or oxidation potential) of the redox moieties. In some cases, the sensor can be configured to measure oxidation potential of an oxidizable functional group of the analyte. A buffer solution can negatively affect activity of the binding unit (e.g., an enzymatic activity of the binding unit), and thus it can be desirable for some binding unit to reduce a concentration of salt in the solution. However, in some cases, a reduced concentration of salt in the buffer can increase resistance of the system, and thereby reduce sensitivity or accuracy of the sensor. In such cases, utilizing a tag with one or more redox moieties and measuring the redox potential of the nucleotide comprising such tag can be advantageous, and improve accuracy.

[0130]

[0129] In some cases, the sensor can be configured to determine a residence time or a duration of time that at least a portion of an analyte is bound on the binding unit (e.g., a polymerase). In some cases, different analytes or different tags coupled to the analytes can exhibit different residence times or the durations of time to the binding unit of the sensor, and the residence time or the duration time can be a unique or additional signature to analyze or identify the analytes. In some cases, the sensor can be configured to determine a residence time or a duration of time that at least a portion of the analyte is in proximity to the binding unit (e.g., a polymerase). In some cases, the sensor can be configured to determine a residence time or a duration of time that at least a portion of the growing strand or at least a portion of a nucleotide is in the nanopore.

[0131]

[0130] The sensor of the present disclosure can comprise an electrical circuit (e.g., CMOS or FET circuit). The electrical circuit can be coupled to a voltage source. A constant voltage can be applied to the electrical circuit, and a change in the current can be measured. Alternatively, a change in voltage necessary to maintain a steady state current can be measured. In some cases, a variable voltage can be applied and the one or more signals can comprise a voltage signal measured from the sensing circuit. The sensor can be in an electrolytic solution (e.g., 0.5 M Potassium Acetate and 10 mM KC1). Alternatively, the sensor may not be in an electrolytic solution. In some cases, the sensor can be in an aqueous solution or gas.

[0132]

[0131] The one or more signals can be a current or voltage measured from the sensing circuit. The one or more signals can be a current and voltage measured from the sensing circuit. The signal can be a tunneling current. Alternatively, the signal may not be a tunneling current. The current can be a Faradaic current. Alternatively, the current may not be a Faradaic current. The current can be at least about 1 picoamp (pA), at least about 10 pA, at least about 100 pA, at least about 1 nanoamp (nA), at least about 10 nA, at least about 100 nA, at least about 1 microamp (mA), at least about 10 mA, at least about 100 mA, or more. The current can be at most about 100 mA, at most about 10 mA, at most about 1 mA, at most about 100 nA, at most about 10 nA, at most about 1 nA, at most about 100 pA, at most about 10 pA, at most about 1 pA, or less. The current can be at least in the picoamp (pA) range, at least in the tens of pA range, at least in the hundreds of pA range, at least in the nanoamp (nA) range, at least in the tens of nA range, at least in the hundreds of nA range, at least in the microamp (mA) range, at least in the tens of mA range, or higher. The current can be at most in the tens of mA range, at most in the mA range, at most in the hundreds of nA range, at most in the tens of nA range, at most in the nA range, at most in the hundreds of pA range, at most in the tens of pA range, at most in the pA range, or lower. The voltage can be at least about 0.1 millivolt (mV), at least about 0.5 mV, at least about 1 mV, at least about 5 mV, at least about 10 mV, at least about 50 mV, at least about 100 mV, at least about 500 mV, or more. The voltage can be at most about 500 mV, at most about 100 mV, at most about 50 mV, at most about 10 mV, at most about 5 mV, at most about 1 mV, at most about 0.5 mV, at most about 0.1 mV, or less. The voltage can be at least in the millivolt (mV) range, at least in the tens of mV range, at least in the hundreds of mV range, or higher. The voltage can be at most in the hundreds of mV range, at most in the tens of mV range, at most in the mV range, or lower.

[0133]

[0132] In some cases, the sensor of the present disclosure can be provided as arrays, such as arrays present on a chip or biochip. The array of sensors can have any suitable number of any sensor of the present disclosure. The array can comprise about 10, about 20, about 50, about 100, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 10000, about 15000, about 20000, about 40000, about 60000, about 80000, about 100000, about 200000, about 400000, about 600000, about 800000, about 1000000, or more sensors.

[0134] Systems and Methods for Analyzing an Analyte

[0135]

[0133] Analysis of an analyte with high accuracy

[0136]

[0134] In an aspect, the present disclosure provides a method for analyzing an analyte. In some cases, the method may comprise providing a nanopore system. In some cases, the method may comprise contacting at least a portion of the analyte with the nanopore system. In some cases, the method may comprise detecting one or more signals (e.g., voltage signals, current signals, impedance signals, and / or signals indicative of an impedance or change thereof) associated with the at least the portion of the analyte. In some cases, the method may comprise using the one or more signals to identify one or more characteristics of the at least the portion of the analyte with an accuracy of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%.

[0135] In some cases, the nanopore system may comprise a fluidic chamber. In some cases, the nanopore system may comprise a nanopore disposed within a membrane. In some cases, the membrane may separate the fluidic chamber to a first side and a second side. In some cases, the nanopore system may comprise a sensor. In some cases, the sensor may comprise a working electrode and a counter electrode.

[0137]

[0136] In some cases, prior to contacting at least a portion of the analyte with the nanopore system, the method may comprise forming a membrane in the fluidic chamber. The membrane can be any suitable membrane as disclosed in the present disclosure. In some cases, the membrane may comprise a lipid bilayer as disclosed in the present disclosure. In some cases, forming the membrane in the fluidic chamber may comprise loading a solution comprising a plurality of lipids (e.g., phospholipids) to the fluidic chamber. In some cases, after loading the plurality of lipids (e.g., phospholipids) to the fluidic chamber, the plurality of lipids (e.g., phospholipids) may assemble to form the lipid (e.g., phospholipid) bilayer. In some cases, the lipid may comprise a phospholipid. In some cases, the phospholipid may comprise diphytanoylphosphatidylcholine. In some cases, the solution may comprise a lipid with a concentration of at least about 1 mg / mL, at least about 2 mg / mL, at least about 5 mg / mL, at least about 10 mg / mL, or at least about 15 mg / mL. In some cases, the solution may comprise a lipid with a concentration of at most about 15 mg / mL, at most about 10 mg / mL, at most about 5 mg / mL, at most about 2 mg / mL, or at most about 1 mg / mL. In some cases, the solution may comprise decane. In some cases, the solution may comprise tridecane. In some cases, the solution may comprise cholesterol. In some cases, the ratio of tridecane / decane may be from about 1 :5 to about 5: 1. In some cases, the ratio of tridecane / decane may be about 1 :3. In some cases, the cholesterol may have a concentration of at least about 1 mg / mL, at least about 2 mg / mL, at least about 5 mg / mL, at least about 10 mg / mL, or at least about 15 mg / mL. In some cases, the cholesterol may have a concentration of at most about 15 mg / mL, at most about 10 mg / mL, at most about 5 mg / mL, at most about 2 mg / mL, or at most about 1 mg / mL. In some cases, prior to contacting at least a portion of the analyte with the nanopore system, the method may comprise inserting or depositing a nanopore in the membrane. In some cases, the method may comprise forming a nucleic acid molecule-polymerase-nanopore complex. In some cases, the method may comprise depositing the nucleic acid molecule-polymerase-nanopore complex to the chamber comprising the formed membrane. In some cases, depositing the nucleic acid molecule-polymerase-nanopore complex to the chamber may insert the nanopore to the membrane. In some cases, depositing or inserting the nanopore (or nanopore-polymerase duplex or nanopore-polymerase-analyte triplex) in the membrane may comprise flowing a nanopore solution comprising one or more nanopores (or nanopore-polymerase duplexes or nanopore- polymerase-analyte triplexes) to the fluidic chamber. In some cases, a nanopore of the one or more nanopores (or nanopore-polymerase duplexes or nanopore-polymerase-analyte triplex) may diffuse into the membrane.

[0138]

[0137] In some cases, the analyte may comprise a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof. In some cases, the one or more characteristics may comprise a presence or absence of the analyte, a size of the analyte, or a concentration of the analyte, or a combination thereof. In some cases, the analyte may comprise a nucleic acid molecule, a protein, or a polypeptide. In some cases, the one or more characteristics may further comprise a sequence information of at least a portion of the analyte, an identity of the analyte, or a mutation of the analyte, or combinations thereof.

[0139]

[0138] In some cases, the accuracy may be at least about 95%. In some cases, the accuracy may be at least about 97%. In some cases, the accuracy may be at least about 99%. In some cases, the accuracy may be at least about 99.5%. In some cases, the accuracy may be at least about 99.9%.

[0140]

[0139] In some cases, the analyte may comprise a nucleic acid molecule. In some cases, the analyte may comprise a circular nucleic acid molecule. In some cases, the analyte may comprise a single stranded circular nucleic acid molecule. In some cases, the single stranded circular nucleic acid molecule may be generated by circularizing a single stranded linear nucleic acid molecule. The circularizing method is disclosed in subsequent section.

[0141]

[0140] In some cases, the method may further comprise hybridizing a primer to at least a portion of the single stranded circular nucleic acid molecule. In some cases, the method may further comprise generating a growing strand comprising sequence complementarity to at least an additional portion of the single stranded circular nucleic acid molecule. In some cases, the generating may comprise coupling a nucleotide to an end of the primer. In some cases, the generating may comprise coupling one or more nucleotide to an end of a growing strand.

[0142]

[0141] In some cases, the nucleotide may comprise one or more tags. In some cases, the one or more tags may induce a change in the one or more signals. In some cases, upon incorporation of the nucleotide to the end of the primer (or a growing strand), the one or more tags may be cleaved off from the nucleotide. In some cases, the method may further comprise directing the one or more tags to the nanopore in a first direction. In some cases, the method may further comprise directing the one or more tags out of the nanopore in a second direction. In some cases, the first direction and the second direction may be the same. In some cases, the first direction and the second direction may be opposite. In some cases, the method may further comprise translocating the one or more tags through the nanopore.

[0143]

[0142] In some cases, the method may comprise contacting the at least the portion of the analyte with the nanopore. In some cases, the method may comprise translocating the at least the portion of the analyte through the nanopore. In some cases, the method may comprise bringing the at least the portion of the analyte in proximity with the nanopore.

[0144]

[0143] In some cases, the method may comprise applying a voltage to the working electrode and the counter electrode. In some cases, the voltage may be at least about 10 mV, at least about 20 mV, at least about 50 mV, at least about 100 mV, at least about 150 mV, at least about 200 mV, at least about 250 mV, at least about 300 mV, or at least about 400 mV. In some cases, the voltage may be at most about 400 mV, at most about 300 mV, at most about 250 mV, at most about 200 mV, at most about 150 mV, at most about 100 mV, at most about 50 mV, or at most about 10 mV. In some cases, the voltage may comprise a direct current (DC) voltage. In some cases, the voltage may comprise an alternating current (AC) voltage. In some cases, the alternating voltage may comprise a first phase and a second phase. In some cases, the first phase and the second phase may have opposite directions. In some cases, the first phase may be a positive phase or a negative phase. In some cases, the second phase may be a positive phase or a negative phase. In some cases, the voltage may have a frequency of at least about 100 Hz, at least about 200 Hz, at least about 500 Hz, at least about 1000 Hz (or 1 kHz), at least about 2 kHz, at least about 5 kHz, or at least about 10 kHz. In some cases, the voltage may have a frequency of at most about 10 kHz, at most about 5 kHz, at most about 2 kHz, at most about 1 kHz, at most about 200 Hz, or at most about 100 Hz.

[0145]

[0144] In some cases, the at least the portion of the analyte may be directed to the nanopore at the first phase. In some cases, the at least the portion of the analyte may be directed to the nanopore at the second phase.

[0146]

[0145] In some cases, the method may comprise obtaining a plurality of measurements of the one or more signals during a cycle in the first or the second phase. In some cases, the plurality of measurements may comprise at least two, at least three, at least four, at least five, at least six, at least ten, or more measurements.

[0147]

[0146] In some cases, the method may comprise determining a first set of the one or more signals during the first phase. In some cases, the first set of the one or more signals may comprise at least two, at least three, at least four, at least five, at least six, at least ten, or more measurements of the one or more signals. In some cases, the method may comprise determining a second set of the one or more signals during the second phase.

[0148]

[0147] In some cases, the method may comprise pre-processing the one or more signals. In some cases, the pre-processing may comprise removing one or more background signals from the one or more signals. In some cases, background signals may comprise higher frequency than a sample signal. In some cases, removing one or more background signals may comprise removing one or more signals that have a frequency greater than a threshold value. In some cases, the threshold value may be at least about 10 Hz, at least about 20 Hz, at least about 25 Hz, at least about 30 Hz, at least about 40 Hz, at least about 50 Hz, at least about 100 Hz, or higher. In some cases, the pre-processing may further comprise removing a baseline portion from the one or more signals, thereby generating one or more pre-processed signals. In some cases, the baseline portion may be obtained when the analyte is not added to the nanopore system (e.g., open nanopore state).

[0149]

[0148] In some cases, the method may further comprise associating at least a portion of the one or more pre-processed signals with the at least the portion of the analyte. In some cases, the method may further comprise associating at least a portion of the one or more signals with the at least the portion of the analyte. In some cases, the method may further comprise associating at least a portion of the one or more signals with one or more nucleotides of the at least the portion of the analyte.

[0150]

[0149] In some cases, a workflow of the detection or analysis may comprise a dry test of the chip. A dry test can comprise number of fluidic chambers on the chip. A dry test may comprise examination the dimension of the chip. A dry test may comprise applying a voltage to the chip and measuring one or more signals from the sensor. The one or more signals may be associated with a resistance or an impedance of a fluidic chamber. If defects in the chip is found, the chip may not be used. In some cases, possible errors may be detected. The possible errors may comprise a GPIO error (a connection error), which can be fixed by pressing the chip harder against the interposer. The interposer may be replaced if error happens for multiple chips. The possible errors may comprise register read / write errors. The possible errors may comprise analog measurement errors. The workflow may comprise a wet test. In the wet test, a fluid may be flown in the chip and one or more signals may be detected. The one or more signals may be associated with a resistance or an impedance of a fluidic chamber and the fluid. If defects in the chip is found, the chip may not be used. The workflow may comprise a membrane (or bilayer) formation as disclosed herein. After the bilayer formation, one or more signals may be detected. The one or more signals may be associated with a resistance, a capacitance, and / or an impedance of a fluidic chamber and the membrane. If defects in the chip is found, the chip may not be used. The workflow may comprise a nanopore formation as disclosed herein. After the nanopore formation, one or more signals may be detected. The one or more signals may be associated with a resistance, a capacitance, and / or an impedance of the fluidic chamber, the nanopore, and the membrane. If defects in the chip is found, the chip may not be used. The workflow may comprise a sequencing. The sequencing may comprise tag reading. The sequencing may comprise event detection (e.g., a tag in proximate to or in a nanopore). The workflow may comprise analyzing the data to analyze the analyte. The analyzing may comprise pre-processing the data (e.g., removing background). In some cases, the analyzing may comprise running low pass filter to remove high frequency data points. In some cases, the analyzing may comprise selecting single pore cell. The analyzing may comprise identifying an event. The analyzing may comprise processing the data. The analyzing may comprise processing calculating resistance ratio (R ratio) of the nanopore and tag to nanopore. In some cases, calculating R ratios may comprise converting raw signal to normalized code (e.g., using 8 bit scale and time of continues signals (10-30 ms for catalytic event per base)). R ratio can be calculated from voltage values. Different R ratio may be used to determine a tag. Different tag may be used to determine the sequence information. In some cases, the analyzing may comprise running clustering algorithm. In some cases, the analyzing may comprise assigning bases to event. In some cases, the analyzing may comprise applying a basecalling algorithm to assign bases. In some cases, the method may further comprise converting the one or more signals via a machine learning algorithm to obtain sequence information of the single stranded circular nucleic acid molecule. In some cases, the machine learning algorithm may comprise a basecalling algorithm. The basecalling algorithm is a neural network based algorithm that detects abnormalities of the electrical signal and uses a recurrent neural network (RNN) to classify the electric signal as bases of the sequence. In addition to sequencing, it also includes several preprocessing steps where the low-quality reads are filtered out. As input, it uses the binary files generated from the device, and creates the fasta / fastq files containing the sequencing results.

[0151]

[0150] FIG. 8 shows an exemplary schematic for detection of an analyte. The analyte may comprise a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof. The analyte may comprise a single stranded circular nucleic acid molecule 111 (or template). A polymerase 112 may be coupled to the nanopore 101. A detection method may comprise hybridizing a primer to the template. The primer can be hybridized to the primer prior to, during, or after the coupling of the template to the polymerase. A plurality of nucleotides may be brought in proximity of the polymerase and template. A growing strand may be generated by coupling a nucleotide to an end of the primer or further an end of the prior added nucleotide. The nucleotide may comprise a tag. The tag (e.g., 113) may be directed into the nanopore prior to, during, or after the coupling to the growing strand. The tag may induce a change in one or more signals in the sensor coupled to the nanopore system. The sensor may detect the one or more signals prior to, during, and / or after the coupling of the nucleotide.

[0152]

[0151] A nanopore cell may be modeled as a capacitor. An ideal capacitor may act like an open circuit in steady state and no current passes through. A capacitor with leakage may comprise a capacitor portion and a resistor portion. Current can pass in steady state through the leakage resistor. When a capacitor is charged, its potential may gradually increase from the initial potential to the final potential. For example, when the potential of the counter electrode (CE) is increased from 1 to 1.1, the nanopore potential can increase from 1 to 1.1 gradually (or exponentially).

[0153]

[0152] FIG. 10A shows an exemplary circuit model of the nanopore cell when no membrane or nanopore are assembled. The circuit may comprise a chip cell capacitor or on-chip capacitor Ccell (part of the chip). The circuit may comprise a working electrode double layer (e.g., at the interface of the working electrode and electrolyte) capacitor CWE, working electrode resistor RWE, counter electrode double layer capacitor CCE, counter electrode resistor RCE, and electrolyte resistor Rs. FIG. 10B shows a heat map in wet test. Tight cluster 1010 confirms assumed CWE and CCE values. Some leakage may be expected due to finite resistance of the buffer solution and finite leakage resistance of CWE and CCE.

[0154]

[0153] FIG. 10C shows an exemplary simplified circuit model of the nanopore cell when membrane is assembled (BLF). The circuit may comprise a chip cell capacitor or on-chip capacitor Ccell. The circuit may comprise a working electrode double layer capacitor CWE, working electrode resistor RWE (not shown), counter electrode double layer capacitor CCE, counter electrode resistor RCE (not shown), and membrane capacitor CMEM. The capacitance of the membrane may increase as the membrane gets thinner. FIG. 10D shows a heat map of BLF. Tight cluster 1011 indicates uniform thickness of the membrane. High CMEM value indicates thinner bilayer. Low RCv7 values at higher RSR values indicate leak free bilayer.

[0155]

[0154] FIG. 10E shows an exemplary simplified circuit model of the nanopore cell when membrane and nanopore are assembled. Nanopore insertion may be modeled as the leakage resistance of the bilayer. The circuit may comprise a chip cell capacitor or on-chip capacitor Ccell. The circuit may comprise a working electrode double layer capacitor CWE, working electrode resistor RWE (not shown), counter electrode double layer capacitor CCE, counter electrode resistor RCE (not shown), membrane capacitor CMEM, and nanopore resistor RpOre. FIG. 10F shows a heat map after nanopore insertion. The tight cluster 1012 at high RCv7 and CMEM value indicate nanopore insertion.

[0156]

[0155] Heatmap is a tool to visualize an individual nanopore cell’s conformity to the electrical model. Each chemical state (e.g., chip prior to membrane insertion, chip after membrane insertion but prior to nanopore insertion, chip after membrane and nanopore insertion, and chip in sequencing) has an ideal membrane capacitance (CMEM2) and voltage on the chip cell capacitor (RCv7) values. Cells that pass the heatmap test from the wet, BLF, and nanopore insertion may be used for the data processing (e.g., with step detection algorithm). FIG. 10G shows an illustration of CMEM2 and RCv7 value regions for wet, membrane, and nanopore. For wet, CMEM2 may be greater than 90 and RCv7 may be less than 40. For membrane, CMEM2 may be between 60 and 80 and RCv7 may be less than 30. For nanopore, CMEM2 may be between 90 and 100 and RCv7 may be between 40 and 100. In some cases, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% nanopore cells may pass the wet, BLF, and nanopore insertion tests. In some cases, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% nanopore cells of the nanopore chip may be used in the data processing.

[0157]

[0156] FIG. 10H shows an exemplary circuit of the sensor for detecting the one or more signals. The circuit 1030 may comprise a counter electrode (CE) 1031. The CE may be disposed in the cis chamber (see FIG. 3). The CE may be configured to apply a potential to the electrolyte in the cis chamber. In some cases, each nanopore cell may have one CE. In some cases, the CE may be shared by a plurality of nanopore cells in the chip. In some cases, the CE may be shared by all nanopore cells in the chip. In some cases, the electrolyte between the counter electrode 1031 and the membrane (e.g., lipid bilayer) may be modeled as a capacitor. In some cases, the electrolyte between the counter electrode 1031 and the membrane may be modeled as a capacitor CCE (1033). The circuit 1030 may comprise a sensing electrode or working electrode (WE) 1032. The WE may be configured to apply a potential to the electrolyte in the trans chamber. In some cases, a WE may be configured to apply a potential to the electrolyte in the trans chamber, independently. In some cases, each nanopore cell may comprise an independent WE. In some cases, a WE in one nanopore cell may apply a first potential to the electrolyte in the nanopore cell and an additional WE in an additional nanopore cell may apply a second potential to the electrolyte in the additional nanopore cell. In some cases, the first potential and the second potential may be same. In some cases, the first potential and the second potential may be different. In some cases, the electrolyte between the working electrode 1032 and the membrane may be modeled as a capacitor. In some cases, the electrolyte between the working electrode 1032 and the membrane and the WE may be modeled as a capacitor CWE (1034). The membrane may be modeled as a capacitor CMEM (1035). The nanopore may be modeled as a resistor Rsut (1036). The circuit may comprise a pass device Ml. Pass device Ml may be configured to connect or disconnect the membrane and the working electrode from the circuit 1030.

[0158]

[0157] The circuit may comprise an on-chip capacitor Cl. The capacitor Cl may be charged or discharged. The circuit may comprise a pass device or switch M2. The circuit may comprise a controller configured to send a signal to the pass device M2 to open or close to disconnect or connect the capacitor Cl to a voltage source (e.g., VRST). When the pass device or switch M2 is closed, the on-chip capacitor Cl may be connected to the voltage source VRST and VRST may provide a voltage to Cl. Cl may be charged by the voltage source. In some cases, the voltage provided by VRST may comprise a constant reference voltage. In some cases, the voltage provided by VRST may not be constant. In some cases, the capacitor Cl may be pre-charged to a voltage level (or a starting or initial voltage). In some cases, the voltage level may be depending on the voltage source V RST. In some cases, the voltage level may be lower than the voltage of VRST. In some cases, the voltage level may be substantially same to the voltage of VRST.

[0159]

[0158] After the capacitor Cl is charged, the switch M2 may be opened to disconnect Cl from the voltage source. When Ml is connected, depending on the relative potential of Cl and CE, Cl may be charged or discharged. If the potential of Cl is lower than CE, Cl may be charged. If the potential of Cl is higher than CE, Cl may be discharged. The frequency and the period of the charge or discharge may depend on the sampling rate of ADC. In some cases, the frequency may be at least 500 Hz, at least 1 kHz, at least 5 kHz, at least 10 kHz, at least 100 kHz, or at least 500 kHz.

[0160]

[0159] In some cases, the capacitor may be re-connected to the voltage source to bring the potential of the capacitor back to the starting or initial voltage. This process may be referred to as a reset. In some cases, the reset may be temporary. After the reset, the capacitor may be disconnected from the voltage source. After the disconnection from the voltage source, the capacitor may charge or discharge.

[0161]

[0160] A RSR can be defined as reset-read gap, which is the time from reset of the circuit to taking of measurement. During the charge of the capacitor, at RSR, the circuit may be reset. After the reset, the potential of the circuit may go back to the initial value and again increase exponentially. An analog to digital convertor (ADC) may be used to quantify continuous analog data into finite digital data. An n bit of ADC may quantify continuous data into 2An discrete quantities. A 2 -bit ADC may quantify 0-4 V voltage to code 0, 1, 2, and 3. A 3 -bit ADC may quantify 0-4 V voltage to 8 discrete values (e.g., code 0, 1, 2, 3, 4, 5, 6, and 7). In some cases, the system disclosed herein may have an 8-bit ADC. The 8-bit ADC may create 256 discrete quantities that can convert an adjustable analog voltage range. For example, for a voltage range of 0.808 to 1.192 V, the resolution may be 1.5 mV. Each change in code (in 256 codes) may correspond to a 1.5 mV increase in voltage. In some cases, an impedance may be dependent on a resistance, a capacitance, and an inductance. In some cases, measuring an impedance or change thereof is advantageous over measuring a current or change thereof of the nanopore cell. In some cases, measuring an impedance or change thereof may provide information related to the nanopore, the analyte in proximate to or in the nanopore, and the membrane. In some cases, measuring an impedance or change thereof may not be limited by the nanopore size. In some cases, measuring an impedance or change thereof is more sensitive than measuring a current signal. In some cases, measuring an impedance or change thereof may not be limited by the electrode size. In some cases, measuring an impedance or change thereof may allow for detecting of an analyte while it is not in the nanopore.

[0162]

[0161] In some cases, a voltage signal (on the on-chip capacitor Cl) may be collected and converted to discrete values by ADC. In some cases, the voltage may be measured by a transistor. In some cases, the circuit may comprise a transistor to measure the voltage signal on Cl. In some cases, the transistor may detect the voltage signal between Cl and the ground. In some cases, the voltage signal may be indicative of an impedance or change thereof of the nanopore cell. The impedance or change thereof may be associated with an open nanopore, at least a portion of an analyte, and / or at least a portion of a tag species. In some cases, the circuit may be connected to a multiplexer (MUX). In some cases, the multiplex may comprise a 2: 1 MUX, a 4: 1 MUX, a 8: 1 MUX, or 16: 1 MUX. In some cases, the multiplex may be configured to convert input signals to a single output. In some cases, the MUX may be connected to the circuit before the ADC. In some cases, the voltage change due to at least a portion of the analyte (e.g., nucleic acid molecule, tag molecule, or a biomolecule) in vicinity of the nanopore or inside the nanopore may be small. In some embodiments, the voltage change may be masked by other voltage signal. In some cases, the circuit may further comprise an amplifier to amplify the voltage signal. In some case, the amplifier can be connected between the on-chip capacitor and the MUX. In some case, the amplifier can be connected between the MUX and the ADC. In some case, the amplifier can be connected between the on-chip capacitor and the ADC, if MUX is absent.

[0163]

[0162] In some cases, there may be a small constant shift in voltage value in the working electrode during sequencing using nanopore, and this voltage characteristic needs to be identifiable from the voltage data to assess impedance characteristics. In some cases, the voltage characteristic may be small and masked by other voltage signals. In some cases, the circuit may comprise an amplifier to amplify the voltage measurements. In some cases, the amplifier may be operatively coupled to the working electrode.

[0164]

[0163] The ADC data may be pre-processed and processed. Pre-processing and processing of the data may comprise background removal, data filtering (e.g., low pass filtering), data compression, data reduction, data subtraction, data averaging, data normalization, and / or event extraction. In some cases, ADC data may be processed into various data frames. The processed data may be usable or used for analyte determination or analysis.

[0165]

[0164] The voltage signal detected by the circuit (or sensor) may be associated with different analyte in proximate or inside the nanopore. When the voltage signal is measured, different states of a nanopore (e.g., open nanopore, analyte in proximate to or in the nanopore) may result in measurements of different voltages. The rate of the voltage decay (e.g., increase by charging or decrease by discharging) may depend at least on the impedance of the nanopore and membrane. The impedance of the nanopore and membrane may depend at least on nanopore resistance and the membrane capacitance. When different analyte is in proximate to or in the nanopore, the impedance of the nanopore / membrane may be different. The impedance of the nanopore / membrane when at least a portion of the analyte is in proximate to or in the nanopore may be different from when no analyte is in proximate to or in the nanopore. When different analyte is in proximate to or in the nanopore, the voltage decay rate may be different. In some cases, the voltage decay curve may be an exponential curve with an RC time constant T (tau)=RC. R is the resistance associated with the nanopore and C is the capacitance associated with the membrane. Tau of the open nanopore may be different from when at least a portion of an analyte is in proximate to or in the nanopore. Tau of one analyte may be different from another analyte. Tau of one tag may be different from another tag. A tau ratio of analyte (e.g., tag) relative to nanopore may be characterized to associate with the identity of the analyte. As the capacitance associated with the membrane may change when the membrane is being charged or discharged, the capacitance of the membrane may not be constant during the detection. In some cases, the capacitance of the membrane with an open nanopore may be different from the capacitance of the membrane with a tag or analyte in the nanopore. In some cases, for a same analyte or tag in the nanopore, the capacitance of the membrane may change over time, e.g., when the on-chip capacitor is being charged or being discharged. A change in tau may be associated with a change in membrane capacitance and / or a change in nanopore resistance. In using a tau ratio at different state of the nanopore (open nanopore, nanopore with an analyte or tag or nanopore with a different analyte or tag) to determine the state of the nanopore, if the change of capacitance of the membrane is kept minimal or negligible, the change in tau may be mainly attributed to the change of resistance of the nanopore. In that case, the change of tau may be directly corresponding to a change of the resistance of the nanopore and the effect of change of the capacitance of the membrane may be neglected.

[0166]

[0165] The circuit may further comprise an amplifier Al coupled to the working electrode. The amplifier may be connected to voltage sources VC1 and / or VC2. VC1 and VC2 may allow to use two voltages if necessary. SELECT and STORE circuit define which voltage to use. P is a connector to switch Ml. In some cases, VC1 may be configured to provide a high voltage and VC2 may be configured to provide a lower voltage than VC1. In some cases, the system may select either VC1 or VC2 for different uses. In some cases, during bilayer formation and / or nanopore formation, the required voltage may be lower than the sequencing process. In some cases, during bilayer formation and / or nanopore formation, a lower voltage may be applied. In some cases, during sequencing, a higher voltage may be applied.

[0167]

[0166] The voltage sources VC1 and VC2 may be configured to select and / or store a potential. The amplifier may be configured to stabilize the discharge of the potential of working electrode. When the pass device Ml is open, the potential of working electrode may reset. With the amplifier, the potential of the working electrode may not reset to the initial value immediately. The voltage source VC1 and / or VC2 may provide a charge potential to the working electrode and membrane, thus stabilizing the capacitance of the membrane. As the decay factor relates to resistance and capacitance of the nanopore, tag, and membrane, during each measurement cycle, the change of capacitance of membrane may affect the sensitivity and accuracy of detection. An amplifier added to stabilize the capacitance of the membrane may improve the accuracy of the detection. In some cases, the voltage sources VC1 or VC2 may be configured to apply a stabilizing potential to the working electrode. In some cases, the stabilizing potential may stabilize the capacitance of the working electrode. In some cases, the stabilizing potential may stabilize the capacitance of the membrane.

[0168]

[0167] FIG. 101 shows an exemplary plot of voltage signals during a measurement cycle. After each reset, the voltage of the working electrode may reduce to zero. As the counter electrode has a higher voltage than the working electrode, the voltage of the working electrode may increase from the initial zero value to a final value. Due to the capacitance of the counter electrode double layer, the membrane, and the capacitance of the working electrode double layer, the voltage of the working electrode does not increase to the final value simultaneously. Instead, the voltage of the working electrode increases to the final value gradually. FIG. 101 shows a voltage curve for an open nanopore and a voltage curve for the nanopore when a tag is in the nanopore. The tau value of the open nanopore may be lower than the tau value of the tag. A first tag may have a different tau value than a second tag. Each of the nucleotide may be coupled to a different tag (with different tau value). When a tag is detected, the tau value may associate to the identity of the tag, then associate to the identity of the added nucleotide. In some cases, multiple measurements (e.g., 6 measurements) may be taken during a positive phase (e.g., with positive amplitude). In some cases, multiple measurements (e.g., 6 measurements) may be taken during a negative phase (e.g., voltage direction reversed from the positive phase) of the applied voltage. After each measurement, the sensing electrode may be reset. In some cases, the multiple measurements may be averaged to generate an average signal value. In some cases, the data may be normalized. In some cases, the data may be compressed.

[0169]

[0168] FIG. 10J shows raw voltage signal. FIG. 10K shows the frequency spectrum of the signal. The voltage signal comprises low frequency region and high frequency region. By applying a low pass filter, high frequency regions (e.g., above 25 Hz) may be filtered out (e.g., removed). FIG. 10L shows the pre-processed signal after removing the high frequency signals.

[0170]

[0169] In some embodiments, the resistance associated with the nanopore in an open-channel state may be in the range of 100 MOhm to 20 GOhm. In some embodiments, the resistance associated with the nanopore in a state where a tag is inside the barrel of the nanopore may be within the range of 200 MOhm to 40 GOhm.

[0171]

[0170] In some cases, time resolved measurements (e.g., voltage, current, or impedance measurement) may be applied to increase the sensitivity and / or accuracy of analyte analysis. In some cases, using single read may not be able to distinguish different analytes or tags. In time resolved measurement, a first measurement can be taken at a first period of time (a first RSR) and a second measurement can be taken at a second period of time (or a second RSR). In some cases, the second period of time may be greater than the first period of time. In some cases, the first period of time may be from about 100 ps to 500 ps. In some cases, the second period of time may be from about 400 ps to 2000 ps. In some cases, the detection of signals may comprise detecting at more than two RSRs for each reset cycle. FIG. HA shows exemplary time-resolved impedance measurement traces for analyte A or tag A. FIG. 11B shows time-resolved impedance measurement traces for analyte B or Tag B. The measurement traces may comprise two reads at different times (e.g., at two RSRs). Reading at different times may generate additional value to distinguish the two tags. In some cases, different tags may have different characteristic figures of codes vs RSR. In some cases, when only one RSR is used in detecting the signal, the different tags may have a same code at the selected RSR, although the traces may have different shapes (e.g., different decay rate). When plotted, the trace of codes vs RSR for tag A may intercept with the trace of codes vs RSR for tag B. If the RSR value at the intercept is used for the detection, the code for tag A and the code for tag B are the same, thus are undifferentiated. However, if more than one RSRs (e.g., RSR1 and RSR2) are used, the codes detected for tag A and tag B at RSR2 are different although the codes detected for tag A and tag B at RSR1 may be same. Because the codes detected at RSR2 are different, tag A and tag B may be differentiated. In some cases, at low RSR values, a tag may have a linear relationship between codes and RSR. In some cases, at low RSR values, a tag may have an exponential relationship between codes and RSR. Reading at different times may generate additional data that lead to a characteristic curve that is not available when only one reading at a reading time. Use of time-resolved impedance may enable detection of tags with the same blocking voltage but with different change of voltage during detection cycle. For tag A, single time read comprises detection at 0.89+ / - 0.01 normalized code. Two times reads may comprise detection at 0.89 + / - 0.01 and 0.82 + / - 0.01 normalized codes. For tag B, single time read comprises detection at 0.89 + / - 0.01 normalized code. Two times reads may comprise detection at 0.89 + / - 0.01 and 0.76 + / - 0 / 01 normalized codes. As the two times reads may comprise detections at additional normalized codes, it may enable the distinguishing of different tags that are otherwise not distinguishable with the sign time read. FIGS. 11C and 11D show detection of 2 level signal with one applied voltage at 2 different read time values (250 ps and 1000 pis). Reading signals at multiple read times in one test may extend the tag sensing range.

[0172]

[0171] FIG. HE shows raw voltage signal during a sample / analyte detection (scatter plot of raw data). FIG. HF shows the raw voltage signal of a baseline (e.g., no sample). FIG. 11G shows the residual signal after subtracting the baseline from the sample signal.

[0173]

[0172] FIG. 11H shows the pre-processed data at a selected tau value. Each dot relates to a measurement cycle. Only one data point is shown in each measurement cycle (e.g., at the selected time value). The pre-processed data may be used to determine if the measurement is a baseline value (e.g., 1111) or an event value (e.g., a tag in a nanopore, e.g., 1112). The event value may be a data that deviates from the baseline. In some cases, the deviated data point may be a single point, in which case, the data point may not be considered as a real event value. In some cases, the deviated data points may be a series of points, in which case, the data points may be considered as real event values. FIG. HI shows exemplary voltage signal curves for open nanopore and nanopore with various tags. Different tag leads to different tau value or decay factor of the nanopore with tag. From the voltage signal, the tau value for nanopore with each tag may be calculated. The tag and the associated nucleotide may be distinguished or determined from the tau values. The sequence information of the template may be therefore determined. In some cases, a ratio of tau value of the nanopore with tag relative to tau value of open nanopore can be used to determine the different tags.

[0174]

[0173] Voltage waveforms and additional details of nanopore circuit

[0175]

[0174] In an aspect, the present disclosure provides a method for analyzing an analyte, the method comprising contacting at least a portion of the analyte with a nanopore system. In some cases, the nanopore system may comprise a nanopore disposed within a membrane, a first electrode disposed adjacent to a first side of the nanopore, and a second electrode disposed adjacent to a second and different side of the nanopore. In some cases, the method may comprise applying a voltage waveform between the first electrode and the second electrode of the nanopore system. In some cases, the method may comprise directing the nanopore system to detect one or more electrical signals associated with the at least the portion of the analyte.

[0176]

[0175] In some cases, the voltage waveform applied between the first electrode and the second electrode. In some cases, the voltage waveform may comprise an alternating current (AC) waveform. In some cases, the voltage waveform may comprise a symmetrical voltage waveform. In some cases, a symmetrical voltage waveform can be a voltage signal that is identical in its positive and negative cycles (e.g., of the AC waveform).

[0177]

[0176] In some cases, the voltage waveform applied between the first electrode and the second electrode may comprise a plurality of voltages (or voltage values). A voltage applied between the first electrode and the second electrode may be a difference in a first potential applied to the first electrode and a second potential applied to the second electrode. In some cases, the first potential and the second potential may be relative to a reference potential. In some cases, the first potential may be referred to as a reference potential. In some cases, the second potential may be referred to as a reference potential.

[0178]

[0177] In some cases, the voltage waveform may comprise an asymmetrical voltage waveform. In some cases, the voltage waveform may comprise a time-varying asymmetrical voltage waveform. In some cases, an asymmetrical voltage waveform can be a voltage signal that is not identical in its positive and negative cycles. Alternatively or in addition to, an asymmetrical voltage waveform can be an asymmetrical, unipolar voltage waveform.

[0179]

[0178] In some cases, at least a portion of the time-varying asymmetrical voltage waveform may alternate between a first voltage and a second voltage of differing absolute magnitudes. In some cases, the absolute magnitudes of the first voltage and the second voltage may differ by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some cases, the absolute magnitudes of the first voltage and the second voltage may differ by at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or at most about 5%. In some cases, the absolute magnitudes of the first voltage and the second voltage may differ by at least about 0.1 volts (V), at least about 0.2 V, at least about 0.4 V, at least about 0.5 V, at least about 0.8 V, at least about 0.9 V, or at least about 1 V. In some cases, the absolute magnitudes of the first voltage and the second voltage may differ by at most about 1 V, at most about 0.9 V, at most about 0.8 V, at most about 0.7 V, at most about 0.6 V, at most about 0.5 V, at most about 0.4 V, at most about 0.3 V, at most about 0.2 V, or at most about 0.1 V. In some cases, a voltage magnitude of one of the first voltage and the second voltage is substantially zero (0 V). In some cases, the first voltage and the second voltage may have different polarities. In some cases, the first voltage may be positive and the second voltage may be negative. In some cases, the first voltage may be positive and the second voltage may be negative with a smaller magnitude (e.g., the first voltage is 1 V and the second voltage is -0.1 V, or the first voltage is 0.1 V and the second voltage is -1 V). In some cases, the second voltage may be positive and the first voltage may be negative. In some cases, the second voltage may be positive and the first voltage may be negative with a smaller magnitude (e.g., the second voltage is 1 V and the first voltage is -0.1 V, or the second voltage is 0.1 V and the first voltage is -1 V). In some cases, the first voltage may be positive and the second voltage may be substantially 0 V. In some cases, the first voltage may be negative and the second voltage may be substantially 0 V. In some cases, the second voltage may be positive and the first voltage may be substantially 0 V. In some cases, the second voltage may be negative and the first voltage may be substantially 0 V. In some cases, at least a portion of the time-varying asymmetrical voltage waveform may comprise a first voltage phase and a second voltage phase. In some cases, the first voltage phase and the second voltage phase may comprise substantially same phase durations. In some cases, the first voltage phase and the second voltage phase may comprise differing phase durations. In some cases, the phase durations of the first voltage phase and the second voltage phase may differ by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some cases, the phase durations of the first voltage phase and the second voltage phase may differ by at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or at most about 5%. In some cases, the phase durations of the first voltage phase and the second voltage phase may differ by at least about 0.1 milliseconds (ms), at least about 0.5 ms, at least about 1 ms, at least about 5 ms, at least about 10 ms, at least about 20 ms, or at least about 30 ms. In some cases, the phase durations of the first voltage phase and the second voltage phase may differ by at most about 30 ms, at most about 20 ms, at most about 10 ms, at most about 5 ms, at most about 1 ms, at most about 0.5 ms, or at most about 0.1 ms. In some cases, the timevarying asymmetrical voltage waveform may comprise a plurality of cycles (e.g., the timevarying asymmetrical voltage waveform in FIG. 12J shows 12 cycles). In some cases, the timevarying asymmetrical voltage waveform may comprise at least 5 cycles, at least 10 cycles, at least 50 cycles, at least 100 cycles, at least 500 cycles, at least 1000 cycles, at least 5000 cycles, or at least 10000 cycles. In some cases, the time-varying asymmetrical voltage waveform may comprise at most 10000 cycles, at most 5000 cycles, at most 1000 cycles, at most 500 cycles, at most 100 cycles, at most 50 cycles, or at most 10 cycles. In some cases, a cycle of the plurality of cycles may comprise a plurality of voltage phases having different voltages. In some cases, the plurality of voltage phases may comprise at least 2, at least 3, at least 4, at least 5, or at least 6 voltage phases. In some cases, the plurality of voltage phases may comprise at most 6, at most 5, at most 4, at most 3, or at most 2 voltage phases. In some cases, the nanopore system may detect one or more electrical signals associated with the at least the portion of the analyte for the plurality of cycles.

[0180]

[0179] In some cases, at least a portion of the time-varying asymmetrical voltage waveform may comprise a unipolar voltage waveform. In some cases, the at least the portion of the unipolar voltage waveform may maintain a constant polarity relative to a reference potential. A unipolar voltage waveform may comprise just one voltage polarity, e.g., the voltage polarity does not change. In a unipolar voltage waveform, the voltage may not change from positive to negative. In a unipolar voltage waveform, the voltage may not change from negative to positive. In some cases, the voltage in the unipolar voltage waveform may change from negative to 0 V. In some cases, the voltage in the unipolar voltage waveform may change from positive to 0 V. In some cases, the voltage in the unipolar voltage waveform may change from a first negative value to a second negative value. In some cases, the voltage in the unipolar voltage waveform may change from a first positive value to a second positive value. In some cases, the voltage in the unipolar voltage waveform may change from 0 V to negative. In some cases, the voltage in the unipolar voltage waveform may change from 0 V to positive. In some cases, at least a portion of the timevarying asymmetrical voltage waveform comprises a bipolar voltage waveform. In some cases, the bipolar voltage waveform may not maintain a constant polarity relative to a reference potential. In some cases, the voltage in the bipolar voltage waveform may change from negative to positive. In some cases, the voltage in the bipolar voltage waveform may change from positive to negative.

[0181]

[0180] In some cases, at least a portion of the time-varying asymmetrical voltage waveform may comprise a plurality of different voltage phases. In some cases, the plurality of different voltage phases may comprise at least 2, at least 3, at least 4, at least 5, or at least 6 voltage phases. In some cases, the plurality of different voltage phases may comprise at most 6, at most 5, at most 4, at most 3, or at most 2 voltage phases. In some cases, at least one of the plurality of different voltage phases may comprise a non-constant voltage phase. In some cases, the non-constant voltage phase may be characterized by a rate of voltage change. In some cases, the non-constant voltage phase may comprise a first period having a first rate of voltage change and a second period having a second rate of voltage change. In some cases, the first rate of voltage change and the second rate of voltage change may be different. In some cases, the rate of voltage change, the first rate of voltage change, and / or the second rate of voltage change may be at least about 0.1 volts per second (V / s), at least about 0.2 V / s, at least about 0.5 V / s, at least about 1 V / s, at least about 2 V / s, at least about 5 V / s, at least about 10 V / s, at least about 20 V / s, or at least about 50 V / s. In some cases, the rate of voltage change, the first rate of voltage change, and / or the second rate of voltage change may be at most about 50 V / s, at most about 20 V / s, at most about 10 V / s, at most about 5 V / s, at most about 2 V / s, at most about 1 V / s, at most about 0.5 V / s, at most about 0.2 V / s, or at most about 0.1 V / s. In some cases, the non-constant voltage phase may be characterized by an increase in voltage over time. In some cases, the non-constant voltage phase may be characterized by a decrease in voltage over time. In some cases, an additional voltage phase of the plurality of different voltage phases may comprise a constant voltage phase. In some cases, the time-varying asymmetrical voltage waveform may be applied to a medium in contact with the nanopore. In some cases, the medium may comprise liquid, electrolyte, or both.

[0181] In some cases, the nanopore system as provided herein may comprise at least one voltage source. In some cases, the nanopore system as provided herein may comprise at least one capacitor electronically coupled to the second electrode. In some cases, the nanopore system as provided herein may comprise at least one electronic switch (or reset switch) for generating a plurality of electrical couplings (e.g., a plurality of sequential temporary couplings) between the at least one voltage source to the at least one capacitor. In some cases, the at least one electronic switch may comprise a single electronic switch. In some cases, the at least one electronic switch may comprise a plurality of electronic switches. In some cases, the nanopore system may comprise at least one pair of the at least one voltage source and the at least one electronic switch, e.g., for charging (or pre-charging) the at least one capacitor. In some cases, the nanopore system may comprise a plurality of pairs (e.g., at least 2, 3, 4, 5, or more pairs) of the at least one voltage source and the at least one electronic switch, e.g., for charging (or pre-charging) the at least one capacitor (same or different capacitors) at the same time or at different times. In some cases, the nanopore system may comprise two or more electronic switches coupled with one voltage source for charging the at least one capacitor. In some cases, the nanopore system may comprise a capacitor (or the on-chip capacitor as disclosed herein) electronically coupled to the second electrode. In some cases, the capacitor may be charged by a voltage source to a starting or initial potential. In some cases, the starting or initial potential may be referred to as a reference potential. In some cases, the starting or initial potential may be normalized as a zero potential. After the electronic switch opens, the capacitor may be disconnected from the voltage source. Due to a potential difference between the capacitor and the first electrode (or between the first electrode and the second electrode), the potential at the capacitor may increase or decrease. In some cases, the nanopore system may comprise an electronic switch (or the pass device as disclosed herein) for generating a plurality of sequential temporary couplings (or a plurality of sequential transient couplings) between the capacitor to a voltage source. In some cases, in a temporary coupling, the capacitor may be temporarily connected to the voltage source. In some cases, the temporary coupling may be for a short duration. In some cases, the duration of temporary coupling may be at most about 0.5 ms, at most about 0.4 ms, at most about 0.3 ms, at most about 0.2 ms, at most about 0.1 ms, or at most about 0.01 ms. In some cases, the duration of temporary coupling may be at least about 0.01 ms, at least about 0.1 ms, at least about 0.2 ms, at least about 0.3 ms, at least about 0.4 ms, or at least about 0.5 ms. Upon the temporary coupling, the potential of the capacitor may be brought back to (e.g., reset to) the starting or initial value. After the temporary coupling, the capacitor may be disconnected from the voltage source. After the disconnection from the voltage source, the capacitor may charge or discharge. During the charge or discharge, the nanopore system may detect a plurality of electrical signals (e.g., voltage signals, current signals, impedance signals, etc.) associated with the at least the portion of the analyte. In some cases, the method may comprise detect a plurality of electrical signals between the plurality of sequential temporary couplings. In some cases, the plurality of electrical signals comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 electrical signals. In some cases, the plurality of electrical signals comprises at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, or at most 2 electrical signals. In some cases, the plurality of electrical signals may be separated in time. In some cases, two adjacent electrical signals of the plurality of electrical signals may be measured within at least about 10 microseconds (ps), at least about 20 ps, at least about 50 ps, at least about 100 ps, at least about 200 ps, at least about 300 ps, at least about 400 ps, at least about 500 ps, at least about 600 ps, at least about 700 ps, at least about 800 ps, at least about 900 ps, at least about 1 ms from one another, or at least one 5 ms from one another. In some cases, two adjacent electrical signals of the plurality of electrical signals may be measured within at most about 5 ms, at most about 1 ms, at most about 800 ps, at most about 700 ps, at most about 600 ps, at most about 500 ps, at most about 400 ps, at most about 300 ps, at most about 200 ps, at most about 100 ps, at most about 50 ps, at most about 20 ps, or at most about 10 ps. In some cases, each of the plurality of electrical signals may comprise a decay in voltage across the capacitor.

[0182]

[0182] In some cases, the plurality of sequential temporary couplings may be different from one another. In some cases, a first sequential temporary coupling and a second sequential temporary coupling may have different durations. In some cases, a duration of the first sequential temporary coupling and a duration of the second sequential temporary coupling may be different by at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 200%, at least about 500%, or at least about 1000%. In some cases, a duration of the first sequential temporary coupling and a duration of the second sequential temporary coupling may be different by at most about 1000%, at most about 500%, at most about 100%, at most about 50%, at most about 20%, at most about 10%, or at most about 5%. In some cases, a duration of the first sequential temporary coupling and a duration of the second sequential temporary coupling may be different by at least about 0.1 ms, at least about 0.2 ms, at least about 0.5 ms, at least about 1 ms, at least about 2 ms, at least about 5 ms, at least about 10 ms, at least about 20 ms, at least about 50 ms, or at least about 100 ms. In some cases, a duration of the first sequential temporary coupling and a duration of the second sequential temporary coupling may be different by at most about 100 ms, at most about 50 ms, at most about 20 ms, at most about 10 ms, at most about 5 ms, at most about 2 ms, at most about 1 ms, at most about 0.5 ms, at most about 0.2 ms, or at most about 0.1 ms.

[0183]

[0183] In some cases, the nanopore system may further comprise an additional voltage source and an additional electronic switch. In some cases, the additional electronic switch may be configured for electrically coupling the additional voltage source to the capacitor or a different capacitor of the nanopore system.

[0184]

[0184] In some cases, the first electrode and the second electrode may be disposed on opposite sides of the nanopore. In some cases, the membrane may separate the fluidic chamber to a cis side and a trans side. In some cases, the first electrode may be disposed adjacent to a cis side of the nanopore. In some cases, the second electrode may be disposed adjacent to a trans side of the nanopore. In some cases, the nanopore system may further comprise a binding unit capable of binding to the at least the portion of the analyte. The binding unit may be any binding unit as disclosed herein. In some cases, the binding unit may comprise a polymerase as disclosed herein. In some cases, the analyte may comprise a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof, as disclosed herein. In some cases, the analyte may comprise a biomolecule, a detection moiety coupled to the biomolecule, or both, as disclosed herein. In some cases, the biomolecule may comprise one or more nucleotide or one or more amino acid residues. In some cases, the biomolecule may comprise one or more biopolymers.

[0185]

[0185] FIG. 12A shows an exemplary circuit model of the sensor for detecting one or more signals. The circuit 1200 may comprise a counter electrode (CE), a working electrode (WE), and a membrane (MEM) comprising a lipid bilayer and a nanopore. The circuit may comprise an on- chip capacitor (CC). The CE may be disposed in the cis chamber. The CE may be configured to apply a potential to the electrolyte in the cis chamber. The electrolyte between the CE and the membrane may be modeled as a capacitor CCE. The WE may be configured to apply a potential to the electrolyte in the trans chamber. The electrolyte between the WE and the membrane may be modeled as a capacitor CWE. The membrane may be modeled as a capacitor CMEM. The nanopore may be modeled as a resistor RP. The circuit 1200 may comprise a voltage source VPRE configured to supply a voltage potential to CC. The circuit may further comprise a reset switch (RESET) configured to connect or disconnect CC to the voltage source VPRE. The circuit may further comprise a reset switch controller configured to send a reset signal to the reset switch. The reset signal may comprise a high voltage signal configured to turn the reset switch on. The reset signal may comprise a low voltage signal configured to turn the reset switch off. When the reset switch (RESET) turns on, CC may be charged to a voltage level of the VPRE. After CC is pre-charged, RESET may be turned off to disconnect CC from VPRE. If the potential of CE is higher than the potential of CC, CC may be further charged. In some cases, CC may be further charged to reach a substantially same potential as CE. If the potential of CE is lower than the potential of CC, CC may be discharged. In some cases, CC may be discharged to reach a same potential as CE. As CE is alternated between a positive and a negative potential, this circuit model may be referred to as a bipolar circuit model.

[0186]

[0186] FIG. 12B shows exemplary simulated voltage levels of the circuit 1200. After the precharged CC is disconnected from VPRE, the voltage of CC is modeled as 0. A square wave of AC voltage is applied to CE. The square wave is modeled as +1 V in a positive phase and -1 V in a negative phase. As CE is alternated between a positive and a negative potential, the circuit model may be referred to as a bipolar model. 1211 shows the simulated CE voltage. 1212 shows the simulated reset voltage signal (VRESET). When VRESET is low (e.g., at 0 V), the reset switch RESET is off and CC is disconnected from VPRE. When VRESET is high (e.g., at 1 V), the rest switch RESET is turned on. When RESET is on, CC is connected to VPRE and VPRE charges the node VCELL (e.g., the connection between CC, WE, and VPRE) to the starting potential (e.g., VPRE or a voltage lower than VPRE). The RESET signal in 1212 comprises a plurality of pulses, configured to apply a plurality of temporary couplings (or transient couplings) between CC and VPRE. At 10 ms, the voltage of CE (VCE) is reduced from 0 to -1 V and maintains at -1 V for 5 ms. As CC has a higher voltage than CE, CC may discharge, and the potential of CC or at CELL node of FIG. 12A (VCELL) may go down gradually from 0 to about -0.9 V. This may be referred to as a voltage decay. 1213 shows the simulated VCELL. The potential at the vicinity of the membrane in the trans side (VWE1) follows the same pattern of VCELL. 1214 shows the simulated VWE1. The potential at the vicinity of the membrane in the cis side (VCE1) is higher than VCE. The voltage across the membrane VBL (VWE1-VCE1) is positive. 1215 shows the simulated VBL. In some cases, VBL can be up to about 1 V. The positive voltage may push or direct at least a portion of the analyte (e.g., a nucleotide, a tag of a nucleotide, a nucleic acid, or a biomolecule) into the nanopore if the portion of the analyte is negatively charged. The threading of the portion of the analyte in the nanopore may be referred to as a bright period. At 10.5 ms, RESET is turned on to bring the potential of CC back to zero. RESET is instantaneously turned off to disconnect CC from VPRE (as shown as a pulse in VRESET 1212). Due to charge accumulation at CWE, the voltage of CWE1 does not reset to 0, but may be at about -0.05 V. As CC has a higher voltage than CE, CC may discharge, and VCELL may go down gradually. From 10.5 ms to 11.5 ms, VCELL decreased from 0 to about - 0.8 V. Due to charge accumulation at the CCE, CMEM, and CWE, the voltage decay of CC may be slower than the first voltage decay from 10 ms to 10.5 ms, and the final voltage (e.g., at 11.5 ms) of VCELL may be a little higher than the first voltage decay (e.g., at 10.5 ms). At 11.5 ms, RESET is turned on to bring the potential of CC back to zero. RESET is instantaneously turned off to disconnect CC from VPRE. Due to charge accumulation at CWE, the voltage of CWE1 does not reset to 0, but may be at about -0.1 V. As CC has a higher voltage than CE, CC may discharge, and VCELL may go down gradually. From 11.5 ms to 12.5 ms, VCELL decreased from 0 to about -0.7 V. Due to charge accumulation at the CCE, CMEM, and CWE, the voltage decay of CC may be slower than the second voltage decay from 10.5 ms to 11.5 ms, and the final voltage (e.g., at 12.5 ms) of VCELL may be a little higher than the first voltage decay (e.g., at 11.5 ms). The reset and decay cycle can be repeat one or more times (e.g., three to nine times). At the end of each decay, the final voltage of VCELL may be a little higher than the previous one. A change of VCELL in each reset and decay cycle is lower than the previous one. A change of VWE1 in each reset and decay cycle is lower than the previous one. A change of VBL in each reset and decay cycle is lower than the previous one. As VBL maintains the positivity, the portion of the analyte may remain in the nanopore for continued detection. In some cases, VCELL may be measured. A decay curve of VCELL is distinguishable between an open nanopore and a nanopore with at least a portion of an analyte or tag in it or between a nanopore with a first analyte or tag and a nanopore with a second analyte or tag. In some cases, a plurality of measurements can be taken in one reset and decay cycle. In some cases, a curve may be constructed based on the plurality of measurements. In some cases, one measurement can be taken in one reset and decay cycle. In some cases, the measurement can be taken at a fixed time point. As a change of VCELL in each reset and decay cycle gets lower than the previous one, after a few cycles, the change of VCELL may become less distinguishable between an open nanopore and a nanopore with at least a portion of an analyte or tag in it or between a nanopore with a first analyte or tag and a nanopore with a second analyte or tag. And after a few cycles, the charges on CC may reduce significantly that it does not provide enough driving force for the voltage decay, and a re-charge of CC is needed.

[0187]

[0187] At 15 ms, VCE is changed from -1 V to 1 V and maintains at 1 V for 5 ms. As CC has a lower voltage than CE, CC may re-charge, and VCELL may go up gradually from -0.5 V to about 0.9 V. VWE1 follows the same pattern of VCELL. VCE1 is lower than VCE. VBL is negative. In some cases, VBL can be up to about -I V. The negative voltage may eject the portion of the analyte out of the nanopore. The period that the portion of the analyte is ejected from the nanopore may be referred to as a dark period. In some cases, after the ejection, the analyte may not re-enter the nanopore for additional detection. In some cases, due to the ejection, the portion of the analyte may have very short residence time within the nanopore to be detected. In some cases, due to the ejection, the portion of the analyte may not be detected. In some cases, due to the ejection, due to the ejection, the detected signal may not be associated with the portion of the analyte. In some cases, keeping at least the portion of the analyte in the nanopore for extended period of time may enable multiple measurements associated with the analyte, which may improve the detection accuracy. At 15.5 ms, RESET is turned on to bring the potential of CC back to zero. RESET is instantaneously turned off to disconnect CC from VPRE. Due to charge accumulation at CWE, the voltage of CWE1 does not reset to 0, but may be at about 0.05 V. As CC has a lower voltage than CE, CC may charge, and VCELL may go up gradually. From 15.5 ms to 16.5 ms, VCELL increased from 0 to about 0.8 V. Due to charge accumulation at the CCE, CMEM, and CWE, the voltage increase of CC may be slower than the first cycle from 15 ms to 15.5 ms, and the final voltage (e.g., at 16.5 ms) of VCELL may be a little lower than the first cycle (e.g., at 15.5 ms). At 16.5 ms, RESET is turned on to bring the potential of CC back to zero. RESET is instantaneously turned off to disconnect CC from VPRE. Due to charge accumulation at CWE, the voltage of CWE1 does not reset to 0, but may be at about 0.1 V. As CC has a lower voltage than CE, CC may charge, and VCELL may go up gradually. From 16.5 ms to 17.5 ms, VCELL increased from 0 to about 0.7 V. Due to charge accumulation at the CCE, CMEM, and CWE, the voltage increase of CC may be slower than the second cycle from 15.5 ms to 16.5 ms, and the final voltage (e.g., at 17.5 ms) of VCELL may be a little lower than the second voltage decay (e.g., at 16.5 ms). The reset and increase cycle can be repeat one or more times (e.g., three to nine times). At the end of each cycle, the final voltage of VCELL may be a little lower than the previous one. A change of VCELL in each cycle is lower than the previous one. A change of VWE1 in each cycle is lower than the previous one. A change of VBL in each cycle is lower than the previous one. As VBL maintains the negativity, the portion of the analyte (or the analyte) may be ejected from the nanopore and may not re-enter the nanopore for continued detection. The ejection of the nanopore may occur at the first cycle, or any cycle when VCE is at -IV. After the ejection of the analyte from the nanopore, subsequently detected signal is not associated with a characteristic of the analyte (but only for the open nanopore), therefore does not provide information to identify the analyte. FIG. 12C shows expanded view of VCELL (1221), VWE1 (1222), and VBL (1223), and a comparison to VCE (square waved traces in 1221 and 1222).

[0188]

[0188] For analyte (e.g., a biomolecule, or a tag coupled to a nucleotide) detection, it is desirable to keep the analyte (or a portion of the analyte) in the nanopore for longer time to collect sufficient data associated with the analyte. If the analyte (or a portion of the analyte) is ejected and does not re-enter the nanopore, the analyte (or a portion of the analyte) may have very limited residence time in the nanopore for detection. In some cases, the analyte (or a portion of the analyte) may not enter the nanopore because it is possible when the nucleotide or analyte is held in the active position of the binding unit or polymerase and the analyte (or a portion of the analyte) is anticipated to be directed into the nanopore, the sensor may apply a negative force on the analyte (or a portion of the analyte) such that it does not enter the nanopore. In such case, the analyte (or a portion of the analyte) may not be detected. If the negative voltage across the membrane is not sufficient to eject the analyte (or a portion of the analyte) completely out of the nanopore, the negative voltage may make the analyte (or a portion of the analyte) move around in the nanopore, which may lead to a change or a fluctuation of resistance of the nanopore. The change or fluctuation of resistance of the nanopore may affect the sensitivity and accuracy of the detection. In addition, the movement of the analyte (or a portion of the analyte) in the nanopore may impact the integrity and / or shape of the nanopore, in return, affecting the sensitivity and accuracy of the detection. For nucleic acid sequencing, prior to or during an incorporation of a nucleotide to a growing strand, at least a portion of the tag coupled to the nucleotide may be directed into the nanopore and be detected in the nanopore. If the tag is ejected and does not reenter the nanopore, the tag may have very limited residence time in the nanopore for detection. If the negative voltage across the membrane is not sufficient to eject the tag completely out of the nanopore, the negative voltage may make the tag move around in the nanopore, which may lead to a change or a fluctuation of resistance of the nanopore. The change or fluctuation of resistance of the nanopore may affect the sensitivity and accuracy of the detection. In addition, the movement of the tag in the nanopore may impact the integrity and / or shape of the nanopore, in return, affecting the sensitivity and accuracy of the detection. Further, ejection of the tag out of the nanopore may increase error during sequencing of homopolymer section (e.g., CGG repeats). The time frame for a nucleotide addition may be a few milliseconds. In a 100 ms period, one to ten nucleotides may be incorporated to a growing strand. If a tag does not enter or is ejected out the nanopore, the sensor may fail to detect a plurality of tags. Therefore, there may be a limitation of the bipolar circuit model.

[0189]

[0189] FIG. 12D shows a comparison of a bipolar CE scheme and a unipolar CE scheme. In the bipolar CE scheme (top panel), VCE is alternated between a positive potential (e.g., +VCE) and a negative potential (e.g., -VCE). In the unipolar CE scheme, VCE is alternated between a zero potential (0 relative to VCC after the pre-charge) and a negative potential (e.g., -VCE). In the unipolar CE scheme, the potential of CE is not increased to a positive level.

[0190] FIG. 12E shows an exemplary circuit model of the sensor for detecting one or more signals. The circuit 1230 may be used for the unipolar CE scheme. The circuit 1230 may be different from the circuit 1200 in that, the circuit may comprise a second a voltage source VPRE2 configured to charge CC and supply a voltage potential to CC. The circuit 1230 may further comprise a second reset switch (RESET2). In some cases, the circuit may comprise a second voltage source VPRE2. RESET2 may be configured to connect or disconnect CC to VPRE2. In some cases, the circuit may not comprise a second voltage source, and RESET2 may be configured to connect or disconnect CC to VPRE. The circuit may further comprise a second reset switch controller configured to send a second reset signal to the second reset switch. The second reset signal may comprise a high voltage signal configured to turn the second reset switch on. The second reset signal may comprise a low voltage signal configured to turn the second reset switch off.

[0190]

[0191] FIG. 12F shows exemplary simulated voltage levels of the unipolar CE scheme. 1231 shows the square waved voltage trace of VCE. 1232 shows the traces of VRESET and VRESET2. VRESET may comprise a plurality of pulses. VRESET2 may comprise a first period that VRESET2 is low (e.g., at 0 V) and a second period that RESET2 is high (e.g., at 1 V). When VRESET2 is low, the second reset switch is off. When VRESET2 is high, the second reset switch is one. In some cases, the pattern of VRESET2 may be same as the pattern of VCE. 1233 shows the trace of VCELL. 1234 shows the trace of VWE1. 1235 shows the trace of VBL. CC is charged to a voltage and disconnected from VPRE. At 10 ms, VCE is changed from 0 V to -1 V and maintains at -1 V for 2.5 ms. RESET is temporarily turned on to recharge or pre-charge CC and immediately turned off to disconnect CC from VPRE (1streset). From 10 ms to 12.5 ms, RESET2 is off. As CC has a higher voltage than CE, CC may discharge, and VCELL may decay gradually from 0 to about -0.9 V. VWE1 follows the same pattern of VCELL and decays to about -0.9 V. VBL is positive. After a second reset (e.g., RESET temporarily couples CC to VPRE and turns off immediately), VCELL decays from 0 V to about -0.85 V. After a third reset, VCELL decays from 0 V to about -0.8 V. After each reset, the potential of WEI increases to a level that is lower than a previous reset. After each reset, VBL increases to a level that is lower than a previous reset. In some cases, VCELL may be measured. The measurement can be performed as disclosed for FIGS. 12A and 12B. As a change of VCELL in each reset and decay cycle gets lower than the previous one, after a few cycles, the change of VCELL may become less distinguishable between an open nanopore and a nanopore with at least a portion of an analyte or tag in it or between a nanopore with a first analyte or tag and a nanopore with a second analyte or tag. And after a few cycles, the charges on CC may reduce significantly that it does not provide enough driving force for the voltage decay, and a re-charge of CC is needed. Following a fourth reset of CC, VBL increases to 0.84 V. Simultaneously, at 12.5 ms, VCE is changed from -1 V to 0 V and maintains at 0 V for 23.5 ms. The time period from 12.5 ms to 36 ms when VCE is at -1 V can be referred to as a dark time. In the dark time, RESET2 may be turned on to re-charge or pre-charge CC. After each pre-charge pulse is done, the CELL node may rise exponentially to its maximum value (or the starting potential). The exponential rate of increase (RC time constant) may a function of the tag or analyte being detected. By doing continuous reset pulses, the system can take more measurements without CE changing state, and possibly get more tag or analyte information. The tag or analyte might not enter the nanopore on the first reset pulse, but it may on a later reset pulse. In some cases, RESET2 may be turned on continuously for a first portion of the dark time, and turned off for the remaining of the dark time. In some cases, RESET2 may be turned on continuously for a first portion of the dark time and turned on and off for a plurality of cycles for the remaining of the dark time. In some cases, a longer duration of the pre-charging in the dark time may provide CC sufficient charge (or charge accumulation) to be discharged when VCE is reduced to -VCE. In the dark time, RESET is turned on and off for a plurality of cycles. VCELL maintains 0 V in the dark time. As there is no potential difference between CC and CE, no charging or discharging of CC will occur. VBL changes from 0.84 V to -0.16 V due to the change of VCE from -1 V to 0 V. Therefore, VBL changes from a positive voltage to a negative voltage, with a magnitude that is about 16% of the magnitude of VCE in the negative phase (1 V). The negative VBL is caused by the difference between CE and accumulated droop voltage. The accumulated charges in CE, WE, and MEM may discharge, and VBL may gradually change from -0.16 V to zero. In some cases, VBL at 16% of VCE may not be sufficient to eject the analyte or tag out of the nanopore. As VCE does not go up to a positive value, VBL may maintain at zero and the analyte or tag may stay in the nanopore for additional detection. At 36 ms, another cycle of VCE change (-1 V for 2.5 ms and 0 V for 23.5 ms) occurs, and this process can be repeated. In some cases, although the magnitude of negative VBL is significantly reduced in comparison to the bipolar CE scheme, where the magnitude of negative VBL may be up to 100% VCE, the negativity of VBL may move the analyte or tag in a direction opposite to the direction that the analyte or tag enters the nanopore, and affect the sensitivity or accuracy of detection. In some cases, VBL at 16% of VCE may be sufficient to eject the analyte or tag out of the nanopore.

[0191]

[0192] FIG. 12G shows expanded view of the simulated voltage levels of the unipolar CE scheme (the period from 36 ms to 46 ms of FIG. 12F). The top plot 1241 shows a trace of VCELL and VCE (square waved trace). The middle plot 1242 shows a trace of VWE1 and VCE (square waved trace). The bottom plot 1243 shows a trace of VBL.

[0192]

[0193] In some cases, VRESET may comprise a first portion comprising a plurality of pulses and a second portion that RESET is off. In some cases, the first portion of VRESET may be the period CE is at -VCE. In some cases, the second portion of VRESET may be the period when CE is at 0 V. In some cases, VRESET2 may comprise a first portion that RESET2 is off and a second portion that RESET2 is on. In some cases, the first portion of VRESET2 may be the period CE is at -VCE. In some cases, the second portion of VRESET2 may be the period when CE is at 0 V. FIG. 12H shows exemplary simulated voltage levels of the circuit 1230. The top plot 1245 shows a trace of VCE (square waved trace). The middle plot 1246 shows a trace of RESET. The bottom plot 1247 shows a trace of RESET2. In some cases, the circuit may comprise one single reset switch which may be configured to perform the functions of RESET and RESET2 as disclosed herein. In some cases, the single reset switch may be configured to apply a plurality of temporary couplings between CC and VPRE when CE is at -VCE. In some cases, the single reset switch may be configured to apply a continuous coupling between CC and VPRE when CE is at 0 V. In some cases, the single reset switch may be configured to apply a plurality of couplings between CC and VPRE when CE is at 0 V. In some cases, a duration of a coupling of the plurality of couplings may be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70, at least about 90%, at least about 100%, at least about 200%, at least about 500%, or at least about 1000%, longer than a duration of a temporary coupling of the plurality of temporary couplings. In some cases, a duration of a coupling of the plurality of couplings may be at most about 1000%, at most about 500%, at most about 200%, at most about 100%, at most about 50%, at most about 10%, or at most about 5%, longer than a duration of a temporary coupling of the plurality of temporary couplings.

[0193]

[0194] To further reduce negative VBL or prevent VBL from going to a negative value, CE compensation may be applied. FIG. 121 shows exemplary simulated voltage levels of the unipolar CE scheme with CE compensation. 1251 shows the trace of RESET and RESET2. 1252 shows the trace of VCELL and VCE compensated. 1253 shows the trace of VWE1 and VCE compensated. 1254 shows the trace of VBL. The circuit is operated same way as in FIG. 12F until the fourth reset of CC. Following the fourth reset, VBL increases to 0.84 V.

[0194] Simultaneously, at 12.5 ms, VCE is changed from -1 V to VWE1 (e.g., about -0.16 V), and then gradually increases to 0 V over a period of 23.5 ms, instead of changing to 0 V at 12.5 ms and maintaining at 0 V for 23.5 ms. The period from 12.5 ms to 36 ms may be referred to as a dark time. The dark time in this case is 23.5 ms. The period from 10 ms to 12.5 ms may be referred to as a bright time. The bright time in this case is 2.5 ms. From 12.5 ms to 36 ms, RESET2 is turned on to re-charge CC. From 12.5 ms to 36 ms, RESET is turned on and off for a plurality of cycles. VCELL maintains 0 V from 12.5 ms to 36 ms. As there is no potential difference between CC and CE, no charge or discharge of CC will occur. VBL changes from 0.84 V to 0 V due to the change of VCE from -1 V to VWE1. In some cases, VBL may drift from 0 V to about -0.028 V as VCE goes from VWE1 to 0 V. The CE compensation further reduces the magnitude of negative VBL to about 2.8% of VCE (1 V), and significantly lowers the possibility of ejection of analyte or tag out of the nanopore, and resistance change or fluctuation in the nanopore. At 36 ms, another cycle of VCE change (-1 V for 2.5 ms and increasing from VWE1 to 0 V over 23.5 ms) occurs, and this process can be repeated.

[0195]

[0195] FIG. 12J shows exemplary simulated voltage levels of the unipolar CE scheme with CE compensation and shorter dark time. 1261 shows the trace of RESET and RESET2. 1262 shows the trace of VCELL and VCE compensated. 1263 shows the trace of VWE1 and VCE compensated. 1264 shows the trace of VBL. The circuit is operated same way as in FIG. 12G until the fourth reset of CC. Following the fourth reset, VBL increases to 0.84 V.

[0196] Simultaneously, at 12.5 ms, VCE is changed from -1 V to VWE1 (e.g., about -0.16 V), and then gradually increases to 0 V over a period of 3 ms (dark time of 3 ms), instead of over a period of

[0197] 23.5 ms (dark time of 23.5 ms). From 12.5 ms to 15.5 ms, RESET2 is turned on to re-charge CC. From 12.5 ms to 15.5 ms, RESET is turned on and off for a plurality of cycles. VCELL maintains 0 V from 12.5 ms to 15.5 ms. As there is no potential difference between CC and CE, no charge or discharge of CC will occur. VBL changes from 0.84 V to 0 V due to the change of VCE from -1 V to VWE1. As 3 ms is not sufficient for VWE1 to recover or return to 0 V, VBL may drift from 0 V to about -0.16 V as VCE goes from VWE1 to 0 V. The short dark time results in DC offset, which in turn produces a negative VBL of about 16% of VCE (1 V). At

[0198] 15.5 ms, another cycle of VCE change (-1 V for 2.5 ms and increasing from VWE1 to 0 V over 3 ms) occurs, and this process can be repeated. As in the dark time, no measurement is taken, a tag or analyte event (tag or analyte in the nanopore) may be missed for long dark time. When the dark time is shortened, the possibility of missing a tag or analyte event may be reduced, however, VBL may be increased as compared to longer dark time. There may be an adjustable dark time that can be optimized to reduce the possibility of missing a tag or analyte event and reduce VBL to a level that does not eject the tag or analyte out of the nanopore. In some cases, the systems and methods provided herein allows for such optimization.

[0199]

[0196] FIG. 12K shows exemplary simulated voltage levels of the unipolar CE scheme without CE compensation but with shorter dark time. 1271 shows the traces of RESET and RESET2. 1272 shows the traces of VCELL and VCE compensated. 1273 shows the traces of VWE1 and VCE compensated. 1274 shows the trace of VBL. No CE compensation is applied, e.g., VCE is changed to -1 V to 0 V instantaneously (no gradual increase). VCE is maintained at 0 V for a dark time of 3 ms. VBL goes to -0.25 V when VCE is changed to 0 V, due to the potential difference between CE and WEI. As 3 ms is not sufficient for VWE1 to recover or return to 0 V, the negative VBL slowly drifts toward 0 V in the dark time, but does not return to 0 V at the end of the dark time. The short dark time results in DC offset, which in turn produces a negative VBL of about 16% of VCE (1 V). At 15.5 ms, another cycle of VCE change (-1 V for 2.5 ms and increasing from VWE1 to 0 V over 3 ms) occurs, and this process can be repeated. A unipolar CE scheme without CE compensation but with shorter dark time results in higher negative VBL in comparison to cases with CE compensation and / or with longer dark time.

[0200]

[0197] The unipolar CE scheme as disclosed herein may have a different reset process (e.g., comprising an additional VPRE and reset set up). The unipolar CE scheme as disclosed herein may comprise a CE compensation mechanism to further reduce the negative VBL. The unipolar CE scheme as disclosed herein may reduce the negative VBL, thus resulting in reduced tag or analyte ejection and more consistent tag or analyte resistance measurement, in comparison to the bipolar CE scheme. The unipolar CE scheme as disclosed herein may improve the detection accuracy and sensitivity, in comparison to the bipolar CE scheme. In sequencing applications, the same nucleotide may create the same RC exponential waveform. As such, the system may output the same electrical signal for the same nucleotide. The system and method provided herein may distinguish a homopolymer unit (e.g., GG repeats or CGG repeats) because two Gs may be separated by time required for second tag to reach polymerase.

[0201]

[0198] FIG. 12L and 12M illustrate exemplary data showing measured signal for pore insertion (top left panel), signal noise check (top right panel), and sequencing (bottom panel) for different nanopore cells in the chip operated with the bipolar CE scheme, green points are signals for tags). FIG. 12N and 120 illustrate exemplary data showing measured signal for pore insertion (top left panel), signal noise check (top right panel), and sequencing (bottom panel) for different nanopore cells in the chip operated with the unipolar CE scheme. A step change of code (correlating to voltage) in the top left panels indicates a state change from membrane to pore insertion. Signals at the top indicate the voltage measured at CE and signals at the bottom indicate the voltage measured at WE. In bipolar CE scheme, as tag or analyte may be ejected, both CE and WE have measured signals. For unipolar CE scheme, as tag or analyte may not be ejected, only one of CE and WE have measured signal, e.g., no signal at negative voltages.

[0202]

[0199] In an aspect, the present disclosure provides a method for analyzing an analyte, comprising providing a nanopore system comprising (i) a nanopore disposed within a membrane, (ii) a first electrode in proximate to and at a first side of the nanopore, and (iii) a second electrode in proximate to and at a second side of the nanopore. In some cases, the second electrode may be operably connected to a capacitor. In some cases, the method may comprise applying a first potential to the first electrode. In some cases, the method may comprise applying a second potential to the capacitor. In some cases, the second potential may be different from the first potential. In some cases, the first potential and the second potential may direct at least a portion of an analyte into the nanopore. In some cases, the method may comprise disconnecting the capacitor from the second voltage source. Due to the difference in the first and second potentials, the capacitor may charge or discharge. In some cases, the method may comprise detecting one or more signals at the capacitor during the charge or discharge of the capacitor. In some cases, the method may comprise re-connecting the capacitor to the second voltage source temporarily to reset the capacitor (e.g., bringing the potential of the capacitor back to the second potential). After the temporary reset, the capacitor may be disconnected from the second voltage source. After the disconnection, the capacitor may charge or discharge due to the difference in the first and second potentials. In some cases, the method may comprise detecting one or more signals at the capacitor during the charge or discharge of the capacitor after the reset. The reset and detection cycle may be repeat one, two, three, four, or more times. In some cases, the method may comprise change the first potential on the first electrode to a value that is substantially same to the second potential that has been applied to the second electrode. In some cases, the first potential on the first electrode may be changed to the value that is substantially same to the second potential instantaneously. In some cases, the first potential on the first electrode may be changed to the value that is substantially same to the second potential gradually. In some cases, the first potential on the first electrode may be changed to the value that is substantially same to the second potential over a period of time. In some cases, the period of time for changing the first potential on the first electrode may be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, or at least about 100% longer than the time the first electrode has been at the first potential. In some cases, the period of time for changing the first potential on the first electrode may be at most about 100%, at most about 90%, at most about 70%, at most about 50%, at most about 30%, at most about 20%, at most about 10%, or at most about 5% longer than the time the first electrode has been at the first potential.

[0203]

[0200] The nucleotide can comprise one or more tags. The nucleotide can comprise at least 1, at least 2, at least 3, at least 4, at least 5, or more tags. The nucleotide can comprise at most 5, at most 4, at most 3, at most 2, or at most 1 tag. The tag can be configured to induce a change in the one or more signals of the sensor. In some cases, a plurality of types of tags can be used for a plurality of nucleotides, and one or more signals of each of the nucleotides with their respective tag can be approximately distinguishable from the others. In some cases, one or more features or characteristics of the nucleotide (e.g., size, shape, charge, vibration, movement within in the fluid, etc.) can further affect the one or more signals of the sensors, thereby further rendering the one or more signals of each of the nucleotides with their respective tag more distinguishable from the others. In some cases, a tag of the present disclosure can be an impedance tag. The impedance tag can be configured to elicit a change in detected impedance in the sensor, e.g., between a working electrode and a reference electrode. Examples of the impedance tag can include, but are not limited to, organic compounds, organometallic compounds, nanoparticles, metals, or functional variants thereof, or combinations thereof.

[0204]

[0201] Alternatively, the nucleotide may not comprise any tag. In such cases, one or more features or characteristics of the nucleotide (e.g., size, shape, charge, vibration, movement within in a fluid, etc.) can further affect the one or more signals of the sensors, thereby further rendering the one or more signals of each of the nucleotides with their respective tag more distinguishable from the others. In some cases, the method may comprise contacting at least a portion of the analyte with the nanopore of the nanopore system. In some cases, the at least a portion of the analyte may be in proximity to the nanopore.

[0205]

[0202] In some cases, the method may comprise detecting one or more signals indicative of an impedance or change thereof associated with the at least the portion of the analyte while the at least the portion of the analyte is in proximity to the nanopore. In some cases, the method may comprise using the one or more signals to identify one or more characteristics of the at least the portion of the analyte with an accuracy of at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, at least about 99.5%, or at least about 99.9%.

[0206]

[0203] Nanopore with multiple chambers

[0207]

[0204] In an aspect, the present disclosure provides a method for analyzing a plurality of samples. In some cases, the method may comprise providing a nanopore system. In some cases, the nanopore system may comprise at least 500,000 fluidic chambers. In some cases, the at least 500,000 fluidic chambers may be capable of independently analyzing a sample. In some cases, the method may comprise contacting a plurality of samples with the at least 500,000 fluidic chambers. In some cases, the method may comprise detecting one or more signals indicative of an impedance or change thereof in the at least 500,000 fluidic chambers. In some cases, the method may comprise using the one or more signals to identify one or more characteristics of the plurality of samples.

[0208]

[0205] As a fluidic chamber may independently analyze a sample, a plurality of samples may be analyzed by the nanopore system. In some cases, the method may be a high throughput method. In some cases, the method may comprise analyzing a plurality of samples simultaneously. In some cases, the method may comprise analyzing a plurality of samples sequentially. In some cases, the method may analyze different types of analytes (e.g., nucleic acid, protein, or small molecule). In some cases, the method may analyze different analytes of the same type.

[0206] In some cases, the nanopore system may comprise at least 600,000, at least 700,000, at least 800,000 at least 900,000, or at least 1,000,000 fluidic chambers. In some cases, the fluidic chambers may be independently addressable. In some cases, the method may comprise contacting a sample of the plurality of samples with a fluidic chamber and contacting an additional sample of the plurality of samples with an additional fluidic chamber. In some cases, the sample and the additional sample may be same. In some cases, the same samples can be analyzed by a plurality of fluidic chambers to increase the accuracy of the analysis (e.g., removing outliners of the plurality analysis result to reduce instrument error and / or procedural error). In some cases, the sample and the additional sample may be different. In some cases, the sample may comprise one or more analytes and the additional sample comprise one or more additional analytes. In some cases, the one or more analytes and the one or more additional analytes may be different. In some cases, the one or more analytes may comprise a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof. In some cases, the one or more additional analytes may comprise a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof. In some cases, a first plurality of the fluidic chambers may be configured to analyze nucleic acids or nucleotides. In some cases, a second plurality of the fluidic chambers may be configured to analyze peptides, polypeptides, or proteins. In some cases, a third plurality of the fluidic chambers may be configured to analyze a small molecule (e.g., a therapeutic drug).

[0209]

[0207] In some cases, a first plurality of the fluidic chambers may be configured to analyze one type of nucleic acids. In some cases, a second plurality of the fluidic chambers may be configured to analyze another type of nucleic acids.

[0210]

[0208] In some cases, a first plurality of the fluidic chambers may be configured to analyze one type of peptides, polypeptides, or proteins. In some cases, a second plurality of the fluidic chambers may be configured to analyze another type of peptides, polypeptides, or proteins.

[0211]

[0209] In some cases, a first plurality of the fluidic chambers may be configured to analyze one type of small molecules. In some cases, a second plurality of the fluidic chambers may be configured to analyze another type of small molecules.

[0212]

[0210] Corresponding nanopore systems

[0213]

[0211] In various aspects, the present disclosure provides a computer processor and a computer memory coupled thereto, wherein the computer memory comprises a machine executable code that, upon execution by the one or more computer processors, implements an one of the methods provided herein. In some cases, the system comprises the nanopore system (or nanopore device) as provided herein.

[0214]

[0212] In an aspect, the present disclosure provides nanopore system. In some cases, the nanopore system may comprise a sensor. In some cases, the sensor may comprise a nanopore disposed within a membrane. In some cases, the sensor may comprise a first electrode disposed adjacent to a first side of the nanopore. In some cases, the sensor may comprise a second electrode disposed adjacent to a second and different side of the nanopore. In some cases, the nanopore system may comprise a processor operatively coupled to the sensor. In some cases, the processor may be configured to direct the sensor to apply a time-varying asymmetrical voltage waveform between the first electrode and the second electrode. In some cases, the processor may be configured to direct the sensor to detect one or more electrical signals associated with the at least the portion of the analyte.

[0215]

[0213] In an aspect, the present disclosure provides a nanopore system. In some cases, the nanopore system may comprise a sensor. In some cases, the sensor may comprise a nanopore disposed within a membrane. In some cases, the sensor may comprise a first electrode disposed adjacent to a first side of the nanopore. In some cases, the sensor may comprise a second electrode disposed adjacent to a second and different side of the nanopore. In some cases, the sensor may comprise at least one capacitor electronically coupled to the second electrode. In some cases, the sensor may comprise at least one electronic switch for generating a plurality of sequential temporary couplings between the at least one capacitor and at least one voltage source. In some cases, the sensor may comprise a processor operatively coupled to the sensor. In some cases, the processor may be configured to direct the sensor to detect a plurality of electrical signals associated with the at least a portion of an analyte between the plurality of sequential temporary couplings. In some cases, the nanopore system may comprise any additional components as disclosed herein to implement any method disclosed herein.

[0216]

[0214] In an aspect, the present disclosure provides a nanopore system (e.g., a nanopore device). In some cases, the nanopore system may comprise a flow cell chip comprising at least 500,000 fluidic chambers. In some cases, the nanopore device may comprise a plurality of sensors configured to measure one or more signals indicative of an impedance or change thereof. In some cases, each fluidic chamber of the at least 500,000 fluidic chambers may be coupled to a working electrode. In some cases, each fluidic chamber may comprise a nanopore embedded in a membrane. In some cases, the flow cell chip may comprise at least 600,000 fluidic chambers. In some cases, the flow cell chip may comprise at least 800,000 fluidic chambers. In some cases, the flow cell chip may comprise at least 1,000,000 fluidic chambers. In some cases, the at least 500,000 fluidic chambers may be independently addressable. In some cases, a sensor of the plurality of sensors may comprise one or more signal generators. In some cases, the one or more signal generators may be configured to apply one or more potentials to one or more electrodes of the sensor. In some cases, the nanopore system may comprise any additional components as disclosed herein to implement any method disclosed herein.

[0217]

[0215] In an aspect, the present disclosure provides a nanopore system. In some cases, the nanopore system may comprise a fluidic chamber. In some cases, the nanopore system may comprise a nanopore disposed within a membrane. In some cases, the membrane may separate the fluidic chamber to a first side and a second side. In some cases, the nanopore system may comprise a sensor. In some cases, the sensor may comprise a working electrode in proximity to the nanopore. In some cases, the sensor may be configured to detecting one or more signals indicative of an impedance or change thereof associated with at least a portion of an analyte. In some cases, the sensor may be further configured to use the one or more signals to identify one or more characteristics of the at least the portion of the analyte with an accuracy of at least about 90%. In some cases, the nanopore system may comprise any additional components as disclosed herein to implement any method disclosed herein.

[0218]

[0216] In an aspect, the present disclosure provides a nanopore system. In some cases, the nanopore system may comprise a nanopore disposed within a membrane. In some cases, the nanopore system may comprise one or more electrodes in proximity to the nanopore. In some cases, the nanopore system may comprise one or more signal generators in electrical communication with the one or more electrodes. In some cases, the one or more signal generators may be configured to apply one or more potentials to the one or more electrodes. In some cases, the one or more signal generators may be configured to stabilize the potential. In some cases, the nanopore system may comprise one or more processors in electrical communication with the one or more electrodes. In some cases, the one or more processors may be independently or collectively configured to detect one or more signals indicative of an impedance or change thereof associated with at least a portion of an analyte. In some cases, the one or more processors may be independently or collectively configured to use the one or more signals to identify one or more characteristics of the at least the portion of the analyte. In some cases, the nanopore system may comprise any additional components as disclosed herein to implement any method disclosed herein.

[0219]

[0217] Additional details

[0220]

[0218] In some cases, the method provided by the present disclosure can be used to determine an identity of an analyte. In some cases, determining an identity of an analyte may comprise comprising one or more signals with a reference signal to determine if the detected analyte has identity to the reference. In some cases, the method provided by the present disclosure can be used to determine a presence of an analyte. In some cases, determining a presence of an analyte may comprise comprising one or more signals with a reference signal to determine if the analyte is present in the sample. In some cases, if the detected signal does not have identity to the reference signal, the analyte may not be present in the sample.

[0221]

[0219] In some cases, the analysis of the analyte may take at least 30 s, at least 1 min, at least 10 min, at least 30 min, at least 1 h, at least 2 h, at least 3 h, at least 5 h, at least 10 h, or at least 12 h. In some cases, the analysis of the analyte may take at most 12 h, at most 10 h, at most 5 h, at most 3 h, at most 2 h, at most 1 h, at most 30 min, at most 10 min, at most 1 min, or at most 30 s.

[0222]

[0220] In some cases, the systems, devices, and methods disclosed herein may have a long read of nucleic acid molecule. In some cases, the systems, devices, and methods disclosed herein may have a long read of at least about 1 kb, at least about 5 kb, at least about 10 kb, at least about 20 kb, at least about 50 kb, at least about 100 kb, at least about 500 kb, or at least about 1 Mb.

[0223]

[0221] In some cases, the systems, devices, and methods disclosed herein may have a high yield. In some cases, the systems, devices, and methods disclosed herein may have a high yield of at least about 0.5 Gb data / run, at least about 1 Gb data / run, at least about 5 Gb data / run, at least about 10 Gb data / run, or at least about 50 Gb data / run.

[0224] Systems and Methods for Processing a Nucleic Acid Molecule

[0225]

[0222] In an aspect, the present disclosure provides a method of processing a single stranded nucleic acid molecule. In some cases, the method may comprise providing a single stranded linear nucleic acid molecule. In some cases, the single stranded linear nucleic acid molecule may be derived from a double stranded linear nucleic acid molecule.

[0226]

[0223] In some cases, the double stranded linear nucleic acid molecule may be obtained from a biological sample (e.g., cells or biological fluids). In some cases, the double stranded linear nucleic acid molecule may be obtained from cultured cells or tissues of a subject. In some cases, the subject can comprise a human, an animal, an insect (e.g., a drosophila), or a plant. In some cases, the double stranded linear nucleic acid molecule may be obtained from clinical samples like plasma and / or tissue biopsies from various cancers and other diseases. In some cases, the double stranded linear nucleic acid molecule can be chemically modified (e.g., cleaved or edited). In some cases, the double stranded linear nucleic acid molecule may not have been chemically modified. In some cases, the double stranded linear nucleic acid molecule can be conjugated. In some cases, two or more double stranded linear nucleic acid molecules can be ligated into a longer double stranded linear molecule. In some cases, the two or more double stranded linear nucleic acid molecules can be ligated by an enzymatic ligation.

[0227]

[0224] In some cases, the double stranded linear nucleic acid molecule may be naturally occurring. In some cases, the double stranded linear nucleic acid molecule may be a synthetic molecule. In some cases, the double stranded linear nucleic acid molecule may not be a synthetic molecule. In some cases, the double stranded linear nucleic acid molecule may be directly obtained from a biological sample of a subject. In some cases, the double stranded linear nucleic acid molecule may comprise a genomic nucleic acid. In some cases, the double stranded linear nucleic acid molecule may comprise a genomic DNA or gDNA. In some cases, the double stranded linear nucleic acid molecule may comprise an amplified population of a double stranded nucleic acid. In some cases, the double stranded linear nucleic acid molecule may comprise an amplicon.

[0228]

[0225] In some cases, the biological sample may comprise circular DNAs, circular RNAs, or linear RNAs. In some cases, the biological sample may be pre-treated to remove the circular DNAs, circular RNAs, or linear RNAs from the biological sample before processing the double stranded linear nucleic acid molecule.

[0229]

[0226] In some cases, the method may further comprise denaturing a double stranded linear nucleic acid molecule to obtain the single stranded nucleic acid molecule. In some cases, the method may further comprise, prior to the denaturing, coupling a double stranded adaptor to an end of the double stranded linear nucleic acid molecule. In some cases, the method may further comprise prior to the denaturing, coupling an additional double stranded adaptor to an additional end of the double stranded linear nucleic acid molecule. In some cases, the at least a portion of the oligonucleotide comprises sequence complementarity to at least a portion of a strand of the double stranded adaptor. In some cases, the at least the additional portion of the oligonucleotide may comprise sequence complementarity to at least a portion of a strand of the additional double stranded adaptor.

[0230]

[0227] In some cases, the method may comprise circularizing the single stranded linear nucleic acid molecule. In some cases, the circularization may be performed in a presence of an oligonucleotide. In some cases, at least a portion of the oligonucleotide may comprise sequence complementarity to at least a portion of an end of the single stranded linear nucleic acid molecule. In some cases, at least an additional portion of the oligonucleotide may comprise sequence complementarity to at least an additional portion of an additional end of the single stranded linear nucleic acid molecule. In some cases, the end of the single stranded linear nucleic acid molecule may be the first end and the additional end of the single stranded linear nucleic acid molecule may be the second end of the single stranded linear nucleic acid molecule.

[0231]

[0228] In some cases, the circularizing may comprise hybridizing the at least the portion of the oligonucleotide with the at least the portion of the end of the single stranded linear nucleic acid molecule. In some cases, the circularizing may comprise hybridizing the additional portion of the oligonucleotide with the at least the additional portion of the additional end of the single stranded linear nucleic acid molecule. In some cases, the circularizing may comprise, via the hybridizing, generating a complex comprising a circularized single stranded nucleic acid molecule hybridized with the oligonucleotide. In some cases, the circularized single stranded nucleic acid molecule may comprise a nick. In some cases, the method may further comprise ligating an end of the circularized single stranded nucleic acid molecule and an additional end of the circularized single stranded nucleic acid molecule to generate a closed circularized single stranded nucleic acid molecule or a single stranded circular (ssc) nucleic acid molecule. In some cases, the method may further comprise removing the oligonucleotide.

[0232]

[0229] In some cases, the method may further comprise coupling a nucleic acid adapter to an end of a double stranded linear nucleic acid molecule. In some cases, the method may further comprise coupling an additional nucleic acid adapter to an additional end of the double stranded linear nucleic acid molecule. In some cases, the nucleic acid adapter and / or the additional nucleic acid adapter may comprise a Y adapter or a double stranded adapter.

[0233]

[0230] In some cases, the method may comprise amplifying the double stranded linear nucleic acid molecule. In some cases, the amplifying may comprise coupling a forward primer comprising complementarity to one strand of the nucleic acid adapter and coupling a reverse primer comprising complementarity to one strand of the additional nucleic acid adapter.

[0234]

[0231] In some cases, the ligation can be performed with an enzyme, e.g., a ligase. In some cases, a ligase can comprise Circligase™ (Epicentre; Madison, Wis.), NAD-dependent ligases such as Taq DNA ligase, Thermus filiformis DNA ligase, Escherichia coliDNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligase (I and II), thermostable ligase, Ampligase thermostable DNA ligase, VanC-type ligase, 9° N DNA Ligase, Tsp DNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, and DNA ligase IV.

[0235]

[0232] After generation of the library molecules, nucleic acid molecules with non-ligated nicks and linear nucleic acid molecules may be eliminated from the library by treatment with an enzyme. In some cases, the enzyme can comprise an exonuclease. In some cases, the enzyme can comprise Exonuclease III.

[0236]

[0233] FIG. 13A shows an exemplary workflow for analyzing a nucleic acid molecule. The workflow may comprise obtaining a sample. The workflow may comprise pre-processing the sample. The sample comprising nucleic acid (e.g., 831) can be pre-processed to generate a library of double stranded nucleic acid molecule 832. In some cases, the pre-processing may include amplification (e.g., PCR). In some cases, the pre-processing may include purification. In some cases, the amplification may enrich a population of nucleic acid molecules. In some cases, the nucleic acids may comprise nucleic acids from bacteria. In some cases, the amplification may comprise generating a library of double stranded nucleic acid molecule with certain region on the nucleic acid molecule. In some cases, the certain region on the nucleic acid molecule may be used to identify a genus and / or a species of a bacterium. The workflow may comprise generating single stranded circular nucleic acid molecule. The double stranded nucleic acid molecule 832 may be denatured to form a single stranded nucleic acid molecule. The single stranded nucleic acid molecule may be circularized in the presence of a bridge oligonucleotide to form a complex (e.g., 833) comprising a circular nucleic acid hybridized with the bridge oligonucleotide. The workflow may comprise a digestion reaction to remove the bridge oligonucleotide to generate a circular nucleic acid molecule for sequencing. The workflow may comprise a purification step to remove any linear nucleic acid molecule. The workflow may comprise a sequencing step to obtain a sequence information of at least a portion of the circular nucleic acid molecule.

[0237]

[0234] FIG. 13B shows an exemplary process for preparing a single stranded circular nucleic acid molecule. A double stranded linear nucleic acid molecule 801 comprises two strands. P refers to a phosphate group at a 5’ end of a strand of the double stranded linear nucleic acid molecule 801. The double stranded linear nucleic acid molecule 801 may be end-repaired to form a double stranded linear nucleic acid molecule 802. The double stranded linear nucleic acid molecule 802 may comprise P at the 5’ end of each strand. The double stranded linear nucleic acid molecule 802 may be A-tailed (e.g., adding at least one base A to the 3’ end of each strand) to form a double stranded linear nucleic acid molecule 803. The double stranded linear nucleic acid molecule 803 may be ligated with two adapters (e.g., 804) to form a chimeric double stranded linear nucleic acid molecule 805. The two adapters may be double stranded linear adapters or Y-adapters. The chimeric double stranded linear nucleic acid molecule 805 may comprise two Y adapters flanked at both ends. The chimeric double stranded linear nucleic acid molecule 805 may be amplified through a polymerase chain reaction (PCR) to generate an amplicon 806 (P shows the phosphate group at 5’ end). The amplicon 806 may be separated (e.g., denatured) to form two single stranded linear nucleic acid molecules (e.g., 807). The single stranded linear nucleic acid molecules 807 may be circularized in the presence of a bridge oligonucleotide (“bridge oligo”) to form a complex 808 comprising a circularized stranded linear nucleic acid molecule 810 hybridized with the bridge oligo 809. The circularized stranded linear nucleic acid molecule 810 may be further ligated to connect the two ends of the circularized stranded linear nucleic acid molecule to form a closed circular. The bridge oligo in the complex 808 may be removed by an enzyme (e.g., Exol or ExoIII), thereby generating a single stranded circular nucleic acid molecule 811. The single stranded circular nucleic acid molecule 811 may be usable or used in subsequent sequencing.

[0235] In some cases, the method may further comprise coupling a primer to the single stranded circular nucleic acid molecule. In some cases, the oligonucleotide may function as a primer.

[0238]

[0236] In some cases, a library of the single stranded circular nucleic acid molecules may be generated. In some cases, the method may further comprise contacting the single stranded circular nucleic acid molecule with a nucleotide under conditions sufficient to incorporate the nucleotide to an end of the primer, thereby generating a growing strand. In some cases, the method may further comprise contacting the closed circularized single stranded nucleic acid molecule with one or more nucleotides under conditions sufficient to incorporate the nucleotide to an end of the growing strand.

[0239]

[0237] In some cases, the growing strand may have sequence complementarity to at least a portion of the single stranded circular nucleic acid molecule. In some cases, the growing strand may have at least one copy of the single stranded circular nucleic acid molecule. In some cases, the growing strand may two or more copies of the single stranded circular nucleic acid molecule.

[0240]

[0238] In some cases, a size of a primer can be at least or up to about 3 nucleotides, at least or up to about 4 nucleotides, at least or up to about 5 nucleotides, at least or up to about 6 nucleotides, at least or up to about 7 nucleotides, at least or up to about 8 nucleotides, at least or up to about 9 nucleotides, at least or up to about 10 nucleotides, at least or up to about 11 nucleotides, at least or up to about 12 nucleotides, at least or up to about 13 nucleotides, at least or up to about 14 nucleotides, at least or up to about 15 nucleotides, at least or up to about 16 nucleotides, at least or up to about 17 nucleotides, at least or up to about 18 nucleotides, at least or up to about 19 nucleotides, at least or up to about 20 nucleotides, at least or up to about 25 nucleotides, or at least or up to about 30 nucleotides, at least or up to about 40 nucleotides, or at least or up to about 50 nucleotides.

[0241]

[0239] In some cases, a primer may comprise a hexamer, heptamer, octamer, nonamer, or decamer. In some cases, the primer can comprise a random er. In some cases, the oligonucleotide primer can comprise a DNA primer. In some cases, the primer can comprise a gene-specific DNA primer.

[0242]

[0240] In some cases, the extension reaction may comprise using a polymerase to add one or more nucleotides to the growing strand. In some cases, the extension reaction may comprise contacting the nucleic acid molecule with a solution comprising one or more nucleotides. In some cases, the solution may comprise a plurality of nucleotides (e.g., A, T, C, and G). In some cases, each type of the nucleotides may be coupled to a different tag as disclosed in the present disclosure. In some cases, each of the tag may have a concentration of at least about 0.05 pM, at least about 0.1 pM, at least about 0.5 pM, at least about 1 pM, at least about 5 pM, at least about 10 pM, at least about 50 pM, at least about 100 pM, at least about 200 pM, at least about 500 pM, at least about 1 mM, or at least about 10 mM. In some cases, each of the tag may have a concentration of at most about 10 mM, at most about 1 mM, at most about 500 pM, at most about 200 pM, at most about 100 gM, at most about 50 gM, at most about 10 gM, at most about 5 pM, at most about 1 pM, at most about 0.5 gM, at most about 0.1 gM, or at most about 0.05 pM.

[0243]

[0241] In some cases, the method may further comprise using the growing strand to analyze a sequence of the at least a portion of the single stranded circular nucleic acid molecule. In some cases, during generation of the growing strand, the method may comprise using a sensor to obtain sequence information of at least a portion of the growing strand. In some cases, the sequence information of the at least the portion of the growing strand may be used to analyze at least a portion of the single stranded circular nucleic acid molecule. In some cases, the method may comprise directly detecting the at least the portion of the growing strand to obtain the sequence information. In some cases, the detecting may be performed while a portion of the growing strand is hybridized to a portion of the single stranded circular nucleic acid molecule. In some cases, the detecting may be performed without generating an amplified copy of the growing strand.

[0244]

[0242] In some cases, the time between generation of the growing strand and measurement of the sequence information of at least a portion of the growing strand may be at most about 5 minutes (min), at most about 4 min, at most about 3 min, at most about 2 min, at most about 1 min, at most about 50 seconds (s), at most about 40 s, at most about 30 s, at most about 20 s, at most about 10 s, at most about 1 s, at most about 900 milliseconds (ms), at most about 800 ms, at most about 700 ms, at most about 600 ms, at most about 500 ms, at most about 400 ms, at most about 300 ms, at most about 200 ms, at most about 100 ms, at most about 50 ms, at most about 10 ms, at most about 1 ms, at most about 900 microseconds (ps), at most about 800 ps, at most about 700 ps, at most about 600 ps, at most about 500 ps, at most about 400 ps, at most about 300 ps, at most about 200 ps, at most about 100 ps, at most about 50 ps, at most about 10 ps, at most about 1 ps, at most about 900 nanoseconds (ns), at most about 800 ns, at most about 700 ns, at most about 600 ns, at most about 500 ns, at most about 400 ns, at most about 300 ns, at most about 200 ns, at most about 100 ns, at most about 90 ns, at most about 80 ns, at most about 70 ns, at most about 60 ns, at most about 50 ns, at most about 40 ns, at most about 30 ns, at most about 20 ns, at most about 10 ns, at most about 9 ns, at most about 8 ns, at most about 7 ns, at most about 6 ns, at most about 5 ns, at most about 4 ns, at most about 3 ns, at most about 2 ns, at most about 1 ns, or less.

[0245]

[0243] In some cases, the sequence information of the at least the portion of the growing strand may be measured substantially in real-time relative to the generation of the growing strand. In some cases, the sequence information of the at least the portion of the growing strand may be measured immediately after or within a short period of time after the generation of the growing strand. In some cases, the short period of time may be at most about 1 ms, at most about 900 ps, at most about 800 ps, at most about 700 ps, at most about 600 ps, at most about 500 ps, at most about 400 ps, at most about 300 ps, at most about 200 ps, at most about 100 ps, at most about 50 ps, at most about 10 ps, at most about 1 ps, at most about 900 ns, at most about 800 ns, at most about 700 ns, at most about 600 ns, at most about 500 ns, at most about 400 ns, at most about 300 ns, at most about 200 ns, at most about 100 ns, at most about 90 ns, at most about 80 ns, at most about 70 ns, at most about 60 ns, at most about 50 ns, at most about 40 ns, at most about 30 ns, at most about 20 ns, at most about 10 ns, at most about 9 ns, at most about 8 ns, at most about 7 ns, at most about 6 ns, at most about 5 ns, at most about 4 ns, at most about 3 ns, at most about 2 ns, at most about 1 ns, or less.

[0246]

[0244] In some cases, the time between (i) addition of a nucleotide to the growing strand during the generation of the growing strand and (ii) measurement, detection, or sequencing of the addition of the nucleotide to the growing strand may be at most about 5 min, at most about 4 min, at most about 3 min, at most about 2 min, at most about 1 min, at most about 50 s, at most about 40 s, at most about 30 s, at most about 20 s, at most about 10 s, at most about 1 s, at most about 900 ms, at most about 800 ms, at most about 700 ms, at most about 600 ms, at most about 500 ms, at most about 400 ms, at most about 300 ms, at most about 200 ms, at most about 100 ms, at most about 50 ms, at most about 10 ms, at most about 1 ms, at most about 900 ps, at most about 800 ps, at most about 700 ps, at most about 600 ps, at most about 500 ps, at most about 400 ps, at most about 300 ps, at most about 200 ps, at most about 100 ps, at most about 50 ps, at most about 10 ps, at most about 1 ps, at most about 900 ns, at most about 800 ns, at most about 700 ns, at most about 600 ns, at most about 500 ns, at most about 400 ns, at most about 300 ns, at most about 200 ns, at most about 100 ns, at most about 90 ns, at most about 80 ns, at most about 70 ns, at most about 60 ns, at most about 50 ns, at most about 40 ns, at most about 30 ns, at most about 20 ns, at most about 10 ns, at most about 9 ns, at most about 8 ns, at most about 7 ns, at most about 6 ns, at most about 5 ns, at most about 4 ns, at most about 3 ns, at most about 2 ns, at most about 1 ns, or less.

[0247]

[0245] In some cases, the addition of a nucleotide to the growing strand and the measurement, detection, or sequencing of the addition of the nucleotide to the growing strand can occur substantially in real-time. In some cases, the measurement, detection, or sequencing of the addition of the nucleotide to the growing strand may be performed immediately after or within a short period of time after the addition of a nucleotide to the growing strand. In some cases, the short period of time may be at most about 1 ms, at most about 900 ps, at most about 800 ps, at most about 700 jus, at most about 600 jus, at most about 500 jus, at most about 400 jus, at most about 300 ps, at most about 200 jus, at most about 100 jus, at most about 50 jus, at most about 10 ps, at most about 1 jus, at most about 900 ns, at most about 800 ns, at most about 700 ns, at most about 600 ns, at most about 500 ns, at most about 400 ns, at most about 300 ns, at most about 200 ns, at most about 100 ns, at most about 90 ns, at most about 80 ns, at most about 70 ns, at most about 60 ns, at most about 50 ns, at most about 40 ns, at most about 30 ns, at most about 20 ns, at most about 10 ns, at most about 9 ns, at most about 8 ns, at most about 7 ns, at most about 6 ns, at most about 5 ns, at most about 4 ns, at most about 3 ns, at most about 2 ns, at most about 1 ns, or less.

[0248]

[0246] In some cases, the extension reaction may generate a concatemer comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more copies / repeats of the sequences that are complementary to the single stranded circular nucleic acid molecule. Consensus sequencing of the multiple copies of the sequences can improve the accuracy of sequencing data. The concatemer sequencing that provides a consensus sequence for the multiple copies / repeats provides a sensitive and accurate analysis of the single stranded circular nucleic acid molecule in a sample.

[0249]

[0247] In some cases, a sensor may be configured to detect one or more signals indicative of incorporation of one or more nucleotides into the at least the portion of the growing strand to obtain the sequence information.

[0250]

[0248] The one or more signals can be current or voltage. The one or more signals can be current and voltage. The one or more signals may be tunneling current. The one or more signals may not be tunneling current. The one or more signals may be ionic current. The one or more signals may not be ionic current. In some cases, the one or more signals may comprise an impedance or change thereof associated with the chimeric nucleic acid molecule, the growing strand, and / or the solution in proximity to the chimeric nucleic acid molecule and / or the growing strand.

[0251]

[0249] In some cases, the sensor may be configured to obtain the sequence information of the at least the portion of the growing strand while a portion of the growing strand is hybridized to a portion of the single stranded circular nucleic acid molecule. In some cases, the sensor may be configured to obtain the sequence information of the at least the portion of the growing strand without generating an amplified copy of the growing strand. In some cases, the sensor may be configured to measure an impedance value or a change thereof in the sensor during the generation of the growing strand to obtain the sequence information. In some cases, the sensor may be configured to directly detect the at least the portion of the growing strand to obtain the sequence information.

[0250] In some cases, the method provided by the present disclosure can be used to determine an identity of a nucleic acid. In some cases, determining an identity of a nucleic acid may comprise aligning one or more sequencing reads with a reference sequence to determine if the detected nucleic acid has sequence identity to the reference sequence.

[0252]

[0251] In some cases, the method provided by the present disclosure can be used to identify one or more mutations in a sequence. In some cases, identifying one or more mutations may comprise aligning one or more sequencing reads with a reference sequence to identify differences between the two. In some cases, the differences may be identified as the mutations.

[0253]

[0252] In some cases, the system may further comprise a polymerase for facilitating an extension reaction to generate a growing strand. In some cases, the polymerase may be coupled to a surface of a sensor. In some cases, the polymerase may be coupled to the surface of the sensor prior to the annealing of a primer to the single stranded circular nucleic acid molecule. In some cases, the polymerase may be coupled to the surface of the sensor simultaneously with the annealing of the primer to the single stranded circular nucleic acid molecule. In some cases, the polymerase may be coupled to the surface of the sensor subsequent to the annealing of the primer to the single stranded circular nucleic acid molecule. In some cases, the polymerase may be coupled to the surface of the sensor prior to the start of the generation of the growing strand. In some cases, the polymerase may be coupled to the surface of the sensor simultaneously with the start of the generation of the growing strand. In some cases, the polymerase may be coupled to the surface of the sensor subsequent to the start of the generation of the growing strand. In some cases, the polymerase may be coupled to a single stranded circular nucleic acid molecule or a single stranded circular nucleic acid molecule -primer complex, generating a polymerase- single stranded circular nucleic acid molecule -primer complex for subsequent sequencing. In some cases, the polymerase may not be coupled to the surface of the sensor. In some cases, the polymerase may be present in a solution that the single stranded circular nucleic acid molecule is disposed at. In some cases, the polymerase may be coupled to single stranded circular nucleic acid molecule or a single stranded circular nucleic acid molecule -primer complex in the solution, generating a polymerase- single stranded circular nucleic acid molecule -primer complex. In some cases, the polymerase- single stranded circular nucleic acid molecule -primer complex may be coupled to a sensor for subsequent sequencing.

[0254]

[0253] In some cases, the polymerase can be coupled to a surface of the sensor through a covalent or non-covalent bond. In some cases, the polymerase is tethered to a surface of the sensor indirectly. In some cases, the polymerase can be tethered to a surface of the sensor through a linker, for example, an alkane linker, an azide linker, or a biotin-streptavidin linker. In some cases, the sensor may comprise a nanopore. In some cases, the nanopore may comprise a spytag peptide. In some cases, the polymerase may comprise a spycatcher peptide that may couple the polymerase to the nanopore through a spycatcher-spytag interaction.

[0255]

[0254] In some cases, the system may further comprise a plurality of nucleotides comprising a tag. In some cases, the plurality of nucleotides can be configured to be incorporated into the at least a portion of a growing strand. The tag can be any suitable tag that is disclosed herein.

[0256]

[0255] Devices and systems for use in methods provided by the present disclosure may accurately detect individual nucleotide incorporation events, such as upon the incorporation of a nucleotide into a growing strand that is complementary to a template. An enzyme such as a polymerase can incorporate nucleotides to a growing polynucleotide chain.

[0257]

[0256] The added nucleotide can be complimentary to the corresponding template polynucleotide strand (e.g., single stranded circular nucleic acid molecule) which is hybridized to the growing strand. A nucleotide can include a tag or tag species that is coupled to any location of the nucleotide including, but not limited to a phosphate such as a y- phosphate, sugar or nitrogenous base moiety of the nucleotide. In some cases, tags may be detected while the tags are associated with a polymerase during the incorporation of nucleotides. The tag may continue to be detected until the tag translocates through the nanopore after nucleotide incorporation and subsequent cleavage and / or release of the tag. Nucleotide incorporation events can release tags from the nucleotides which pass through a nanopore and are detected. A tag can be released by the polymerase, or cleaved / released in any suitable manner including but without limitation cleavage by an enzyme located near the polymerase. In this way, the incorporated base may be identified (i.e., adenine, cytosine, guanine, thymine or uracil) because a unique tag is released from each type of nucleotide (i.e., adenine, cytosine, guanine, thymine or uracil). In nucleotide incorporation events that do not release a tag, a tag coupled to an incorporated nucleotide may be detected with the aid of a nanopore. In some examples, the tag can move through or in proximity to the nanopore and be detected with the aid of the nanopore.

[0258]

[0257] Methods and systems of the disclosure can enable the detection of polynucleotide incorporation events, such as at a resolution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10000, at least 50000, at least 100000, or more polynucleotide bases within a given time period. For example, a nanopore system can be used to detect individual polynucleotide incorporation events, with each event being associated with an individual nucleic acid base. In other examples, a nanopore system can be used to detect an event that is associated with a plurality of bases. For example, a signal sensed by the nanopore device can be a combined signal from at least 2, at least 3, at least 4, or at least 5 bases.

[0258] In some cases, tags do not pass through the nanopore. In some cases, the tags can be detected by the nanopore and exit the nanopore without passing through the nanopore such as exiting from the inverse direction from which the tag entered the nanopore. A sequencing device can be configured to actively expel the tags from the nanopore.

[0259]

[0259] In some cases, tags are not released upon nucleotide incorporation events. Nucleotide incorporation events can present tags to a nanopore without releasing the tags. The tags can be detected by the nanopore without being released from the nucleotide. The tags may be attached to the nucleotides by a linker of sufficient length to present the tag to the nanopore for detection.

[0260]

[0260] Nucleotide incorporation events may be detected in real-time as they occur by a nanopore. An enzyme such as a polymerase attached to or in proximity to a nanopore can facilitate the flow of a polynucleotide through or adjacent to a nanopore. A nucleotide incorporation event, or the incorporation of a plurality of nucleotides, may release or present one or more tags, which may be detected by a nanopore. Detection can occur as the tags flow through or adjacent to the nanopore, as the tags reside in the nanopore and / or as the tags are presented to the nanopore. In some cases, an enzyme attached to or in proximity to the nanopore may aid in detecting tags upon the incorporation of one or more nucleotides.

[0261]

[0261] A tag can be an atom, a molecule, a collection of atoms, or a collection of molecules. A tag may provide an optical, electrochemical, magnetic, or electrostatic such as an inductive or capacitive, signature, which signature may be detected with the aid of a nanopore.

[0262]

[0262] In some cases, the tag can have a size that is sufficiently large to induce a change in the electrochemical property (e.g., capacitance, impedance, etc.) of the electrochemical cell (e.g., a nanopore sensor, a sensor without a nanopore, etc.) as disclosed herein, when the nucleotide is sufficiently close to a sensor moiety of the nanopore systems. In some cases, the change in the electrochemical property can occur and can be detectable prior to, during, or subsequent to release of the tag from the nucleotide. For example, the analyte can be brought to the nanopore sensor, e.g., via the polymerase that is extending the growing strand, and such complexation of the nucleotide to the polymerase, the growing strand, and / or the nucleotide to be analyzed may be sufficient to induce the change in the electrochemical property (e.g., change in capacitance, resistance, and / or impedance of the nanopore system). In some cases, because the size of the tag may be responsible for the detection or analysis of the analyte, the tag may not need to be a fluorescent molecule.

[0263]

[0263] In some cases, the tag may comprise a polynucleotide sequence that does not exhibit complementarity to at least a portion of the analyte. In some cases, the polynucleotide sequence can exhibit less than or equal to about 90%, less than or equal to about 80%, less than or equal to about 70%, less than or equal to about 60%, less than or equal to about 50%, less than or equal to about 40%, less than or equal to about 30%, less than or equal to about 20%, less than or equal to about 10%, less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2%, less than or equal to about 1%, less than or equal to about 0.5%, or less than or equal to about 0.1% sequence identity to the polynucleotide sequence of the analyte.

[0264]

[0264] The polynucleotide sequence of the tag can have a length of at least about 5 bases, at least about 10 bases, at least about 15 bases, at least about 20 bases, at least about 25 bases, at least about 30 bases, at least about 35 bases, at least about 40 bases, at least about 45 bases, at least about 50 bases, at least about 55 bases, at least about 60 bases, at least about 65 bases, at least about 70 bases, at least about 75 bases, at least about 80 bases, at least about 85 bases, at least about 90 bases, at least about 95 bases, at least about 100 bases, at least about 110 bases, at least about 120 bases, at least about 130 bases, at least about 140 bases, at least about 150 bases, at least about 160 bases, at least about 170 bases, at least about 180 bases, at least about 190 bases, at least about 200 bases, or more. The length of the polynucleotide sequence of the tag can be at most about 200 bases, at most about 190 bases, at most about 180 bases, at most about 170 bases, at most about 160 bases, at most about 150 bases, at most about 140 bases, at most about 130 bases, at most about 120 bases, at most about 110 bases, at most about 100 bases, at most about 95 bases, at most about 90 bases, at most about 85 bases, at most about 80 bases, at most about 75 bases, at most about 70 bases, at most about 65 bases, at most about 60 bases, at most about 55 bases, at most about 50 bases, at most about 45 bases, at most about 40 bases, at most about 35 bases, at most about 30 bases, at most about 25 bases, at most about 20 bases, at most about 15 bases, at most about 10 bases, at most about 5 bases, or less.

[0265]

[0265] In some cases, the polynucleotide sequence of the tag can comprise a polyN (e.g., T40, A40, A10, or T10). The polyN can be characterized by having (i) two or more of a same base (e.g., TTTT) or (ii) two or more of a same set of bases (e.g., a poly dinucleotide, such as

[0266] AT AT AT AT) that are contiguous. The same set of bases can comprise at least two different bases, at least three different bases, at least four different bases, at least five different bases, or more. The same set of bases can comprise at most five different bases, at most four different bases, at most three different bases, or at most two different bases. A length of the same set of bases can be at least about 2 bases, at least about 3 bases, at least about 4 bases, at least about 5 bases, at least about 6 bases, at least about 7 bases, at least about 8 bases, at least about 9 bases, at least about 10 bases, or more. The length of the same set of bases can be at most about 10 bases, at most about 9 bases, at most about 8 bases, at most about 7 bases, at most about 6 bases, at most about 5 bases, at most about 4 bases, at most about 3 bases, or at most about 2 bases. Non-limiting examples of the polyN can comprise poly A, polyT, polyC, polyG, polyU, or polydinucleotide (e.g., poly AT, polyCG, polyAA, polyTT, polyCC, polyGG, polyUU). The polyN can have a length of at least about 5 bases, at least about 10 bases, at least about 15 bases, at least about 20 bases, at least about 25 bases, at least about 30 bases, at least about 35 bases, at least about 40 bases, at least about 45 bases, at least about 50 bases, at least about 55 bases, at least about 60 bases, at least about 65 bases, at least about 70 bases, at least about 75 bases, at least about 80 bases, at least about 85 bases, at least about 90 bases, at least about 95 bases, at least about 100 bases, at least about 110 bases, at least about 120 bases, at least about 130 bases, at least about 140 bases, at least about 150 bases, at least about 160 bases, at least about 170 bases, at least about 180 bases, at least about 190 bases, at least about 200 bases, or more. The polyN can have a length of at most about 200 bases, at most about 190 bases, at most about 180 bases, at most about 170 bases, at most about 160 bases, at most about 150 bases, at most about 140 bases, at most about 130 bases, at most about 120 bases, at most about 110 bases, at most about 100 bases, at most about 95 bases, at most about 90 bases, at most about 85 bases, at most about 80 bases, at most about 75 bases, at most about 70 bases, at most about 65 bases, at most about 60 bases, at most about 55 bases, at most about 50 bases, at most about 45 bases, at most about 40 bases, at most about 35 bases, at most about 30 bases, at most about 25 bases, at most about 20 bases, at most about 15 bases, at most about 10 bases, at most about 5 bases, or less.

[0267]

[0266] In some cases, the tag can comprise radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Non-limiting examples of an identifier moiety (e.g., a fluorescent label) may include fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6- carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy- X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS), [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein- 15 -d ATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein- 15 -2 '-d ATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY- TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein- 12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5- dUTP, and Texas Red- 12-dUTP.

[0268]

[0267] In some cases, the tag can comprise polymers that are not polypeptide or polynucleotide. In some embodiment, the polymers are substantially soluble in aqueous conditions. Non-limiting examples of polymers (e.g., a polymer chain or a portion thereof that does not comprise a polynucleotide sequence or a polypeptide sequence) comprise polyethylene glycol, polyethylenimine, polyacrylamide, polyacrylic acid, polyvinyl alcohol, or ionic polymers. In some cases, the polymers can be homopolymers. In some cases, the polymers can be copolymers.

[0269]

[0268] In some cases, the molecular weight of the tag can be from about 50 dalton (Da) to about 500 Da, from about 50 Da to about 1 kilodalton (kDa), from about 50 Da to about 2 kDa, from about 50 Da to about 5 kDa, from about 50 Da to about 10 kDa, from about 50 Da to about 15 kDa, from about 50 Da to about 20 kDa, from about 50 Da to about 25 kDa, from about 50 Da to about 30 kDa, from about 50 Da to about 35 kDa, from about 50 Da to about 40 kDa, from about 50 Da to about 50 kDa, from about 50 Da to about 60 kDa, from about 50 Da to about 70 kDa, from about 50 Da to about 80 kDa, from about 50 Da to about 90 kDa, from about 50 Da to about 100 kDa, from about 100 Da to about 10 kDa, from about 100 Da to about 15 kDa, from about 100 Da to about 20 kDa, from about 100 Da to about 25 kDa, from about 100 Da to about 30 kDa, from about 100 Da to about 35 kDa, from about 100 Da to about 40 kDa, from about 100 Da to about 50 kDa, from about 100 Da to about 60 kDa, from about 100 Da to about 70 kDa, from about 100 Da to about 80 kDa, from about 100 Da to about 90 kDa, from about 100 Da to about 100 kDa, from about 200 Da to about 10 kDa, from about 200 Da to about 15 kDa, from about 200 Da to about 20 kDa, from about 200 Da to about 25 kDa, from about 200 Da to about 30 kDa, from about 200 Da to about 35 kDa, from about 200 Da to about 40 kDa, from about 200 Da to about 50 kDa, from about 200 Da to about 60 kDa, from about 200 Da to about 70 kDa, from about 200 Da to about 80 kDa, from about 200 Da to about 90 kDa, from about 200 Da to about 100 kDa, from about 500 Da to about 10 kDa, from about 500 Da to about 15 kDa, from about 500 Da to about 20 kDa, from about 500 Da to about 25 kDa, from about 500 Da to about 30 kDa, from about 500 Da to about 35 kDa, from about 500 Da to about 40 kDa, from about 500 Da to about 50 kDa, from about 500 Da to about 60 kDa, from about 500 Da to about 70 kDa, from about 500 Da to about 80 kDa, from about 500 Da to about 90 kDa, from about 500 Da to about 100 kDa, from about 1 kDa to about 10 kDa, from about 1 kDa to about 15 kDa, from about 1 kDa to about 20 kDa, from about 1 kDa to about 25 kDa, from about 1 kDa to about 30 kDa, from about 1 kDa to about 35 kDa, from about 1 kDa to about 40 kDa, from about 1 kDa to about 50 kDa, from about 1 kDa to about 60 kDa, from about 1 kDa to about 70 kDa, from about 1 kDa to about 80 kDa, from about 1 kDa to about 90 kDa, from about

[0270] 1 kDa to about 100 kDa, from about 2 kDa to about 10 kDa, from about 2 kDa to about 15 kDa, from about 2 kDa to about 20 kDa, from about 2 kDa to about 25 kDa, from about 2 kDa to about 30 kDa, from about 2 kDa to about 35 kDa, from about 2 kDa to about 40 kDa, from about

[0271] 2 kDa to about 50 kDa, from about 2 kDa to about 60 kDa, from about 2 kDa to about 70 kDa, from about 2 kDa to about 80 kDa, from about 2 kDa to about 90 kDa, from about 2 kDa to about 100 kDa, from about 5 kDa to about 10 kDa, from about 5 kDa...

Claims

1. CLAIMSWHAT IS CLAIMED IS:

1. A method comprising:(a) contacting at least a portion of an analyte with a nanopore system, wherein said nanopore system comprises (i) a fluidic chamber, (ii) a nanopore disposed within a membrane, wherein said membrane separates said fluidic chamber to a first side and a second side, and (iii) a sensor comprising a first electrode and a second electrode;(b) detecting one or more signals indicative of an impedance or change thereof associated with said at least said portion of said analyte; and(c) using said one or more signals to identify one or more characteristics of said at least said portion of said analyte with an accuracy of at least about 90%.

2. The method of claim 1, further comprising applying a time-varying asymmetrical voltage waveform between said first electrode and said second electrode.

3. The method of claim 2, wherein at least a portion of said time-varying asymmetrical voltage waveform alternates between a first voltage and a second voltage of differing absolute magnitudes.

4. The method of claim 3, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 5%.

5. The method of claim 3, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 50%.

6. The method of claim 3, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 0.1 volts (V).

7. The method of claim 3, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 0.5 V.

8. The method of claim 3, wherein a voltage magnitude of one of said first voltage and said second voltage is substantially zero (0 V).

9. The method of claim 2, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises a first voltage phase and a second voltage phase of differing phase durations.

10. The method of claim 9, wherein said phase durations of said first voltage phase and said second voltage phase differ by at least about 5%.

11. The method of claim 9, wherein said phase durations of said first voltage phase and said second voltage phase differ by at least about 50%.

12. The method of claim 9, wherein said phase durations of said first voltage phase and said second voltage phase differ by at least about 1 millisecond (ms).

13. The method of any one of claims 2-12, wherein said time-varying asymmetrical voltage waveform comprises a plurality of cycles, a cycle of said plurality of cycles comprising a plurality of voltage phases having different voltages, wherein said nanopore system detects one or more electrical signals associated with said at least said portion of said analyte for said plurality of cycles.

14. The method of any one of claims 2-13, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises a unipolar voltage waveform.

15. The method of claim 14, wherein said at least said portion of said unipolar voltage waveform maintains a constant polarity relative to a reference potential.

16. The method of claim 2, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises a bipolar voltage waveform.

17. The method of claim 2, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises at plurality of different voltage phases, wherein at least one of said plurality of different voltage phases comprises a non-constant voltage phase.

18. The method of claim 17, wherein said non-constant voltage phase is characterized by a rate of voltage change of at least about 0.1 volts per second (V / s).

19. The method of claim 17, wherein said non-constant voltage phase is characterized by an increase in voltage over time.

20. The method of claim 17, wherein said non-constant voltage phase is characterized by a decrease in voltage over time.

21. The method of claim 17, wherein an additional voltage phase of said plurality of different voltage phases comprises a constant voltage phase.

22. The method of any one of claims 1-21, wherein said nanopore system further comprises at least one voltage source, at least one capacitor electronically coupled to said second electrode, and at least one electronic switch for generating a plurality of sequential temporary couplings between said at least one voltage source to said at least one capacitor.

23. The method of claim 22, further comprising detecting a plurality of electrical signals associated with said at least said portion of said analyte between said plurality of sequential temporary couplings.

24. The method of claim 23, wherein two adjacent electrical signals of said plurality of electrical signals are measured within at most about at most about 500 microseconds from one another.

25. The method of claim 23 or 24, wherein each of said plurality of electrical signals comprise a decay in voltage across said capacitor.

26. The method of any one of claims 1-25, wherein said nanopore system comprises at least 500,000 fluidic chambers.

27. The method of claim 26, wherein said at least 500,000 fluidic chambers are capable of independently analyzing a sample.

28. The method of any one of claims 1-27, wherein said nanopore system further comprises an amplifier electrically coupled to said second electrode, wherein said amplifier is electrically coupled to a stabilizing voltage source.

29. The method of claim 28, further comprising, via said stabilizing voltage source and said amplifier, applying a stabilizing potential on said second electrode during a discharge of said second electrode to stabilize a capacitance of said second electrode.

30. The method of claim 29, wherein said stabilizing potential stabilizes a capacitance of said membrane.

31. The method of any one of claims 1-30, wherein said analyte comprises a nucleic acid molecule.

32. The method of claim 31, wherein said analyte comprises a single stranded circular nucleic acid (sscNA) molecule.

33. The method of claim 32, further comprising, prior to (a), circularizing a linear single stranded nucleic acid molecule, thereby generating said sscNA molecule.

34. The method of claim 33, wherein said circularizing comprises (i) hybridizing at least a portion of an oligonucleotide with at least a portion of an end of said linear single stranded nucleic acid molecule, and (ii) hybridizing at least an additional portion of said oligonucleotide with at least an additional portion of an additional end of said linear single stranded nucleic acid molecule, thereby generating a complex comprising a circularized single stranded nucleic acid molecule hybridized with said oligonucleotide, wherein said circularized single stranded nucleic acid molecule comprises a nick.

35. The method of claim 34, further comprising ligating an end of said circularized single stranded nucleic acid molecule and an additional end of said circularized single stranded nucleic acid molecule to generate said single stranded circular nucleic acid molecule.

36. The method of claim 35, further comprising removing said oligonucleotide.

37. The method of any one of claims 31-36, further comprising (i) hybridizing a primer to at least a portion of said sscNA molecule, and (ii) generating a growing strand comprising sequence complementarity to at least an additional portion of said sscNA molecule.

38. The method of any one of claims 1-37, further comprising, prior to (a), forming said membrane in said fluidic chamber.

39. The method of any one of claims 1-38, wherein said membrane comprises a lipid bilayer.

40. The method of claim 39, wherein said forming said membrane comprises loading a solution comprising a plurality of lipids to said fluidic chamber, wherein said plurality of phospholipids assembles to form said lipid bilayer.

41. The method of any one of claims 1-40, further comprising, depositing said nanopore in said membrane.

42. The method of claim 41, wherein said depositing comprises flowing a solution comprising one or more nanopores to said fluidic chamber, wherein a nanopore of said one or more nanopores diffuses into said membrane.

43. The method of any one of claims 1-42, wherein in (a), said contacting further comprises bringing said at least said portion of said analyte in proximity with said nanopore.

44. The method of any one of claims 1-43, further comprising, prior to (c), applying a voltage to said first electrode and said second electrode.

45. The method of claim 44, wherein said voltage comprises an alternating voltage.

46. The method of claim 45, wherein said alternating voltage comprises a first phase and a second phase.

47. The method of claim 46, wherein said first phase and said second phase have opposite directions.

48. The method of claim 46, wherein (b) comprises obtaining a plurality of measurements of said one or more signals during a cycle in said first phase.

49. The method of claim 46, wherein (b) comprises determining a first set of said one or more signals during said first phase.

50. The method of claim 49, further comprising determining a second set of said one or more signals during said second phase.

51. The method of any one of claims 1-50, further comprising, prior to (c), pre-processing said one or more signals.

52. The method of claim 51, wherein said pre-processing comprises removing one or more background signals from said one or more signals.

53. The method of claim 52, wherein said removing one or more background signals comprises removing one or more signals that have a frequency greater than a threshold value.

54. The method of claim 51, wherein said pre-processing further comprises removing a baseline portion from said one or more signals, thereby generating one or more pre-processed signals.

55. The method of claim 54, wherein said baseline portion is obtained when said analyte is not added to said nanopore system.

56. The method of claim 1, wherein said analyte comprises a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof.

57. The method of any one of claims 1-56, wherein said one or more signals are current or voltage.

58. The method of any one of claims 1-57, wherein said sensor further comprises a processor in electrical communication with said first electrode and / or said second electrode of said sensor.

59. The method of any one of claims 1-58, wherein said one or more characteristics comprise a presence or absence of said analyte, a size of said analyte, or a concentration of said analyte, or a combination thereof.

60. The method of any one of claims 1-59, wherein said analyte comprises a nucleic acid molecule, a protein, or a polypeptide, and wherein said one or more characteristics further comprise a sequence information of at least a portion of said analyte, an identity of said analyte, or a mutation of said analyte, or combinations thereof.

61. The method of any one of claims 1-60, wherein said accuracy is at least about 95%.

62. A method comprising:(a) contacting at least a portion of an analyte with a nanopore system, said nanopore system comprising (i) a nanopore disposed within a membrane, (ii) a first electrode disposed adjacent to a first side of said nanopore, and (iii) a second electrode disposed adjacent to a second and different side of said nanopore;(b) applying a time-varying asymmetrical voltage waveform between said first electrode and said second electrode of said nanopore system; and(c) directing said nanopore system to detect one or more electrical signals associated with said at least said portion of said analyte.

63. The method of claim 62, wherein at least a portion of said time-varying asymmetrical voltage waveform alternates between a first voltage and a second voltage of differing absolute magnitudes.

64. The method of claim 63, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 5%.

65. The method of claim 63, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 50%.

66. The method of claim 63, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 0.1 volts (V).

67. The method of claim 63, wherein said absolute magnitudes of said first voltage and said second voltage differ by at least about 0.5 V.

68. The method of claim 63, wherein a voltage magnitude of one of said first voltage and said second voltage is substantially zero (0 V).

69. The method of claim 62, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises a first voltage phase and a second voltage phase of differing phase durations.

70. The method of claim 69, wherein said phase durations of said first voltage phase and said second voltage phase differ by at least about 5%.

71. The method of claim 69, wherein said phase durations of said first voltage phase and said second voltage phase differ by at least about 50%.

72. The method of claim 69, wherein said phase durations of said first voltage phase and said second voltage phase differ by at least about 0.1 milliseconds (ms).

73. The method of claim 62, wherein said time-varying asymmetrical voltage waveform comprises a plurality of cycles, a cycle of said plurality of cycles comprising a plurality of voltage phases having different voltages, wherein said nanopore system detects one or more electrical signals associated with said at least said portion of said analyte for said plurality of cycles.

74. The method of claim 62, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises a unipolar voltage waveform.

75. The method of claim 74, wherein said at least said portion of said unipolar voltage waveform maintains a constant polarity relative to a reference potential.

76. The method of claim 62, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises a bipolar voltage waveform.

77. The method of claim 62, wherein at least a portion of said time-varying asymmetrical voltage waveform comprises at plurality of different voltage phases, wherein at least one of said plurality of different voltage phases comprises a non-constant voltage phase.

78. The method of claim 77, wherein said non-constant voltage phase is characterized by a rate of voltage change of at least about 0.1 volts per second (V / s).

79. The method of claim 77, wherein said non-constant voltage phase is characterized by an increase in voltage over time.

80. The method of claim 77, wherein said non-constant voltage phase is characterized by a decrease in voltage over time.

81. The method of claim 77, wherein an additional voltage phase of said plurality of different voltage phases comprises a constant voltage phase.

82. The method of any one of claims 62-81, wherein said time-varying asymmetrical voltage waveform is applied to a medium in contact with said nanopore.

83. The method of claim 82, wherein said medium comprises liquid, electrolyte, or both.

84. The method of any one of claims 62-83, wherein said nanopore system further comprises at least one voltage source, at least one capacitor electronically coupled to said second electrode, and at least one electronic switch for generating a plurality of sequential temporary couplings between said at least one voltage source to said at least one capacitor.

85. The method of claim 84, further comprising detecting a plurality of electrical signals associated with said at least said portion of said analyte between said plurality of sequential temporary couplings.

86. The method of claim 85, wherein two adjacent electrical signals of said plurality of electrical signals are measured within at most about 500 microseconds from one another.

87. The method of claim 86, wherein each of said plurality of electrical signals comprise a decay in voltage across said at least one capacitor.

88. The method of any one of claims 84-87, wherein said plurality of sequential temporary couplings are different from one another.

89. The method of claim 88, wherein a first sequential temporary coupling and a second sequential temporary coupling have different durations.

90. The method of claim 89, wherein a duration of said first sequential temporary coupling and a duration of said second sequential temporary coupling are different by at least about 5%.

91. The method of claim 89, wherein a duration of said first sequential temporary coupling and a duration of said second sequential temporary coupling are different by at least about 1 ms.

92. The method of any one of claims 84-91, wherein said at least one electronic switch comprises a single electronic switch.

93. The method of any one of claims 84-91, wherein said at least one electronic switch comprises a plurality of electronic switches.

94. The method of any one of claims 84-93, wherein said analyte comprises a biomolecule, a detection moiety coupled to said biomolecule, or both.

95. The method of claim 94, wherein said biomolecule comprises one or more nucleotide or one or more amino acid residues.

96. The method of any one of claims 84-95, wherein said analyte comprises a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof.

97. A method for analyzing a plurality of samples, comprising:(a) contacting a plurality of samples with a nanopore system comprising at least 500,000 fluidic chambers, wherein said at least 500,000 fluidic chambers are capable of independently analyzing a sample;(b) detecting one or more signals indicative of an impedance or change thereof in said at least 500,000 fluidic chambers; and(c) using said one or more signals to identify one or more characteristics of said plurality of samples.

98. The method of claim 97, wherein said nanopore system comprises at least 600,000 fluidic chambers.

99. The method of claim 97, wherein said nanopore system comprises at least 1,000,000 fluidic chambers.

100. The method of claim 97, wherein said at least 500,000 fluidic chambers are independently addressable.

101. The method of claim 97, wherein (b) comprises contacting a sample of said plurality of samples with a fluidic chamber and contacting an additional sample of said plurality of samples with an additional fluidic chamber.

102. The method of claim 101, wherein said sample and said additional sample are same.

103. The method of claim 101, wherein said sample and said additional sample are different.

104. The method of claim 101, wherein said sample comprises one or more analytes and said additional sample comprise one or more additional analytes.

105. The method of claim 104, wherein said one or more analytes and said one or more additional analytes are different.

106. The method of claim 104, wherein said one or more analytes comprise a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof.

107. The method of claim 104, wherein said one or more additional analytes comprise a small molecule, a nucleotide, a nucleic acid, an amino acid, a peptide, or a polypeptide, or a variant thereof, or a combination thereof.

108. A method, comprising:(a) contacting at least a portion of an analyte with a nanopore system, wherein said nanopore system comprises (i) a nanopore disposed within a membrane, (ii) one or more electrodes in proximity to said nanopore, and (iii) one or more signal generators in electrical communication with said one or more electrodes;(b) applying a potential to said one or more electrodes, thereby detecting one or more signals indicative of an impedance or change thereof associated with said at least said portion of said analyte, wherein said one or more signal generators stabilize said potential; and(c) using said one or more signals to identify one or more characteristics of said at least said portion of said analyte.

109. The method of claim 108, wherein a signal generator of said one or more signal generators is electrically coupled to a sensing electrode via an amplifier.

110. The method of claim 109, wherein (b) further comprises, via said signal generator and said amplifier, applying a stabilizing potential on said sensing electrode during a discharge of said sensing electrode to stabilize a capacitance of said sensing electrode.

111. The method of claim 110, wherein said stabilizing potential stabilizes a capacitance of said membrane.

112. The method of claim 108, wherein said one or more signals comprise current or voltage.

113. The method of claim 108, wherein said detecting comprises said one or more signals between a sensing electrode and a counter electrode of said nanopore system.

114. The method of claim 113, wherein (b) further comprises applying an alternating voltage to said sensing electrode and said counter electrode.

115. The method of claim 114, wherein said one or more signals comprise a signal delay.

116. The method of claim 108, further comprising detecting one or more additional signals while said at least said portion of said analyte is not in said nanopore system.

117. The method of claim 116, wherein (c) comprises determining a relationship between said one or more signals and said one or more additional signals.

118. The method of claim 117, wherein said relationship is associated with a characteristic of said at least said portion of said analyte.

119. The method of claim 108, wherein said one or more signals are associated with (i) a resistance of said at least said portion of said analyte, (ii) a resistance of said nanopore, and / or (iii) a capacitance of said membrane.

120. The method of claim 119, wherein said one or more additional signals are associated with (i) said resistance of said nanopore, and (ii) said capacitance of said membrane.

121. The method of claim 108, wherein stabilizing said potential stabilizes said capacitance of said membrane, thereby improving detection accuracy.

122. A system comprising: a computer processor and a computer memory coupled thereto, wherein the computer memory comprises a machine executable code that, upon execution by the one or more computer processors, implements the method of any one of the preceding claims.

123. The system of claim 122, further comprising the nanopore system.

124. A method, comprising:(a) providing a single stranded nucleic acid molecule;(b) circularizing said single stranded nucleic acid molecule, in a presence of an oligonucleotide,wherein at least a portion of said oligonucleotide comprises sequence complementarity to at least a portion of an end of said single stranded nucleic acid molecule, and at least an additional portion of said oligonucleotide comprises sequence complementarity to at least an additional portion of an additional end of said single stranded nucleic acid molecule, wherein said circularizing comprises hybridizing said at least said portion of said oligonucleotide with said at least said portion of said end of said single stranded nucleic acid molecule and hybridizing said additional portion of said oligonucleotide with said at least said additional portion of said additional end of said single stranded nucleic acid molecule, thereby generating a complex comprising a circularized single stranded nucleic acid molecule hybridized with said oligonucleotide, wherein said circularized single stranded nucleic acid molecule comprises a nick; and(c) ligating an end of said circularized single stranded nucleic acid molecule and an additional end of said circularized single stranded nucleic acid molecule to generate a closed circularized single stranded nucleic acid molecule.

125. A nanopore system, comprising: a sensor comprising:(i) a fluidic chamber;(ii) a nanopore disposed within a membrane, wherein said membrane separates said fluidic chamber to a first side and a second side; and(iii) a sensor comprising a sensing electrode in proximity to said nanopore; a processor operatively coupled to said sensor, wherein said processor is configured to direct said sensor to (a) detect one or more signals indicative of an impedance or change thereof associated with at least a portion of an analyte, and (b) use said one or more signals to identify one or more characteristics of said at least said portion of said analyte with an accuracy of at least about 90%.

126. A nanopore system, comprising: a sensor comprising:(i) a nanopore disposed within a membrane;(ii) a first electrode disposed adjacent to a first side of said nanopore; and(iii) a second electrode disposed adjacent to a second and different side of said nanopore; a processor operatively coupled to said sensor, wherein said processor is configured to direct said sensor to:(a) apply a time-varying asymmetrical voltage waveform between said first electrode and said second electrode; and(b) detect one or more electrical signals associated with said at least said portion of said analyte.

127. A nanopore system, comprising: a flow cell chip comprising at least 500,000 fluidic chambers; and a plurality of sensors configured to detect one or more signals indicative of an impedance or change thereof, wherein each fluidic chamber of said at least 500,000 fluidic chambers is coupled to a sensing electrode, wherein said each fluidic chamber comprises a nanopore embedded in a membrane.

128. A nanopore system, comprising: a nanopore disposed within a membrane; one or more electrodes in proximity to said nanopore; one or more signal generators in electrical communication with said one or more electrodes; wherein said one or more signal generators are configured to (a) apply one or more potentials to said one or more electrodes, and (b) stabilize said one or more potentials; and one or more controllers operatively coupled to said one or more electrodes, wherein said one or more controllers are individually or collectively configured to (1) detect one or more signals indicative of an impedance or change thereof associated with at least a portion of an analyte, and (2) use said one or more signals to identify one or more characteristics of said at least said portion of said analyte.

129. A nanopore system, comprising: a sensor comprising:(i) a nanopore disposed within a membrane;(ii) a first electrode disposed adjacent to a first side of said nanopore;(iii) a second electrode disposed adjacent to a second and different side of said nanopore;(iv) a capacitor electronically coupled to said second electrode; and(v) an electronic switch for generating a plurality of sequential temporary couplings between said capacitor and a voltage source; a processor operatively coupled to said sensor, wherein said processor is configured to direct said sensor to detect a plurality of electrical signals associated with said at least a portion of an analyte between said plurality of sequential temporary couplings.

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