Faraday system and method for self-limiting protein pore insertion in a membrane

Abstract

Described herein are systems and methods for inserting a single pore (316) into a membrane (314) under faradaic conditions. A stepped or ramped voltage waveform can be applied across the membrane of a cell of an array. Here, the voltage waveform begins at a first voltage and increases over a period of time to a second voltage. The voltage waveform has a polarity that maintains the first species of the redox couple (308) in its current oxidation state. The first voltage is selected to be low enough to reduce the risk of damaging the membrane, while the rate of voltage increase is selected to provide sufficient time for the pore to insert into the membrane. Once the pore is inserted into the membrane, the voltage drops rapidly across the membrane. Therefore, further increases in the voltage applied between the electrodes reduce the risk of damaging the membrane.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 705,097, filed on June 10, 2020, the entire content of which is incorporated herein by reference for all purposes.

[0002] This application can be related to International Patent Application No. PCT / EP2019 / 084581, filed on December 11, 2019, which claims the priority of U.S. Provisional Application No. 62 / 777,976, filed on December 11, 2018, each of which is incorporated herein by reference in its entirety for all purposes.

[0003] Incorporation by Reference All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Background Art

[0004] Nanopore - based sequencing chips are analytical tools that can be used for DNA sequencing. These devices can incorporate a very large number of sensor cells configured as an array. For example, a sequencing chip can include an array of 1 million cells having, for example, 1000 rows × 1000 columns of cells. Each of the cells of the array can include a membrane and a protein pore having a pore size on the order of 1 nanometer in inner diameter. Such nanopores have been shown to be effective in the rapid sequencing of nucleotides.

[0005] When a potential is applied across a nanopore immersed in a conductive fluid, a small ion current can exist due to the conduction of ions across the nanopore. The size of the current is susceptible to the pore size and the type of molecules disposed within the nanopore. The molecules can be specific tags bound to specific nucleotides. Accordingly, it becomes possible to detect nucleotides at specific positions of nucleic acids. As a method for measuring the resistance of a molecule, a voltage or other signal in a circuit including a nanopore can be measured (e.g., by an integrating capacitor), thereby making it possible to detect what molecules are in the nanopore.

[0006] For a sequencing chip to function properly, generally only one pore should be inserted in the membrane for a given cell. When multiple pores are inserted into a single membrane, the interpretation of the electrical signature generated by nucleotides passing through the multiple pores simultaneously becomes much more difficult.

[0007] The application of a voltage across the membrane during the pore insertion step may perhaps facilitate the process of pore insertion by lowering the stability of the membrane and making the pore itself more easily insertable into the membrane. However, the application of an excessive voltage across the membrane can cause significant damage to the membrane. As a result, the cell becomes unusable.

[0008] Accordingly, it is preferable to provide a system and method for reliably inserting a single pore into a membrane while reducing the risk of excessive damage to the membrane. SUMMARY OF THE INVENTION

[0009] Various embodiments provide techniques and systems related to the insertion of a single pore into a membrane in a cell of a nanopore-based sequencing chip. In some embodiments, the insertion of the pore into the membrane reduces the likelihood of further insertion of pores into the membrane.

[0010] Other embodiments relate to systems and computer-readable media associated with the methods described herein.

[0011] In some embodiments, a method of forming an array of nanopore sensor cells is provided. The method includes introducing a nanopore proximate to a cell, the cell having a working electrode and a membrane sealing the cell, the working electrode being powered by an alternating current (AC) connected power source; applying a voltage waveform across the membrane of the cell, the voltage waveform beginning at a first voltage and increasing over a period to a second voltage; and inserting the nanopore into the membrane while applying the voltage waveform.

[0012] In some embodiments, the first voltage is between about 0 and 100 mV. The second voltage is between about 100 and 2000 mV.

[0013] In some embodiments, the working electrode is a capacitive electrode.

[0014] In some embodiments, the voltage waveform includes a plurality of incremental steps between the first voltage and the second voltage.

[0015] In some embodiments, the plurality of incremental steps increment by about 1 to 100 mV each.

[0016] In some embodiments, the plurality of incremental steps increment by about 1 to 25 mV each.

[0017] In some embodiments, each of the incremental steps has a period between about 0.1 and 60 seconds.

[0018] In some embodiments, the period of the incremental steps is variable.

[0019] In some embodiments, the duration of the incremental step at a lower voltage is longer than the duration of the incremental step at a higher voltage.

[0020] In some embodiments, the duration of the incremental step is constant.

[0021] In some embodiments, the voltage waveform includes a slope between a first voltage and a second voltage.

[0022] In some embodiments, the slope is between about 0.1 and 2.0 V per minute.

[0023] In some embodiments, the slope has a constant slope.

[0024] In some embodiments, the slope has a variable slope.

[0025] In some embodiments, the slope has a slope at a lower voltage that is less than the slope at a higher voltage.

[0026] In some embodiments, applying the voltage waveform is applied to an unthinned film.

[0027] In some embodiments, the method further includes thinning an unthinned film using the applied voltage waveform.

[0028] In some embodiments, a system for sequencing molecules is provided. The system includes an array of cells on a substrate, each cell having a working electrode and an aperture configured to be sealed by a membrane, the working electrode being powered by an alternating current (AC) connected power supply; an array of cells on the substrate; a counter electrode; a power supply that is AC connected to each of the working electrodes; and a controller programmed to supply a voltage waveform, starting at a first voltage and increasing over a period to a second voltage, to the cells using the working electrode and the counter electrode.

[0029] In some embodiments, the working electrode is a capacitive electrode.

[0030] In some embodiments, the voltage waveform includes a plurality of incremental steps between the first voltage and the second voltage.

[0031] In some embodiments, the voltage waveform includes a slope between the first voltage and the second voltage.

[0032] In some embodiments, the controller is further programmed to supply the voltage waveform to an unthinned membrane.

[0033] In some embodiments, a method of forming an array of nanopore sensor cells is provided. The method includes introducing a nanopore proximate to the cell, where the cell has a working electrode and a membrane that seals the cell, and the working electrode is powered by an electrically connected power source; applying a voltage waveform across the membrane of the cell, where the voltage waveform begins at a first voltage and increases over a period to a second voltage, the voltage waveform includes an alternating current (AC) modulation component, and the AC modulation component is configured such that an electrical measurement can be obtained through the working electrode while the voltage waveform is applied across the membrane of the cell; and inserting the nanopore into the membrane while applying the voltage waveform.

[0034] In some embodiments, the AC modulation component has an amplitude of less than 100 mV. In some embodiments, the AC modulation component has a frequency between 10 Hz and 1000 Hz.

[0035] In some embodiments, a method of forming a cell covered with a membrane is provided. The method includes flowing a membrane-forming material over the cell, where the cell has a working electrode and the working electrode is powered by an electrically connected power source; disposing a layer of the membrane-forming material over the cell; applying a voltage waveform across the layer of the membrane-forming material using the working electrode and a counter electrode on the opposite side of the layer of the membrane-forming material, where the voltage waveform includes an AC modulation component, and the AC modulation component is configured such that an electrical measurement can be obtained through the working electrode while the voltage waveform is applied across the layer of the membrane-forming material; and thinning the layer of the membrane-forming material into a membrane, where the membrane is configured to receive a nanopore.

[0036] In some embodiments, the AC modulation component has an amplitude of less than 100 mV. In some embodiments, the AC modulation component has a frequency between 10 Hz and 1000 Hz.

[0037] A good understanding of the characteristics and advantages of the embodiments of the present invention can be obtained by referring to the following detailed description and the accompanying drawings.

[0038] In some embodiments, a method of forming an array of nanopore sensor cells is provided. The method includes introducing a nanopore closest to a cell in a solution containing a first species of a redox pair but not containing a second species of the redox pair, the cell having a working electrode and a membrane that seals the cell, the working electrode being powered by an electrically connected power source, introducing the nanopore; applying a voltage waveform across the membrane of the cell, the voltage waveform starting at a first voltage and increasing over a period to a second voltage, the voltage waveform having a polarity that maintains the first species of the redox pair in its current oxidation state, applying the voltage waveform across the membrane of the cell; and inserting the nanopore into the membrane while applying the voltage waveform.

[0039] In some embodiments, the redox pair is water-soluble. In some embodiments, the redox pair is ferricyanide and ferrocyanide.

[0040] In some embodiments, the method further includes applying a second voltage waveform having a polarity that oxidizes or reduces the first species to the second species.

[0041] In some embodiments, the method includes inserting a molecule through the pore and applying a sequencing voltage to sequence the molecule under Faradaic conditions.

[0042] In some embodiments, a system for sequencing a molecule is provided. The system includes an array of cells on a substrate, each of the cells having a working electrode and an aperture configured to be sealed by a membrane having a nanopore, an array of cells on a substrate; a counter electrode; a power source electrically connected to each of the working electrodes; and a controller programmed to deliver a voltage waveform that starts at a first voltage and increases over a period to a second voltage to the cells using the working electrode and the counter electrode, the voltage waveform having a polarity that maintains a first species of a redox pair in its current oxidized state.

[0043] In some embodiments, the working electrode is configured to selectively operate as both a capacitively coupled electrode and a resistively coupled electrode

[0044] In some embodiments, the controller is further programmed to deliver a second voltage waveform having a polarity that oxidizes or reduces the first species to a second species.

[0045] In some embodiments, the system further includes a solution that includes a first species of a redox pair but does not include a second species of the redox pair, the solution being configured to be disposed within the cells of the array. In some embodiments, the redox pair is water-soluble. In some embodiments, the redox pair is ferricyanide and ferrocyanide.

[0046] In some embodiments, the controller applies a voltage to insert the molecule into the pore and is further programmed to apply a sequencing voltage to sequence the molecule under Faradaic conditions.

Brief Description of the Drawings

[0047] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description of exemplary embodiments in which the principles of the invention are utilized, and from the accompanying drawings thereof:

[0048]

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[0049] The term Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods, devices, and materials similar to or equivalent to those described herein can be used in the practice of the disclosed technology. The following terms are provided to facilitate understanding of certain terms that are frequently used and are not meant to limit the scope of the present disclosure. Abbreviations used herein have their ordinary meanings in the fields of chemistry and biology.

[0050] "Nanopore" refers to a pore, channel, or passage formed or otherwise provided within a membrane. The membrane can be an organic membrane such as a lipid bilayer or a synthetic membrane such as a membrane made of a polymeric material. The nanopore can be located adjacent to or in proximity to a sensing circuit such as a complementary metal oxide semiconductor (CMOS) or field effect transistor (FET) circuit, or an electrode connected to such a sensing circuit. In some examples, the nanopore has a characteristic width or diameter on the order of about 0.1 nanometers (nm) to about 1000 nm. In some implementations, the nanopore can be a protein.

[0051] "Nucleic acid" refers to deoxyribonucleotides, or ribonucleotides, and their polymers, which can be either single-stranded or double-stranded. This term encompasses nucleic acids containing known nucleotide analogs, or modified backbone chain residues or linkages. These include synthetic substances, naturally occurring ones, and non-naturally occurring ones. They have binding properties similar to those of reference nucleic acids. They are metabolized in a manner similar to that of reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substituents), as well as complementary sequences, and the explicitly stated sequences. Specifically, degenerate codon substituents can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid can be used interchangeably with genes, complementary deoxyribonucleic acid (cDNA), messenger ribonucleic acid (mRNA), oligonucleotides, and polynucleotides.

[0052] In addition to referring to naturally occurring ribonucleotide or deoxyribonucleotide monomers, the term "nucleotide" is understood to refer to their related structural variants, including functionally equivalent derivatives and analogs, with respect to the particular context in which the nucleotide is used (such as hybridization to complementary bases), unless the context clearly indicates otherwise.

[0053] The term "tag" refers to a detectable moiety, which can be an atom or molecule, or an aggregate of atoms or molecules. The tag can provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive or capacitive) signature. This signature can be detected with the aid of a nanopore. Typically, when a nucleotide is attached to the tag, the tag is called a "tagged nucleotide". The tag can be attached to the nucleotide via a phosphate moiety.

[0054] The term "template" refers to a single-stranded nucleic acid molecule that is copied into a complementary strand of DNA nucleotides for DNA synthesis. In some cases, the template can refer to the DNA sequence that is copied during mRNA synthesis.

[0055] The term "primer" refers to a short nucleic acid sequence that provides a starting point for DNA synthesis. Enzymes that act as catalysts in DNA synthesis, such as DNA polymerase, can add new nucleotides to the primer for DNA replication.

[0056] The term "polymerase" refers to an enzyme that synthesizes polynucleotides directed towards a template. This term encompasses both the entire polypeptide and the domain having polymerase activity. DNA polymerases are well known to those skilled in the art and include, but are not limited to, DNA polymerases excised from or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima, or modified versions thereof. These include both DNA-dependent polymerases and RNA-dependent polymerases such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, most of which are classified into families A, B, and C. There is little or no sequence similarity between the various families. Most of the family A polymerases can be single-stranded proteins that contain multiple enzyme functions including polymerase, 3'-to-5' exonuclease activity, and 5'-to-3' exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase and 3'-to-5' exonuclease activity, as well as accessory elements. Family C polymerases are typically multi-subunit proteins with polymerization and 3'-to-5' exonuclease activity. In Escherichia coli (E. coli), three types of DNA polymerases have been found: DNA polymerase I (family A), DNA polymerase II (family B), and DNA polymerase III (family C). In eukaryotic cells, three different family B polymerases, DNA polymerase α, δ, and ε, are involved in nuclear replication. Polymerase γ, one of the family A polymerases, is used for mitochondrial DNA replication. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, as well as bacterial RNA polymerases, and also phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

[0057] The term "on period" generally refers to the period during which the tag of a tagged nucleotide is pushed into the nanopore by an electric field applied through an AC signal. The term "off period" generally refers to the period during which the tag of a tagged nucleotide is pushed out of the nanopore by an electric field applied through an AC signal. An AC cycle can include an on period and an off period. In different embodiments, the polarity of the voltage signal applied to the nanopore cell can be varied to make the nanopore cell be in the on period (or the off period).

[0058] The term "signal value" refers to the value of a sequencing signal output from a sequencing cell. According to a particular embodiment, the sequencing signal is an electrical signal measured at and / or output from a point in the circuit of one or more sequencing cells. For example, the signal value is (or represents) a voltage or a current. The signal value can represent the result of a direct measurement of voltage and / or current, and / or can represent an indirect measurement value. For example, the signal value can be the measured period until a voltage or a current reaches a specified value. The signal value is related to the resistivity of the nanopore and can represent any measurable quantity from which the resistivity and / or conductance of the nanopore (inserted and / or not inserted) can be derived. As another example, the signal value can correspond to, for example, the intensity of light from a phosphor bound to a nucleotide added to a nucleic acid using a polymerase.

[0059] The term "osmolality" is also known as osmotic concentration and refers to the unit of measurement of solute concentration. Osmolality measures the number of osmoles of solute particles per unit volume of a solution. An osmole is a unit of measurement of the number of moles of solute contributing to the osmotic pressure of a solution. Osmolality enables the measurement of the osmotic pressure of a solution and the determination of how a solvent disperses across a semipermeable membrane (osmosis) separating two solutions with different osmotic concentrations.

[0060] The term "osmolite" refers to any soluble compound that, when dissolved in a solution, raises the osmolality of that solution.

Best Mode for Carrying Out the Invention

[0061] According to certain embodiments, the techniques and systems disclosed below relate to the insertion of a single pore into a membrane of a cell of a nanopore-based sequencing chip. In some embodiments, the insertion of the pore into the membrane reduces the likelihood of further insertion of pores into the membrane. This thus makes pore insertion more self-limiting and reduces or eliminates the need for active feedback during the insertion step.

[0062] Examples of nanopore systems, circuits, and sequencing operations are first described. Subsequently, examples of techniques for replacing nanopores in a DNA sequencing cell are described. Embodiments of the present invention can be implemented in many ways. These ways include a computer program product embodied on a process, system, and computer-readable storage medium, and / or a processor configured to execute instructions stored on and / or provided by a memory connected to the processor.

[0063] I. Nanopore-Based Sequencing Chip FIG. 1 is a plan view of an embodiment of a nanopore sensor chip 100 having an array 140 of nanopore cells 150. Each of the nanopore cells 150 includes a control circuit integrated on a silicon substrate of the nanopore sensor chip 100. In some embodiments, sidewalls 136 are included in the array 140 to separate groups of nanopore cells 150 such that each of the groups can receive different samples for characterization. Each of the nanopore cells can be used to determine the sequence of a nucleic acid. In some embodiments, the nanopore sensor chip 100 includes a cover plate 130. In some embodiments, the nanopore sensor chip 100 also includes a plurality of pins 110 that interact with other circuits such as a computer processor.

[0064] In some embodiments, the nanopore sensor chip 100 includes multiple chips in the same package such as a multi-chip module (MCM) or a system-in-package (SiP). The chips can include, for example, memory, a processor, a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), a data converter, a high-speed I / O interface, and the like.

[0065] In some embodiments, the nanopore sensor chip 100 is connected (e.g., docked) to a nanotip workstation 120 that can include various components for performing (e.g., automatically performing) various embodiments of the processes disclosed herein. These processes can include, for example, an analyte delivery mechanism such as a pipette for supplying a lipid suspension or other membrane structure suspension, an analyte solution, and / or other liquid, suspension, or solid. The components of the nanotip workstation can further include a robotic arm, one or more computer processors, and / or memory. Multiple polynucleotides can be detected on the array 140 of nanopore cells 150. In some embodiments, each of the nanopore cells 150 can be individually addressable.

[0066] II. Nanopore Sequencing Cell The nanopore cell 150 in the nanopore sensor chip 100 can be implemented in many different ways. For example, in some embodiments, tags of different sizes and / or chemical structures are attached to different nucleotides in the nucleic acid molecules to be sequenced. In some embodiments, nucleotides tagged with different polymers may be hybridized with a template so that a complementary strand may be synthesized on the template of the nucleic acid molecule to be sequenced. In some implementations, both the nucleic acid molecule and the attached tag move through the nanopore, and the ionic current passing through the nanopore can indicate the nucleotides within the nanopore based on the specific size and / or structure of the tag attached to the nucleotide. In some implementations, only the tag is moved into the nanopore. Different tags within the nanopore can also be detected in many different ways.

[0067] A. Nanopore Sequencing Cell Structure FIG. 2 shows an exemplary embodiment of a nanopore cell 200 in a nanopore sensor chip, such as the nanopore cell 150 in the nanopore sensor chip 100 of FIG. 1, which can be used to evaluate the properties of polynucleotides or polypeptides. The nanopore cell 200 can include a well 205 formed from dielectric layers 201 and 204, a membrane such as a lipid bilayer 214 formed on the well 205, and a sample chamber 215 separated from the well 205 by the lipid bilayer 214 on the lipid bilayer 214. The well 205 can contain a large amount of electrolyte 206, and the sample chamber 215 can hold a bulk electrolyte 208 containing nanopores, such as soluble protein nanopore transmembrane molecular complexes (PNTMCs), and an analyte of interest (e.g., a nucleic acid molecule to be sequenced).

[0068] The nanopore cell 200 can include a working electrode 202 at the bottom of the well 205 and a counter electrode 210 disposed within the sample chamber 215. The signal source 228 can apply a voltage signal between the working electrode 202 and the counter electrode 210. A single nanopore (e.g., PNTMC) can be inserted into the lipid bilayer 214 by an electroporation process induced by the voltage signal, thereby forming a nanopore 216 within the lipid bilayer 214. Individual membranes within the array (e.g., lipid bilayer 214 or other membrane structures) can be made not to be chemically or electrically connected to each other. Thus, each of the nanopore cells in the array can act on the target analyte and can be an independent sequencing machine that generates data unique to a single polymer molecule associated with a nanopore that modulates the ionic current through an otherwise impermeable lipid bilayer.

[0069] Additional embodiments of systems and methods for pore insertion are described in Section III below. In particular, these systems and methods describe self-limiting pore insertion that efficiently achieves single pore insertion in the membrane of the cell.

[0070] As shown in FIG. 2, the nanopore cell 200 can be formed on a substrate 230 such as a silicon substrate. The dielectric layer 201 can be formed on the substrate 230. Dielectric materials used to form the dielectric layer 201 can include, for example, glass, oxides, nitrides, and the like. An electrical circuit 222 for controlling electrical stimulation and processing signals detected from the nanopore cell 200 can be formed on the substrate 230 and / or within the dielectric layer 201. For example, a plurality of patterned metal layers (e.g., metal 1 to metal 6) can be formed within the dielectric layer 201, and a plurality of active devices (e.g., transistors) can be fabricated on the substrate 230. In some embodiments, the signal source 228 is included as part of the electrical circuit 222. The electrical circuit 222 can include, for example, amplifiers, integrators, analog-to-digital converters, noise filters, feedback control logic, and / or various other components, and the like. The electrical circuit 222 can be further connected to a processor 224 connected to a memory 226, where the processor 224 can analyze the sequencing data and determine the sequence of the polymer molecules sequenced in the array.

[0071] The working electrode 202 can be formed on the dielectric layer 201 and can at least form a part of the bottom of the well 205. In some embodiments, the working electrode 202 is a metal electrode. For non-Faradaic conduction, the working electrode 202 can be made of a metal or other material resistant to corrosion and oxidation, such as, for example, platinum, gold, titanium nitride, and graphite. For example, the working electrode 202 can be a platinum electrode electroplated with platinum. In another example, the working electrode 202 can be a working electrode made of titanium nitride (TiN). The working electrode 202 can be porous, thereby increasing its surface area and providing a capacitance associated with the working electrode 202. Since the working electrodes of the nanopore cells can be independent of the working electrodes of other nanopore cells, the working electrodes can be referred to as cell electrodes in the present disclosure.

[0072] The dielectric layer 204 can be formed on the dielectric layer 201. The dielectric layer 204 forms a wall surrounding the well 205. Dielectric materials used to form the dielectric layer 204 can include, for example, glass, oxides, silicon nitride (SiN), polyimide, or other suitable hydrophobic insulating materials. The upper surface of the dielectric layer 204 can be silanized. The silanization can form a hydrophobic layer 220 above the upper surface of the dielectric layer 204. In some embodiments, the hydrophobic layer 220 has a thickness of about 1.5 nanometers (nm).

[0073] The well 205 formed by the dielectric layer wall 204 contains a large amount of electrolyte 206 above the working electrode 202. The large amount of electrolyte 206 can be neutralized and can contain one or more of the following: lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2). In some embodiments, the large amount of electrolyte 206 has a thickness of about 3 micrometers (μm).

[0074] Also, as shown in FIG. 2, a membrane can be formed above the dielectric layer 204, and this membrane extends across the well 205. In some embodiments, this membrane includes a lipid monolayer 218 formed above the hydrophobic layer 220. When this membrane reaches the opening of the well 205, the lipid monolayer 208 can transition into a lipid bilayer 214 that extends across the opening of the well 205. The lipid bilayer can include, or can consist of, lipids such as phospholipids. The lipids can be selected, for example, from: dipalmitoyl-phosphatidylcholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanyl-sn-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, GM1 ganglioside, lysophosphatidylcholine (LPC), or any combination thereof. Other phospholipid derivatives may also be used.For example, phosphatidic acid derivatives (e.g., DMPA, DDPA, DSPA), phosphatidylcholine derivatives (e.g., DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol derivatives (e.g., DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine derivatives (e.g., DMPE, DPPE, DSPE, DOPE), phosphatidylserine derivatives (e.g., DOPS), PEG lipid derivatives (e.g., mPEG lipid, polyglycerol lipid, functional lipid, terminal active lipid), and dilauroyl lipids (e.g., DPhPC, DOPhPC, DPhPE, and DOPhPE). In some embodiments, the bilayer can be formed using, for example, the following non-lipid-based materials: amphiphilic block copolymers (e.g., poly(butadiene)-block-poly(ethylene oxide), PEG diblock copolymer, PEG triblock copolymer, PPG triblock copolymer, and poloxamer), and other amphiphilic copolymers that can be nonionic or ionic. In some embodiments, the bilayer can be formed from a combination of a lipid-based material and a non-lipid-based material. In some embodiments, the bilayer material can be supplied in a solvent phase containing one or more organic solvents such as alkanes (e.g., decane, tridecane, hexadecane, etc.) and / or one or more silicone oils (e.g., AR-20).

[0075] As shown, the lipid bilayer 214 is embedded in a single nanopore 216 formed, for example, by a single PNTMC. As described above, the nanopore 216 can be formed by inserting a single PNTMC into the lipid bilayer 214 by electroporation. The nanopore 216 can be sized to allow at least one site of the analyte of interest and / or small ions (e.g., Na + , K + , Ca 2+ , Cl - ) to pass between the two sides of the lipid bilayer 214.

[0076] The sample chamber 215 is above the lipid bilayer 214 and can hold a solution of the analyte to be characterized. The solution is an aqueous solution containing the bulk electrolyte 208, neutralized to an optimal ion concentration, and can be maintained at an optimal pH to keep the nanopore 216 open. The nanopore 216 traverses the lipid bilayer 214 and provides the only path for ion flow from the bulk electrolyte 208 to the working electrode 202. In addition to the nanopore (e.g., PNTMC) and the analyte of interest, the bulk electrolyte 208 can further contain one or more of the following: lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2).

[0077] The counter electrode (CE) 210 can be an electrochemical potential sensor. In some embodiments, the counter electrode 210 is shared among a plurality of nanopore cells and can thus be referred to as a common electrode. In some cases, the common potential and the common electrode can be common to all nanopore cells or at least all nanopore cells within a particular grouping. The common electrode can be configured to apply a common potential to the bulk electrolyte 208 in contact with the nanopore 216. The counter electrode 210 and the working electrode 202 can be connected to a signal source 228 to provide an electrical stimulus (e.g., a voltage bias) across the lipid bilayer 214 and can be used to sense the electrical properties (e.g., resistance, capacitance, and ion current flow) of the lipid bilayer 214. In some embodiments, the nanopore cell 200 can also include a reference electrode 212.

[0078] In some embodiments, various checks are performed during the formation of the nanopore cell as part of the calibration. Once the nanopore cell is formed, additional calibration steps can be performed, for example, to identify a nanopore cell that operates as desired (e.g., one nanopore within the cell). Such calibration checks can include physical checks, voltage calibration, open channel calibration, and identification of a cell having a single nanopore.

[0079] B. Detection Signals of Nanopore Sequencing Cells Nanopore cells in a nanopore sensor chip, such as nanopore cell 150 in nanopore sensor chip 100, can enable parallel sequencing using single molecule nanopore-based sequencing by synthesis (Nano-SBS) technology.

[0080] FIG. 3 shows an embodiment of a nanopore cell 300 that performs nucleotide sequencing using Nano-SBS technology. In Nano-SBS technology, a template 332 to be sequenced (e.g., a nucleotide acid molecule or another analyte of interest) and a primer can be introduced into the bulk electrolyte 308 within the sample chamber of the nanopore cell 300. By way of example, the template 332 can be circular or linear. The nucleic acid primer can be hybridized to a site on the template 332 to which nucleotides 338 tagged with four different polymers can be added.

[0081] In some embodiments, an enzyme (e.g., polymerase 334 such as a DNA polymerase) is associated with a nanopore 316 for use in synthesizing a complementary strand onto a template 332. For example, polymerase 334 can be covalently attached to nanopore 316. Polymerase 334 can act as a catalyst in the incorporation onto a primer that uses a single-stranded nucleic acid molecule of nucleotide 338 as a template. Nucleotide 338 can include a tag species (a “tag”) that uses nucleotides of one of four different types: A, T, G, or C. When a tagged nucleotide is properly complexed with polymerase 334, this tag can be drawn (e.g., loaded) into the nanopore by an electrical force such as a force generated by an electric field applied across lipid bilayer 314 and / or nanopore 316 in the presence of a voltage. The end of the tag can be positioned within the barrel of nanopore 316. A tag held within the barrel of nanopore 316 can generate a unique ion blockade signal 340 based on the distinct chemical structure and / or size of that tag, thereby electronically discriminating the added base to which the tag binds.

[0082] As used herein, a “loaded” or “threaded” tag is positioned within and / or remains near a nanopore over a suitable period, such as from 0.1 milliseconds (ms) to 10,000 ms. In some cases, the tag is loaded into the nanopore before being released from the nucleotide. In some examples, the probability that a loaded tag passes through (and / or is detected by) the nanopore after being released after a nucleotide incorporation event is suitably high, such as from 90% to 99%.

[0083] In some embodiments, prior to the polymerase 334 being connected to the nanopore 316, the conductance of the nanopore 316 is high, such as about 300 picosiemens (300 pS). When a tag is loaded in the nanopore, a unique conductance signal (e.g., signal 340) is generated due to the distinct chemical structure and / or size of the tag. For example, the conductance of the nanopore can be about 60 pS, 80 pS, 100 pS, or 120 pS, each of which corresponds to one of four types of tagged nucleotides. And the polymerase can incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule through isomerization and phosphate transfer reactions.

[0084] In some cases, some of the tagged nucleotides may not match (complementary bases) the current position of the nucleic acid molecule (template). Tagged nucleotides that are not base-paired with the nucleic acid molecule can also pass through the nanopore. These unpaired nucleotides can be repelled by the polymerase on a time scale shorter than the time scale on which correctly paired nucleotides remain associated with the polymerase. Tags directed at unpaired nucleotides can pass quickly through the nanopore and can be detected over a short period (e.g., less than 10 ms). On the other hand, tags directed at paired nucleotides are loaded into the nanopore and can be detected over a long period (e.g., at least 10 ms). Thus, unpaired nucleotides can be identified by a downstream processor based at least in part on the time during which the nucleotide is detected within the nanopore.

[0085] The conductance (or equivalently, the resistance) of a nanopore containing a loaded (inserted) tag is measured via a signal value (e.g., a voltage or current passing through the nanopore), thereby providing identification of the tag species and thus the nucleotide at the current position. In some embodiments, a direct current (DC) signal is applied to the nanopore cell (e.g., so that the direction in which the tag moves through the nanopore is not reversed). However, using DC to operate the nanopore sensor over a long period of time can change the composition of the electrodes, make the ion concentration across the nanopore unbalanced, and have other undesirable effects that can adversely affect the lifespan of the nanopore cell. Applying an alternating current (AC) waveform can reduce electron migration, avoid these undesirable effects, and can have certain advantages as described below. The nucleic acid sequencing method utilizing tagged nucleotides described herein has sufficient affinity for the applied AC voltage, and thus an AC waveform can be used to achieve those advantages.

[0086] The ability to recharge the electrodes during the AC detection cycle can be suitable when electrodes that change molecular characteristics in the electrification reaction (e.g., electrodes containing silver), or sacrificial electrodes that are electrodes where the molecular characteristics in the electrification reaction change, are used. When a DC signal is used, the electrodes can be consumed during the detection cycle. Recharging can prevent the electrodes from reaching a consumption limit, such as complete depletion, which can be a problem when the electrodes are small (e.g., when the electrodes are small enough to provide an array of electrodes having at least 500 electrodes per square millimeter). The electrode lifespan, in some cases, increases with the width of the electrodes and at least partially depends on the width of the electrodes.

[0087] Suitable conditions for measuring the ionic current passing through the nanopores are known to those skilled in the art, and examples thereof are provided herein. The measurement can be performed using a voltage applied across the membrane and the pores. In some embodiments, the voltage used is in the range of -400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV, and 0 mV and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV, and +400 mV. The voltage used can be more preferably in the range of 100 mV to 240 mV, and most preferably in the range of 160 mV to 240 mV. Nanopores using an increased applied potential can increase the discrimination between different nucleotides. Nucleic acid sequencing using an AC waveform and tagged nucleotides is described in U.S. Patent Application Publication No. 2014 / 0134616, filed Nov. 6, 2013, entitled "Nucleic Acid Sequencing Using Tags", which is hereby incorporated by reference in its entirety. In addition to the tagged nucleotides described in U.S. Patent Application Publication No. 2014 / 0134616, sequencing can be performed using nucleotide analogs having a sugar moiety or an acyclic moiety such as (S)-glycerol nucleoside triphosphates (gNTPs) of the five common nucleobases of adenine, cytosine, guanine, uracil, and thymine (Horhota et al., Organic Letters, 8:5345-5347

[2006] ).

[0088] C. Electrical Circuit of the Nanopore Sequencing Cell FIG. 4 shows an embodiment of an electrical circuit 400 (which can include each part of the electrical circuit 222 in FIG. 2) within a nanopore cell, such as nanopore cell 400. As described above, in some embodiments, the electrical circuit 400 can be shared among multiple or all of the nanopore cells in a nanopore sensor chip, and thus includes a counter electrode 410, which can also be referred to as a common electrode. The common electrode is configured to apply a common potential to a bulk electrolyte (e.g., bulk electrolyte 208) that contacts a lipid bilayer (e.g., lipid bilayer 214) in the nanopore cell by connecting to a voltage source V LIQ 420. In some embodiments, an AC non-Faradaic mode is utilized, and a voltage V LIQ is modulated using an AC signal (e.g., a rectangular wave) and applied to the bulk electrolyte that contacts the lipid bilayer in the nanopore cell. In some embodiments, V LIQ is a rectangular wave having a magnitude of ±200 to 250 mV and a frequency, for example, between 25 and 400 Hz. The bulk electrolyte between the counter electrode 410 and the lipid bilayer (e.g., lipid bilayer 214) can be modeled, for example, by a large capacitor (not shown) of 100 μF or more.

[0089] FIG. 4 also shows an electrical model 422 that represents the electrical properties of the working electrode 402 (e.g., working electrode 202) and the lipid bilayer (e.g., lipid bilayer 214). The electrical model 422 includes a capacitor 426 (C 二分子層 ) that models the capacitance associated with the lipid bilayer, and a resistor 428 (R 細孔 ) that models the variable resistance associated with the nanopore, which can vary based on the presence of a specific tag in the nanopore. The electrical model 422 also has a double-layer capacitance (C 二重層 ) and includes a capacitor 424 that represents the electrical properties between the working electrode 402 and the well 205. The working electrode 402 can be configured to apply a distinct potential independent of the working electrodes in other nanopore cells.

[0090] The pass device 406 is a switch that can be used to connect or disconnect the lipid bilayer and the working electrode to the electrical circuit 400. The pass device 406 can be controlled by a control line 407 to enable or disable a voltage stimulus applied across the lipid bilayer in the nanopore cell. Before the lipid accumulates to form the lipid bilayer, the impedance between these two electrodes may be very low because the well of the nanopore cell is not sealed, and thus, to avoid a short - circuit situation, the pass device 406 can be maintained in an open state. After the lipid solvent has deposited in the nanopore cell and the well of the nanopore cell is sealed, the pass device 406 can be closed.

[0091] The circuit 400 can further include an on - chip integrated capacitor 408(n cap ). The integrated capacitor 408 can be pre - charged by closing the switch 401 using a reset signal 403 such that the integrated capacitor 408 is connected to a voltage source V PRE 405. In some embodiments, the voltage source V PRE 405 provides a specific reference voltage, such as 900 mV in magnitude. When the switch 401 is closed, the integrated capacitor 408 can be pre - charged to the reference voltage level of the voltage source V PRE 405.

[0092] After pre - charging the integrated capacitor 408, the switch 401 can be opened using the reset signal 403 such that the connection from the voltage source V PRE 405 of the integrated capacitor 408 is disconnected. At this point, depending on the level of the voltage source V LIQ , the potential of the counter electrode 410 can be at a higher level than that of the working electrode 402 (and the integrated capacitor 408), and vice versa. For example, during the positive phase of a rectangular wave from the voltage source V LIQ (e.g., the bright or dark period of an AC voltage source signal cycle), the potential of the counter electrode 410 is at a higher level than the potential of the working electrode 402. During the voltage source V LIQDuring the negative phase of the rectangular wave from PRE PRE , for example, the dark period or the bright period of the AC voltage source signal cycle, the potential of the counter electrode 410 is at a level lower than the potential of the working electrode 402. Thus, in some embodiments, the integration capacitor 408 can be further charged from the pre-charged voltage level of the voltage source V

[0093] 405 to a higher level during the bright period, and can be discharged to a lower level due to the potential difference between the counter electrode 410 and the working electrode 402 during the dark period. In other embodiments, the charging and discharging are performed during the dark period and the bright period, respectively.

[0094] After being sampled by the ADC 435, the integration capacitor 408 can be pre-charged by closing the switch 401 using the reset signal 403 so that the integration capacitor 408 is connected to the voltage source V PRE 405 again. The steps of pre-charging the integration capacitor 408, waiting for a certain period during which the integration capacitor 408 is charged or discharged, and sampling and converting the voltage level of the integration capacitor by the ADC 435 can be repeated in the cycle through the array determination process.

[0095] The digital processor 430 can process the ADC output data, for example, for normalization, data buffering, data filtering, data compression, data reduction, event extraction, or the assembly of the ADC output data into various data frames from an array of nanopore cells. In some embodiments, the digital processor 430 further performs downstream processing such as base determination. The digital processor 430 can be implemented as hardware (e.g., in a graphics processing unit (GPU), FPGA, ASIC, etc.) or as a combination of hardware and software.

[0096] Accordingly, the voltage signal applied across the nanopore can be used to detect a particular state of the nanopore. One possible state of the nanopore is the open-channel state when the tag-bound polyphosphate disappears from the barrel of the nanopore, which is also referred to herein as the state of the nanopore through which no insertion occurs. The other four possible states of the nanopore each correspond to the state when one of the four different types of tag-bound polyphosphate nucleotides (A, T, G, or C) is retained within the barrel of the nanopore. Yet another possible state of the nanopore is the state when the lipid bilayer is ruptured.

[0097] When the voltage level on the integrating capacitor 408 is measured after a certain period, different states of the nanopore can result in different measured values of the voltage level. This is because the rate of voltage decay (decrease due to discharge or increase due to charging) on the integrating capacitor 408 (i.e., the slope of the voltage on the integrating capacitor 408 with respect to the time plot) is related to the nanopore resistance (e.g., resistor R 細孔This is because it depends on the resistance of 428. In particular, in different states, since the resistance associated with the nanopore varies due to the distinct chemical structure of the molecule (tag), different corresponding rates of voltage decay can be observed and can be used to distinguish different states of the nanopore. The voltage decay curve can be an exponential function curve with an RC time constant τ = RC, where R is the resistance associated with the nanopore (i.e., R 細孔 is the resistance associated with resistor 428), and C is the capacitance associated with the membrane (i.e., C 二分子層 is the capacitance associated with capacitor 426) in parallel with R. The time constant of the nanopore cell can be, for example, in the range of about 200 - 500 ms. The decay curve may not exactly match the exponential function curve due to the detailed implementation of the bilayer, but the decay curve is similar to the exponential function curve and is monotonic, and thus can enable the detection of tags.

[0098] In some embodiments, in the open-channel state, the resistance associated with the nanopore is in the range of 100 MOhm to 20 GOhm. In some embodiments, when the tag is inside the barrel of the nanopore, the resistance associated with the nanopore can be in the range of 200 MOhm to 40 GOhm. In other embodiments, since the voltage to the ADC 435 still changes due to the voltage decay in the electrical model 422, the integrating capacitor 408 is omitted.

[0099] The rate of voltage decay on the integration capacitor 408 can be determined in different ways. As described above, the rate of voltage decay can be determined by measuring the voltage decay over a certain period of time. For example, the voltage on the integration capacitor 408 can first be measured by the ADC 435 at time t1, and subsequently, the voltage can be measured again by the ADC 435 at time t2. If the slope of the voltage on the integration capacitor 408 with respect to the time curve is steeper, the voltage difference is larger, and if the slope of the voltage curve is not steeper, the voltage difference is smaller. Therefore, the voltage difference can be used as a measurement criterion for determining the rate of voltage decay on the integration capacitor 408, and thus, the state of the nanopore cell.

[0100] In other embodiments, the rate of voltage decay is determined by measuring the period required for a selected amount of voltage decay. For example, the time required for the voltage to drop, or the time required for the voltage to rise from a first voltage level V1 to a second voltage level V2 can be measured. If the slope of the voltage is steeper with respect to the time curve, the required time is shorter, and if the slope of the voltage is not steeper with respect to the time curve, the required time is longer. Therefore, the measured required time can be used as a measurement criterion for determining the rate of voltage decay on the integration capacitor n cap 408, and thus, the state of the nanopore cell. One skilled in the art will understand the various circuits that can be used to measure the resistance of a nanopore, including signal value measurement techniques such as voltage or current measurement.

[0101] In some embodiments, the electrical circuit 400 does not include an on-chip made pass device (e.g., pass device 406) and an additional capacitor (e.g., integration capacitor 408(n cap ))), thereby facilitating the reduction of the size of the nanopore-based sequencing chip. Due to the thinness of the membrane (lipid bilayer), the membrane (e.g., capacitor 426(C 二分子層The capacitance associated with ()) can, by itself, without the need for additional on-chip capacitance, be sufficient to create the required RC time constant. Thus, capacitor 426 can be used as an integrating capacitor and charged in advance by voltage signal V PRE and subsequently discharged or charged by voltage signal V LIQ . In an electrical circuit, otherwise, the elimination of the additional capacitor and the pass device fabricated on-chip can significantly reduce the mounting area of a single nanopore cell in the nanopore sequencing chip, thereby facilitating the scaling of the nanopore sequencing chip to include more cells (e.g., having millions of cells in the nanopore sequencing chip).

[0102] D. Sampling of Data in Nanopore Cells To perform nucleic acid sequencing, the voltage level of the integrating capacitor (e.g., integrating capacitor 408 (n cap ) or capacitor 426 (C 二分子層 )) can be sampled and converted by an ADC (e.g., ADC 435) while the tagged nucleotide is being added to the nucleic acid. For example, if the applied voltage is such that V LIQ is lower than V PRE , the tag of the nucleotide can be pushed into the barrel of the nanopore by the electric field across the nanopore applied through the counter electrode and the working electrode.

[0103] 1. Insertion An insertion event is a case where a tagged nucleotide binds to a template (e.g., a single strand of nucleic acid) and its tag enters and exits the barrel of the nanopore and moves. This movement can occur multiple times during the insertion event. When the tag is inside the barrel of the nanopore, the resistance of the nanopore can be made higher and the current that can flow through the nanopore can be made smaller.

[0104] During array determination, the tag may not be inside the nanopore depending on the AC cycle (referred to as the open channel state), in which case the current is maximum because the resistance of the nanopore is smaller. When the tag is drawn into the barrel of the nanopore, the nanopore enters the bright mode. When the tag is pushed out of the barrel of the nanopore, the nanopore enters the dark mode.

[0105] 2. Bright and dark periods During the AC cycle, the voltage on the integrating capacitor can be sampled multiple times by the ADC. For example, in one embodiment, an AC voltage signal is applied to the entire system at, for example, about 100 Hz, and the acquisition speed of the ADC can be about 2000 Hz per cell. Therefore, about 20 data points (voltage measurements) can be acquired for each AC cycle (cycle of the AC waveform). The plurality of data points corresponding to one cycle of the AC waveform can be called a set. For one set of data points for an AC cycle, for example, it can correspond to the bright mode (period) when the tag is pushed into the barrel of the nanopore, V LIQ when V PRE is lower than V LIQ a subset can be acquired. Another subset is, for example, when V PRE is higher than V

[0106] 3. Measured voltage For each of the data points, when the switch 401 is opened, for example, when V LIQ is higher than V PRE as the rise from V PRE to V LIQ or, when V LIQ is lower than V PRE as the drop from V PRE to V LIQ as a result of the charging / discharging at V LIQ the integrating capacitor (for example, integrating capacitor 408 (n cap ) or capacitor 426 (C 二分子層) The voltage in () changes so as to decay. Due to the charging of the working electrode, the final voltage value can deviate from V LIQ It can deviate from. The rate of change of the voltage level on the integration capacitor can include nanopores, and thus can be controlled by the value of the resistance of the bilayer that can include molecules (e.g., tags of tagged nucleotides) in the nanopores. The voltage level can be measured at a predetermined time after switch 401 is opened.

[0107] Switch 401 can operate at the data acquisition rate. Switch 401 can typically close for a relatively short period between two acquisitions of data, immediately after measurement by the ADC. The switch enables the acquisition of multiple data points over each sub-period (bright or dark) of each AC cycle of V LIQ If switch 401 remains open, the voltage level on the integration capacitor, and thus the output value of the ADC, is sufficiently decayed and remains in that state. Instead, when switch 401 is closed, the integration capacitor is recharged (up to V PRE ), and is ready for another measurement. Thus, switch 401 enables the acquisition of multiple data points over each sub-period (bright or dark) of each AC cycle. Such multiple measurements can enable a higher resolution than by a fixed ADC (e.g., from 8 bits to 14 bits due to a large number of measurements, which may be averaged). Multiple measurements can also provide motion information about the molecules inserted into the nanopores. The timing information can enable the determination of the period during which the insertion occurs. This can also be used to assist in determining whether the sequences of multiple nucleotides added to the nucleic acid strand have been determined.

[0108] Figure 5 shows an example of data points acquired from the nanopore cell during the bright and dark periods of the AC cycle. In Figure 5, the change in the data points is emphasized for illustrative purposes. The voltage (V PRE) is, for example, at a constant level such as 900 mV. The voltage signal 510 (V applied to the counter electrode of the nanopore cell LIQ ) is an AC signal shown as a rectangular wave, and in this case, the duty cycle can be any suitable value of 50% or less, such as, for example, about 40%.

[0109] During the illumination period 520, due to different voltage levels applied to the working electrode and the counter electrode (e.g., due to the flow of charge and / or ions on the tag), the voltage signal 510 (V applied to the counter electrode is such that the tag can be pushed into the barrel of the nanopore by the electric field caused by the different voltage levels applied to the working electrode and the counter electrode LIQ ) is lower than the voltage V applied to the working electrode PRE . When the switch 401 is opened, the voltage at the node in front of the ADC (e.g., at the integrating capacitor) drops. After the voltage data point is acquired (e.g., after a specified period), the switch 401 can be closed, the voltage at the measurement node rises, and it returns to V PRE again. This process of measuring multiple voltage data points can be repeated. In this way, multiple data points can be acquired during the illumination period.

[0110] As shown in FIG. 5, the first data point 522 (also referred to as the first point delta (FPD)) in the illumination period after the change in the sign of the V LIQ signal can be lower than the subsequent data point 524. This can be because there is no tag in the nanopore (open channel), and thus it has a low resistance and a high discharge rate. In some examples, the first data point 522, as shown in FIG. 5, is V LIQIt can exceed the level. This can be caused by the capacitance of the bilayer connecting the signal to the on-chip capacitor. Data point 524 can be acquired after an insertion event occurs, i.e., after the tag is pushed into the barrel of the nanopore. In this case, the resistance of the nanopore, and thus the rate of discharge of the integrating capacitor, depends on the specific type of tag pushed into the barrel of the nanopore. Data point 524 can be slightly reduced for each measurement by the charge accumulated at C 二重層 424.

[0111] During the dark period 530, the voltage signal 510 (V LIQ ) applied to the counter electrode is higher than the voltage (V PRE ) applied to the working electrode so that any tag is pushed out of the barrel of the nanopore. When switch 401 is opened, the voltage level of the voltage signal 510 (V LIQ ) is higher than V PRE , causing the voltage at the measurement node to rise. After a voltage data point is acquired (e.g., after a specified period), switch 401 can be closed, causing the voltage at the measurement node to drop and return to V PRE again. This process of measuring multiple voltage data points can be repeated. Thus, a plurality of data points including the first point delta 532 and subsequent data points 534 can be acquired during the dark period. As described above, during the dark period, any nucleotide tag is pushed out of the nanopore, and thus, in addition to being used in normalization, a minimum amount of information about any nucleotide tag is acquired.

[0112] FIG. 5 also shows that during the light period 540, the voltage signal 510 (V LIQ ) applied to the counter electrode is the voltage (V PREEven if it is lower than , it indicates that no insertion event occurs (open channel). Therefore, the resistance of the nanopore is low, and the discharge rate of the integrating capacitor is high. As a result, the acquired data points, including the first data point 542 and the subsequent data point 544, indicate a low voltage level.

[0113] The voltage measured during the light or dark period may be expected to be approximately the same for each measurement of a constant resistance of the nanopore (e.g., during the bright mode of a given AC cycle while one tag is within the nanopore). However, this may not be the case when charge (C 二重層 ) is accumulated in the double-layer capacitor 424. This charge accumulation can lengthen the time constant of the nanopore cell. As a result, the voltage level can be shifted, and thus, for each data point in a cycle, the measured value is decreased. Therefore, as shown in FIG. 5, within a cycle, the data points may change slightly from one data point to another.

[0114] Further details regarding the measurement can be found, for example, in U.S. Patent Application Publication No. 2016 / 0178577 entitled "Nanopore-Based Sequencing With Varying Voltage Stimulus", U.S. Patent Application Publication No. 2016 / 0178554 entitled "Nanopore-Based Sequencing With Varying Voltage Stimulus", U.S. Patent Application No. 15 / 085,700 entitled "Non-Destructive Bilayer Monitoring Using Measurement Of Bilayer Response To Electrical Stimulus", and U.S. Patent Application No. 15 / 085,713 entitled "Electrical Enhancement Of Bilayer Formation", the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0115] 4. Normalization and Base Calling For each nanopore cell of the nanopore sensor chip that can be used, a production mode for sequencing nucleic acids can be executed. The ADC output data obtained during sequencing can be normalized to provide higher accuracy. The normalization can consist of offset effects such as cycle shape, gain drift, charge injection offset, and baseline shift. In some implementations, the signal values of the on-period cycles corresponding to the translocation events can be flattened such that a single signal value (e.g., the average value) is obtained for each cycle, or adjustments can be made to the measured signals to reduce intra-cycle attenuation (one type of cycle shape effect). Gain drift generally spreads across the entire signal and changes on the order of seconds from 100s to 1,000s. As an example, gain drift can be triggered by changes in the solution (pore resistance) or changes in the bilayer capacitance. Baseline shift occurs with a time scale of about 100 ms and relates to the voltage offset at the working electrode. Baseline shift can be promoted by a change in the effective rectification ratio from translocation as a result of the need to maintain charge balance in the sequencing cell from the on-period to the off-period.

[0116] After normalization, embodiments can determine clusters of voltages for the inserted channels, with each cluster corresponding to a different tag species and thus a different nucleotide. The clusters can be used to determine the probability of a given voltage corresponding to a given nucleotide. As another example, the clusters can be used to determine cutoff voltages for distinguishing between different nucleotides (bases).

[0117] III. Self-Limiting Pore Insertion After the pore is inserted into the membrane of the cell, the voltage across the membrane begins to rapidly drop due to the relatively high conductance of the pore. The drop in voltage across the membrane reduces the driving force for further pore insertion in the membrane.

[0118] FIG. 6 shows an embodiment of a circuit diagram 600 for a nanopore sensor cell. Here, some of the various voltages and components of the sensor cell that may be relevant to the systems and methods described herein are highlighted. Examples of these include: the voltage (Vapp) 602 applied between the working electrode and the counter electrode, the voltage (Vbly) 604 across the bilayer, the voltage (Vpre) 606 used for pre-charging the working electrode (C-double layer) 608 and the integrating capacitor (NCAP) 610, and the voltage (Vliq) 612 applied to the counter electrode.

[0119] What is described herein are methods and systems that utilize this property of controlling single pore insertion by inserting protein pores without active feedback during the insertion step. In some embodiments of this pore insertion method, an AC-connected voltage is applied through a capacitive working electrode. This voltage is maintained across the membrane due to the low conductance of the poreless membrane. In some embodiments, the voltage can be applied across the entire array of cells regardless of the current state of pore insertion. In some embodiments, the voltage can be applied to cells having a membrane. The applied voltage waveform can be stepped up in magnitude as a ramp, as a plurality of expansion steps, or as other shapes designed to lower the probability of additional protein pore insertion while also reducing the risk of membrane damage. This can be achieved by limiting the voltage application transient characteristics, such as by using small voltage steps, a gentle rate of voltage increase in a voltage ramp, etc.

[0120] For example, in some embodiments, as shown in FIG. 7, the pore insertion voltage (Vapp) can be applied as a stepped voltage waveform 700 that begins at 0 mV and increases in 100 mV increments every 5 seconds up to a maximum voltage of 600 mV. In some embodiments, the initial voltage can be about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mV. In some embodiments, the step increase can be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mV. In some embodiments, the duration of each step can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 seconds. In some embodiments, the steps can have variable durations. For example, in some embodiments, some or all of the steps at lower voltages can have longer durations than those at higher voltages. In some embodiments, the maximum voltage is about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mV. In some embodiments, one or more elements of the pore insertion voltage waveform can be predetermined, such as the initial starting voltage, the magnitude of the voltage step increase, the duration of each step, and / or the maximum voltage.

[0121] In some embodiments, as shown in FIG. 8A, the pore insertion voltage can be applied as a sloped voltage waveform 800 that begins at 0 mV and rises at a rate of 1 V per minute to a maximum voltage of 600 mV. In some embodiments, the initial voltage can be about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mV. In some embodiments, the rate of voltage rise is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 V per minute. In some embodiments, the maximum voltage is about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mV. In some embodiments, one or more elements of the pore insertion voltage waveform can be predetermined, such as the initial start voltage, the rate of voltage rise, and / or the maximum voltage.

[0122] In some embodiments, one or more elements of the pore insertion voltage waveform can be determined based on measured electrical and / or physical properties of components of the cell, such as the membrane seal resistance, which is the resistance across the membrane after the membrane has formed a seal across the cell. In some embodiments, these measurements can be obtained before the voltage waveform is applied and the waveform is fully determined before it is applied. This is different from an active feedback-based method that uses measurements obtained during stimulation to change one or more stimulation parameters. Since the poration method described herein is self-limiting, there is no need to utilize an active poration method that includes measuring changes in the electrical or physical properties of a system, or components of a system, derived from inserting a pore into a membrane, and subsequently adjusting the poration voltage in response to this to prevent insertion of a second pore into the membrane.

[0123] In some embodiments, the methods described herein can be applied to an array of sensors having capacitive electrodes at the base of the microwells with the suspended membrane and a counter electrode on the opposite side of the membrane. These sensors can be used to detect the presence of pores after the application of an insertion drive voltage has been removed from all cells. It is possible to detect the presence of pores during the application of the voltage, but this is not necessary in this method. Pores can be inserted without feedback on the application of the voltage on any individual sensor in the array or in the aggregate.

[0124] This method effectively scans the voltages necessary to overcome pore insertion activation barriers that can vary between individual membranes within the array, between narrow or wide regions on the array, or between an array from one device to another array from a second device. Additionally, the poration voltage can vary between mutant pores and between the composition and structure of the membranes, including lipid bilayers, block copolymers, or other implementations. By scanning or sweeping the voltage over a narrow to wide range, a single voltage waveform can be made strong enough to function effectively in a very large number of different types of pore arrays or in the same type of pore array with a certain amount of variability.

[0125] In addition, by sweeping from a low voltage to a high voltage, the bilayer can have a higher likelihood of pores being inserted in the membrane before reaching the critical voltage level that damages the membrane. In addition, as shown in FIGS. 9A and 9B, when pores are inserted, the pores can dissipate the increased voltage across the membrane, and thus, after the pores are inserted, when the voltage further increases, it is possible to both reduce the risk of damage to the membrane and reduce the likelihood of further pore insertion. As long as the magnitude of the voltage step, or the rate of increase of the voltage slope, is not too large, the pores can effectively dissipate the excessive voltage increase across the membrane, thus reducing the risk of membrane damage and the likelihood of further pore insertion. On the other hand, it is desirable to increase the magnitude of the voltage step or increase the rate of increase of the voltage slope in order to shorten the time to complete the poration step.

[0126] In some embodiments, the upper limit of the voltage waveform can be determined by comparing the dynamics and / or probability of pore insertion as a function of voltage and time with the dynamics and / or probability of membrane damage as a function of voltage and time. For example, FIG. 10A shows a plot of the number of pore insertions in an array as a function of voltage. FIG. 10B shows a plot of the number of deactivations / shorts typically resulting from membrane breakage and damage as a function of voltage. From these two plots, an optimal maximum voltage can be determined that is balanced with a large number of pore insertions and a small number of deactivations / shorts.

[0127] In some embodiments, during the pore insertion step, the concentration of pores in the solution is low enough to reduce passive insertion of pores into the membrane, while still being high enough to allow voltage-assisted insertion of pores into the membrane. Passive insertion of pores refers to the insertion of pores into the membrane without the application of a voltage across the membrane for assistance in pore insertion. In some embodiments, the percentage of pores inserted through passive insertion is less than 50%, 40%, 30%, 20%, or 10%. The percentage of pores inserted through voltage-assisted insertion is at least 50%, 60%, 70%, 80%, or 90%. Reducing the rate of passive pore insertion can reduce the likelihood that multiple pores are inserted into a single membrane.

[0128] In some embodiments, the leakage current can cause an increase in voltage in one or more cells in the array after the membrane is placed on the cell. This trapped charge can vary in magnitude over time between cells. This makes it difficult to apply a uniform voltage across the entire membrane of the cell when inducing poration. For example, when trapped charge is present in cells in the array in varying amounts, applying a uniform voltage (Vapp) to all of the cells can result in the cells receiving different amounts of effective voltage during the poration step. This can lead to a high level of variability in the number of cells with a single pore insertion and / or, in some cells, the voltage can be applied in an excessive amount. This can cause damage to the membrane. Using a stepped or sloped voltage waveform can solve these problems.

[0129] In some embodiments, the formation of the membrane over the cell opening is achieved by flowing a solvent and a membrane material, such as a lipid or a block copolymer, over the cell opening. Subsequently, for example, when a lipid is used, the membrane can be thinned to a bilayer by applying a voltage across the membrane and / or by manipulating an osmotic concentration imbalance across the membrane, as further described in U.S. Patent Publication No. 20170283867A1 and International Patent Publication No. WO2018001925, respectively. The entire contents of each of these are incorporated herein by reference for all purposes. As described below, a thinned membrane is a membrane that is sufficiently thin (i.e., for example, having a thickness less than the length of the pore) such that pores can be inserted into the membrane. On the other hand, a non-thinned membrane is a membrane that has a thickness that is too thick to allow for the insertion of pores (i.e., for example, having a thickness greater than the length of the pore). In some embodiments, the formation of the thinned membrane (i.e., lipid bilayer) over the cells in the array can be completed before initiating the poration process and before inserting pores into the membrane. In other embodiments, the process of thinning the membrane can be combined with the process of inserting pores into the membrane by using the same voltage waveform, such as any of the voltage waveforms described herein, for both the thinning process and the poration process. The pore complex can be flowed over the membrane during the combined process of thinning and poration. In some embodiments, the combined process of thinning and poration can be applied after a non-thinned membrane has been formed across the cells in the array by applying a voltage during the dispensing of the membrane material onto the cells. The initial formation of the non-thinned membrane can result in non-uniform confinement of charge. Additionally, a non-uniform osmotic pressure can be achieved across the membrane during the combined process of thinning and poration. Combining the thinning step and the poration step can substantially reduce the time taken to prepare an array-like pore sensor. Thus, improving the throughput of the sensor array system.

[0130] The method described herein provides a number of advantages, including improving the rate of good single pore insertion, reducing the rate of multiple pore insertions, and reducing the potential for membrane damage.

[0131] IV. Self-Limiting Pore Insertion in Faradaic Systems and Methods As described above, in systems and methods where voltage application is capacitively coupled, such as non-Faradaic electrochemical systems, the voltage driving pore insertion decays at a rate proportional to the membrane conductance. Thus, when the conductance increases by several orders of magnitude (e.g., from a sealed membrane to a single pore membrane), the voltage maintained across the membrane rapidly decreases and remains low for subsequent voltage applications without the need to change the voltage applied to the porous membrane. In this way, self-limiting pore insertion can be driven under various conditions without changing the active stimulation intended to prevent additional pore insertion, using an array of electrodes with suspended membranes.

[0132] However, in systems where voltage application is resistively coupled, such as Faradaic electrochemical systems, the voltage across the membrane does not decay as a first-order effect of changes in membrane conductance. As long as the resistive coupling between the electrode and the electrolyte is maintained, the voltage across the membrane is first-order constant. By first dispensing a fluid that is capable of resistive coupling but is in a state that prevents it, capacitive coupling can be established temporarily and reversibly to enable self-limiting perforation. After pore insertion, the fluid can be modified by solution exchange or voltage application to enable resistive coupling again.

[0133] For example, a reversible electrochemical system A + e = B (where "e" is an electron and A and B are oxidation states of chemical species) can be prevented from proceeding by first removing all species "A" so that the solution contains only species "B". In such a state, the system behaves as capacitive coupling under a negative (reductive) voltage bias. By applying a positive (oxidative) voltage bias, species A can be regenerated and the system resumes its Faradaic (resistive coupling) characteristics.

[0134] Similarly, when species “B” is alternatively excluded such that the solution contains only species “A”, the solution behaves as a capacitive junction under a positive (oxidizing) voltage bias. By applying a negative (reducing) voltage bias, species “B” is regenerated and the system reverts to its Faradaic (resistive junction) characteristics.

[0135] This enables the system and method to be selectively and reversibly operated as either a Faradaic or non-Faradaic system and method, as desired. As described herein, temporary or selective operation as a non-Faradaic system enables utilization of a system and method for self-limiting pore insertion. For example, a sloped voltage waveform can be applied to facilitate single pore insertion, and after the pore solution is removed and replaced with a solution containing the sample with the molecule to be sequenced, the system and method can operate under the Faradaic conditions described above, for example, by providing only one half of a redox pair of oxidizing and reducing species and operating under appropriate oxidation or reduction conditions.

[0136] In some embodiments, the solution containing the pore used during pore insertion does not contain a redox pair or couple (i.e., contains neither “A” nor “B”, or contains only one of “A” or “B”). In some embodiments, the solution containing the molecule to be sequenced (i.e., which is a nucleic acid, polymer, derivative molecule of a nucleic acid, etc.) can contain a redox pair or couple, or can contain one of the species of the redox pair. In some embodiments, a voltage is used to regenerate the missing species of the redox pair.

[0137] Examples of redox pairs include ferrocyanide and ferricyanide, ferrocenecarboxylic acid (FCA) and ferroceneacetic acid (FAA), [Co(bpy)] 2+ / 3+ , and other water-soluble redox pairs.

[0138] A. Sequencing Systems and Methods Particularly Suitable for Use Under Faradaic Conditions In some sequencing systems, particularly when large molecules are inserted into pores, it may be desirable to operate under Faraday conditions. This is because a constant voltage can be applied across the pore to drive the molecules passing through the pore and / or continuous measurement readings can be obtained over a long period of time. Examples of nanopore sequencing systems and methods for inserting sample molecules into pores during sequencing include sequencers from Oxford Nanopore (i.e., U.S. Pat. Nos. 9,758,823 and 10,416,117, each of which is incorporated by reference in its entirety) and Stratos Genomics (i.e., U.S. Pat. Nos. 7,939,259 and 9,771,614, each of which is incorporated by reference in its entirety).

[0139] For example, Stratos Genomics sequences nucleic acids by creating "Xpandomers" from nucleic acid templates. This is achieved by encoding nucleic acid information on extended-length surrogate polymers that are easier to detect. The surrogate polymer (referred to herein as "Xpandomer") is formed by template-directed synthesis that preserves the original genetic information of the target nucleic acid while increasing the linear separation of the individual elements of the sequence data.

[0140] In one embodiment, a method for sequencing a target nucleic acid, comprising: a) providing a daughter strand produced by template-directed synthesis, the daughter strand comprising a plurality of subunits coupled with a sequence corresponding to all or part of the contiguous nucleotide sequence of the target nucleic acid, each subunit comprising a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond, to provide the daughter strand; b) cleaving at least one selectively cleavable bond to generate an Xpandomer having a length longer than the plurality of subunits of the daughter strand, the Xpandomer comprising a tether and a reporter element for analyzing genetic information in a sequence corresponding to all or part of the contiguous nucleotide sequence of the target nucleic acid, to generate the Xpandomer; and c) detecting the reporter element of the Xpandomer.

[0141] In a more specific embodiment, the reporter element for analyzing genetic information may be related to the tether of the Xpandomer, the daughter strand before cleavage of at least one selectively cleavable bond, and / or the Xpandomer after cleavage of at least one selectively cleavable bond. The Xpandomer may further comprise all or part of at least one probe or nucleobase residue, and the reporter element for analyzing genetic information may be related to at least one probe or nucleobase residue or may be the probe or nucleobase residue itself. Further, the selectively cleavable bond may be a covalent bond, an intratether bond, a bond between or within the probes or nucleobase residues of the daughter strand, and / or a bond between the probes or nucleobase residues of the daughter strand and the target template.

[0142] In a further embodiment, an oligomer substrate construct for use in template-directed synthesis for sequencing a target nucleic acid is disclosed. The oligomer substrate construct is a first probe portion linked to a second probe portion, wherein each of the first and second probe portions has a terminal group suitable for template-directed synthesis, a first probe portion, and a tether having a first end and a second end, at least the first end of the tether being linked to at least one of the first and second probe portions, and the oligomer substrate construct, when used in template-directed synthesis, includes a constrained Xpandomer and can form a daughter strand having a plurality of subunits coupled in a sequence corresponding to all or part of the contiguous nucleotide sequence of the target nucleic acid, and the individual subunits include a tether, the first and second probe portions, and at least one selectively cleavable bond.

[0143] In another embodiment, a monomer substrate construct for use in template-directed synthesis for sequencing a target nucleic acid is disclosed. The monomer substrate construct includes a nucleobase residue having a terminal group suitable for template-directed synthesis and a tether having a first end and a second end, at least the first end of the tether being linked to the nucleobase residue, and the monomer substrate construct, when used in template-directed synthesis, includes a constrained Xpandomer and can form a daughter strand having a plurality of subunits coupled in a sequence corresponding to all or part of the contiguous nucleotide sequence of the target nucleic acid, and the individual subunits include a tether, the nucleobase residue, and at least one selectively cleavable bond.

[0144] In yet a further embodiment, a template-daughter strand duplex including a daughter strand duplexed with a template strand, and a method for forming it from the template strand and the oligomer or monomer substrate construct are disclosed.

[0145] V. AC Modulation of Voltage Waveform In some embodiments, as shown in FIG. 7, the pore insertion waveform 700 can be a voltage waveform with AC modulation. As shown here, the pore insertion waveform 702 is stepped. The components of the AC modulation 702 can be superimposed on top of the voltage waveform 700 to provide rapid voltage fluctuations at each of the stepped voltages. The voltage fluctuations or changes enable electrical measurements to be taken while the pore insertion waveform 700 is being applied during the electroporation step during pore insertion. These electrical measurements can be used to check the integrity of the membrane (i.e., detect membrane defects such as short conditions), check the leakiness of the membrane (i.e., the resistance and / or conductance of the membrane), and check the insertion of pores, and generally can be used to monitor the progress of the electroporation step. In some embodiments, measurements cannot be taken while the voltage waveform is being applied. In these embodiments, measurements are generally taken before, after, or between applications of the voltage waveform. Superimposing the AC modulation component on the voltage waveform enables simultaneous application of the voltage waveform and acquisition of measurements.

[0146] In some embodiments, an AC modulation component is also superimposed on other voltage waveforms. For example, the film formation waveform can be AC modulated so that electrical measurements can be taken while the film formation waveform is being applied during the step of forming a film over a well. These electrical measurements can be used to check the integrity of the membrane (i.e., detect membrane defects such as short conditions) to check whether the well is covered with the film-forming material, check the leakiness of the membrane (i.e., the resistance and / or conductance of the membrane), check whether the membrane is of a suitable thickness for pore insertion, and generally can be used to monitor the progress of the film formation step.

[0147] In some embodiments, another voltage waveform that can be AC modulated is the translocation voltage waveform that can be used to translocate molecules through pores.

[0148] In some embodiments, the amplitude of the AC modulation component can be minimized in order to reduce the influence of that AC modulation component on the main function of the voltage waveform (i.e., film formation or pore insertion), while still allowing accurate measurements to be obtained. A relatively large amplitude AC modulation component may significantly affect the membrane during the film formation step and / or the pore insertion step, resulting in a higher transient voltage than expected. This can, for example, cause membrane defects or multiporation. Thus, in some embodiments, the amplitude of the AC modulation component may be less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 mV. In some embodiments, the amplitude of the AC modulation component may be less than 50, 40, 30, 20, or 10% of the amplitude of the voltage waveform being modulated. In other embodiments, the amplitude of the AC modulation component can scale with the amplitude of the voltage waveform being modulated.

[0149] In some embodiments, the frequency of the AC modulation component can be minimized while still allowing accurate measurements. In some embodiments, the frequency of the AC modulation can be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 times the maximum sampling frequency. In some embodiments, the frequency is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 Hz. In some embodiments, the frequency is less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 Hz. In some embodiments, the frequency is between 10 and 1000 Hz, or between 25 and 750 Hz, or between 50 and 500 Hz.

[0150] FIG. 8B shows an embodiment of the self-limiting pulse waveform shown in FIG. 8A to which an AC modulation component 802 is added for measurement. In this embodiment, the amplitude of the AC modulation component is 100 mV, which is shown as the line thickness on the graph.

[0151] An AC modulation component can be superimposed on any other voltage waveform so that a measured value can be obtained while the voltage waveform is being applied.

[0152] VI. COMPUTER SYSTEM Any of the computer systems described herein can utilize any suitable number of subsystems, many of which may be optional. An example of such a subsystem is shown in FIG. 11 in computer system 1110. In some embodiments, the computer system includes a single computer device, and the subsystems may be components of the computer device. In other embodiments, the computer system includes a plurality of computer devices, each of which is a subsystem having internal components. The computer system can include desktop and laptop computers, tablets, mobile phones, and other mobile devices.

[0153] The subsystems shown in FIG. 11 are interconnected via a system bus 1180. Additional subsystems are shown, such as a printer 1174, a keyboard 1178, a memory device(s) 1179, a monitor 1176 connected to a display adapter 1182, etc. Peripheral devices and input / output (I / O) devices connected to an I / O controller 1171 can be connected to the computer system using any of a number of means known to those skilled in the art, such as an I / O port 1177 (e.g., Universal Serial Bus (USB), FireWire®). For example, an I / O port 1177 or an external interface 1181 (e.g., Ethernet®, Wi-Fi, etc.) can be used to connect the computer system 1110 to a wide area network such as the Internet, a mouse input device, or a scanner. The interconnection via the system bus 1180 not only enables information exchange between subsystems, but also enables the central processor 1173 to communicate with each subsystem and control the execution of multiple instructions from the system memory 1172 or a memory device 1179 (e.g., a fixed disk such as a hard drive or an optical disk). The system memory 1172 and / or a memory device(s) 1179 can embody a computer-readable medium. Another subsystem is a data collection device 1175, such as a camera, a microphone, an accelerometer, or other sensors. It is possible to output any data described herein from one component to another and to the user.

[0154] A computer system can include a plurality of the same components or subsystems connected together, for example, via an external interface 1181, via an internal interface, or via a removable storage device that can be connected and removed from one component to another. In some embodiments, the computer system, subsystem, or device communicates via a network. In such cases, one computer can be regarded as a client and another computer can be regarded as a server, and each can be part of the same computer system. Each of the client and the server can include a plurality of systems, subsystems, or components.

[0155] Various aspects of the embodiments can be implemented in the form of control logic using hardware circuitry (e.g., APSIC or FPGA) and / or using computer software by a generally modular or integrated programmable processor. As used herein, a processor can include a single-core processor, a multi-core processor on the same integrated chip, or a plurality of processing units located on a single circuit board or networked, as well as dedicated hardware. Based on the disclosure and teachings provided herein, one of ordinary skill in the art will know and understand other ways and / or methods for implementing the embodiments of the present invention using hardware and combinations of hardware and software.

[0156] Any of the software components or functions described in this application can be implemented as software code executed by a processor using any suitable computer language, such as Java®, C, C++, C#, Objective C, Swift, etc., or a script language using conventional or object-oriented techniques, such as Perl or Python. The software code can be stored as a series of instructions or commands on a computer-readable medium for storage and / or transmission. Suitable non-transitory computer-readable media can include random access memory (RAM), read-only memory (ROM), magnetic media such as hard drives or floppy disks, or optical media such as compact discs (CDs) or digital versatile discs (DVDs), flash memory, etc. The computer-readable media can be any combination of such storage or transmission devices.

[0157] Such programs can also be transmitted using a carrier signal adapted for transmission via wired networks, optical networks, and / or wireless networks that comply with various protocols, including the Internet. Thus, a computer-readable medium can be created using a data signal encoded with such a program. A computer-readable medium encoded with program code can be packaged using compatible devices or provided separately from other devices (e.g., can be downloaded via the Internet). Any such computer-readable medium can be provided on or within a single computer product (e.g., a hard drive, CD, or an entire computer system), or can exist on or within different computer products within a system or network. A computer system can include a monitor, printer, or other suitable display for providing any of the results described herein to a user.

[0158] Any of the methods described herein can be performed, in whole or in part, using a computer system that includes one or more processors configured to perform the steps. Accordingly, embodiments can be directed to a computer system configured to perform the steps of any of the methods described herein, perhaps having different components for performing each respective step or each respective group of steps. Although the steps of the methods herein are presented as ordered steps, they may be performed simultaneously, at different times, or in a different order. Additionally, portions of these steps can be used in conjunction with portions of other steps from other methods. Also, all or portions of one step can be optional. Further, any step of any method can be performed by a module, unit, circuit, or other means of the system for performing these steps.

[0159] The specific details of particular embodiments can be combined in any suitable manner without departing from the spirit and scope of the embodiments of the present invention. However, other embodiments of the present invention can be directed to specific embodiments related to each individual aspect, or specific combinations of those individual aspects.

[0160] The foregoing embodiments have been described in some detail for purposes of clarity of understanding, but the present invention is not limited to the details provided. There are many alternative ways to implement the present invention. The disclosed embodiments are for purposes of illustration and not limitation. The above description of the exemplary embodiments of the present invention is presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the above teachings.

[0161] When a feature or element is referred to herein as being "above" another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly" on another feature or element, there are no intervening features or elements. When a feature or element is referred to as being "connected", "attached", or "coupled" to another feature or element, it can be directly connected, attached, or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached", or "directly coupled" to another feature or element, there are no intervening features or elements. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be understood by those of ordinary skill in the art that references to a structure or feature being "adjacent" to another feature can have portions that overlap with or are beneath the adjacent feature.

[0162] The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the terms "comprises" and / or "comprising" specify the presence of the stated features, steps, acts, elements, and / or components but do not preclude the presence or addition of one or more other features, steps, acts, elements, components, and / or groups thereof. It is further understood that as used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be omitted with " / "

[0163] Spatially relative terms, such as "under", "below", "lower", "over", "upper", etc., may be used herein to describe the relationship of one element or feature to another element or feature shown in a different element or figure for ease of explanation. It will be understood that spatially relative terms are intended to encompass different directions of the device in use or operation in addition to the directions shown in the figures. For example, if the device in the figure is turned over, an element described as "under" or "beneath" another element or feature will be "over" the other element or feature. Thus, the exemplary term "under" can encompass both upward and downward directions. The device may be oriented in other ways (e.g., rotated 90 degrees or in other directions), and the spatially relative descriptors used herein may be interpreted accordingly. Similarly, terms such as "upwardly", "downwardly", "vertical", "horizontal", etc. are used herein only for the purpose of explanation unless otherwise specified.

[0164] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context otherwise indicates. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element described below could be termed a second feature / element, and similarly, a second feature / element described below could be termed a first feature / element without departing from the teachings of the present invention.

[0165] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are meant to cover the inclusion of a stated method or item (e.g., a composition, and an apparatus and method including the apparatus) without excluding any other method or item. For example, the term "comprising" is understood to imply the inclusion of any element or step recited herein, but not the exclusion of any other element or step.

[0166] As used herein, including those used in the examples, and as used in the specification and claims, unless specifically designated otherwise, all numbers can be read as if the term began with the word "about" or "approximately" even if the term is not explicitly stated. The phrases "about" or "approximately" are used when describing size and / or position to indicate that the value and / or position being described is within a reasonable expectation range of the value and / or position. For example, a numerical value can have a value of + / -0.1% of the recited value (or range of values), + / -1% of the recited value (or range of values), + / -2% of the recited value (or range of values), + / -5% of the recited value (or range of values), + / -10% of the recited value (or range of values), and so on. Any numerical value shown herein should also be understood to include "about" or "approximately" that value unless the context indicates otherwise. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range described herein is intended to include all sub-ranges subsumed therein. Also, as would be appropriately understood by those skilled in the art, when a value is disclosed as "less than", it is also understood that "greater than or equal to" the value and the possible ranges between the values are also disclosed. For example, if the value "X" is disclosed, "less than or equal to X" as well as "greater than or equal to X" (e.g., X is a numerical value) are also disclosed. Also, throughout this patent application, data is provided in many different forms, and it is understood that this data represents ranges of endpoints and starting points, and any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that values greater than, more than, less than, less than or equal to, and equal to 10 and 15 are disclosed along with the values between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0167] Although various exemplary embodiments are described above, various changes can be made to the various embodiments without departing from the scope of the invention, as set forth in the claims. For example, the order in which the various method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments, one or more method steps may be completely skipped. Any feature of the various apparatus and system embodiments may or may not be included in some embodiments. Accordingly, the foregoing description has been provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention, as set forth in the claims.

[0168] The examples and figures included herein are illustrative rather than limiting, showing specific embodiments in which the subject matter may be practiced. As noted above, other embodiments may be utilized and derived therefrom, and structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such embodiments of the subject matter of the present invention, where multiple are actually disclosed, may be referred to herein individually or collectively by the term "invention" merely for convenience and without any intention of voluntarily limiting the scope of this patent application to any single invention or inventive concept. Accordingly, while specific embodiments have been illustrated and described herein, any configuration calculated to achieve the same purpose may be used in place of the specific embodiments shown. The present disclosure is intended to embrace any and all adaptations or variations of the various embodiments. Combinations of the above embodiments and other embodiments not specifically described herein will be apparent to those skilled in the art upon review of the above description.

[0169] All patents, patent applications, publications, and descriptions mentioned in this specification are hereby incorporated by reference in their entirety for all purposes. None of them are admitted to be prior art.

Claims

1. A method of forming an array of nanopore sensor cells, comprising: introducing nanopores into a solution disposed within the cells of the array, the solution containing a first species of a redox pair and not containing a second species of the redox pair, wherein the cells each have a working electrode and a membrane that seals the cell, and the working electrode is powered by an electrically connected power source; applying a voltage waveform across the membrane of the cell, wherein the voltage waveform begins at a first voltage and increases over a period to a second voltage, and the voltage waveform has a polarity that maintains the first species of the redox pair in its current oxidized state, thereby capacitively coupling between the working electrode and the solution; inserting the nanopores into the membrane while applying the voltage waveform, and further comprising applying a second voltage waveform having a polarity that oxidizes or reduces the first species to the second species, thereby resistively coupling between the working electrode and the solution. The method as described above.

2. The method according to claim 1, wherein the redox pair is water-soluble.

3. The method according to claim 1, wherein the redox pair is ferricyanide and ferrocyanide.

4. inserting a molecule through the nanopore, and further comprising applying a sequencing voltage to sequence the molecule under Faradaic conditions, the method according to any one of claims 1 to 3.

5. A system for sequencing a molecule, comprising: an array of cells on a substrate, each of the cells having a working electrode and an aperture configured to be sealed by a membrane having a nanopore; a counter electrode; a power source electrically connected to each of the working electrodes; a controller, a controller programmed to deliver a voltage waveform to the cells using the working electrode and the counter electrode, the voltage waveform beginning at a first voltage and increasing over a period to a second voltage, and the voltage waveform having a polarity that maintains a first species of a redox pair in its current oxidized state, thereby capacitively coupling between the working electrode and the solution. The controller is further programmed to deliver a second voltage waveform having a polarity that oxidizes or reduces the first species to the second species, thereby enabling resistive coupling between the working electrode and the solution, and, further comprising a solution that includes the first species of the redox pair but does not include the second species of the redox pair, the solution being configured to be disposed within the cell of the array, the system comprising. **Claim 6** The system according to claim 5, wherein the redox pair is water-soluble. **Claim 7** The system according to claim 5, wherein the redox pair is ferricyanide and ferrocyanide. **Claim 8** The controller is applying a voltage to insert the molecule into the pore; The system according to any one of claims 5 to 7, further programmed to apply a sequencing voltage to sequence the molecule under Faraday conditions.

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