Systems and methods for self-limiting protein pore insertion in membrane

Abstract

To provide systems and methods for inserting a single pore into a membrane.SOLUTION: A stepped or ramped voltage waveform can be applied across respective membranes of cells of an array. The voltage waveform starts at the first voltage and increases in magnitude over a period of time to the second voltage. The first voltage is selected to be low enough to reduce a risk of damaging the membrane. Meanwhile, the rate of voltage increase is selected to provide sufficient time for each of the pores to insert into each of the membranes. Once a pore is inserted into the membrane, the voltage rapidly drops across the membrane. Therefore, the risk of damaging the membrane is reduced even if the voltage applied between electrodes is further increased.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 777,976, filed December 11, 2018, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Incorporation by Reference All publications and patent applications mentioned in this specification are herein incorporated 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 technology]

[0003] Nanopore-based sequencing chips are analytical tools that can be used for deoxyribonucleic acid (DNA) sequencing. These devices can incorporate very large numbers of sensor cells arranged in an array. For example, a sequencing chip can contain an array of one million cells, such as 1000 rows by 1000 columns of cells. Each cell of the array can contain a membrane and a protein pore with a pore size on the order of one nanometer in inner diameter. Such nanopores have been shown to be effective in rapid sequencing of nucleotides.

[0004] When an electric potential is applied across a nanopore immersed in a conducting fluid, there can be a small ionic current due to the conduction of ions across the nanopore. The size of the current is sensitive to the pore size and the type of molecule placed in the nanopore. The molecule can be a specific tag attached to a specific nucleotide. Thus, detection of the nucleotide at a specific position in the nucleic acid is possible. As a way to measure the resistance of the molecule, the voltage or other signal in the circuit containing the nanopore can be measured (e.g., at an integrating capacitor). Thus, it is possible to detect what molecule is in the nanopore.

[0005] For a sequencing chip to function properly, typically only one pore should be inserted in the membrane for a given cell: if multiple pores are inserted into a single membrane, interpretation of the electrical signatures generated by nucleotides passing through multiple pores simultaneously becomes much more difficult.

[0006] Application of a voltage across the membrane during the pore insertion step may facilitate the pore insertion process, possibly by reducing the stability of the membrane and allowing the pores to more easily insert themselves into the membrane. However, application of too much voltage across the membrane may cause catastrophic destruction of the membrane, rendering the cell unusable.

[0007] It would therefore be advantageous 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

[0008] Various embodiments provide techniques and systems for inserting a single pore into a membrane in a cell of a nanopore-based sequencing chip. In some embodiments, the insertion of a pore into the membrane reduces the likelihood of inserting additional pores into the membrane.

[0009] Other embodiments include systems and computer-implemented methods associated with the methods described below. It is aimed at reading media.

[0010] In some embodiments, a method of forming an array of nanopore sensor cells is provided, the method including 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 building up over a period of time to a second voltage, and inserting the nanopore into the membrane during the step of applying the voltage waveform.

[0011] In some embodiments, the first voltage is between about 0 and 100 mV, and the second voltage is between about 100 to 2000 mV.

[0012] In some embodiments, the working electrode is a capacitive electrode.

[0013] In some embodiments, the voltage waveform includes multiple incremental steps between the first voltage and the second voltage.

[0014] In some embodiments, the multiple incremental steps are increments of about 1 to 100 mV.

[0015] In some embodiments, the multiple incremental steps are increments of about 1 to 25 mV.

[0016] In some embodiments, each incremental step has a duration of between about 0.1 and 60 seconds.

[0017] In some embodiments, the duration of the incremental steps is variable.

[0018] In some embodiments, the duration of the incremental steps at the lower voltage is longer than the duration of the incremental steps at the higher voltage.

[0019] In some embodiments, the duration of the incremental steps is constant.

[0020] In some embodiments, the voltage waveform includes a ramp between the first voltage and the second voltage.

[0021] In some embodiments, the ramp is between about 0.1 and 2.0 V per minute.

[0022] In some embodiments, the incline has a constant slope.

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

[0024] In some embodiments, the ramp has a slope at the lower voltage that is less than the slope at the higher voltage.

[0025] In some embodiments, the applying of the voltage waveform is applied to a film that is not thinned.

[0026] In some embodiments, the method further comprises thinning the unthinned film with an applied voltage waveform.

[0027] In some embodiments, a system for molecular sequencing is provided that 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, the working electrodes being powered by an alternating current (AC) connected power source, a counter electrode, a power source, the power source being AC connected to each of the working electrodes, and a controller programmed to supply a voltage waveform to the cells using the working and counter electrodes that begins at a first voltage and increases over a period of time to a second voltage.

[0028] In some embodiments, the working electrode is a capacitive electrode.

[0029] In some embodiments, the voltage waveform includes multiple incremental steps between the first voltage and the second voltage.

[0030] In some embodiments, the voltage waveform includes a ramp between the first voltage and the second voltage.

[0031] In some embodiments, the controller is further programmed to apply a voltage waveform to the unthinned membrane.

[0032] In some embodiments, a method of forming an array of nanopore sensor cells is provided that can include introducing a nanopore proximate to a cell having a working electrode and a membrane sealing the cell, the working electrode being powered by an electrically connected power source, applying a voltage waveform across the membrane of the cell, the voltage waveform beginning at a first voltage and building up over a period of time to a second voltage, the voltage waveform including an alternating current (AC) modulation component, the AC modulation component configured such that electrical measurements can be taken 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.

[0033] 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.

[0034] In some embodiments, a method of forming a membrane-covered cell is provided, the method including flowing a membrane-forming material over a cell, the cell having a working electrode, the working electrode being powered by an electrically connected power source, disposing a layer of membrane-forming material over the cell, applying a voltage waveform across the layer of membrane-forming material using the working electrode and a counter electrode opposite the layer of membrane-forming material, the voltage waveform including an alternating current (AC) modulation component, the AC modulation component configured such that electrical measurements may be taken through the working electrode while the voltage waveform is being applied across the layer of membrane-forming material, applying a voltage waveform across the layer of membrane-forming material using the working electrode and a counter electrode opposite the layer of membrane-forming material, and thinning the layer of membrane-forming material into a membrane, the membrane configured to receive a nanopore.

[0035] 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.

[0036] A better understanding of the features and advantages of the embodiments of the present invention may be obtained by reference to the following detailed description and the accompanying drawings. can be obtained by referring to the

[0037] 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 present invention can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 is a top view of an embodiment of a nanopore sensor chip having an array of nanopore cells. [Diagram 2]FIG. 2 shows an embodiment of a nanopore cell in a nanopore sensor chip that can be used to assess the properties of a polynucleotide or polypeptide. [Diagram 3] FIG. 3 shows an embodiment of a nanopore cell for sequencing nucleotides using nanopore-based sequencing-by-synthesis (Nano-SBS) technology. [Figure 4] FIG. 4 shows an embodiment of an electrical circuit in a nanopore cell. [Diagram 5] FIG. 5 shows example data points acquired from a nanopore cell during the light and dark phases of an AC cycle. [Figure 6] FIG. 6 shows an embodiment of a circuit diagram of a nanopore sensor cell. [Figure 7] FIG. 7 shows a stepped voltage waveform that can be used to promote pore insertion. [Figure 8] A and B show ramped voltage waveforms that can be used to promote pore insertion. [Figure 9] A and B show that in some embodiments, when a pore is inserted, the pore can dissipate the increased voltage across the membrane itself, thus both reducing the risk of damage to the membrane as the voltage increases further after the pore is inserted, and reducing the likelihood of further pore insertion. [Figure 10] A shows a plot of the number of pore insertions in the array as a function of voltage and time, and B shows a plot of the number of deactivations / shorts resulting from membrane rupture as a function of voltage and time. [Figure 11] FIG. 11 is a computer system according to certain embodiments of the present disclosure.

[0039] term Unless otherwise defined, 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 or equivalent to those described herein may be used in the practice of the disclosed technology. The following terms are provided to facilitate understanding of certain frequently used terms and are not meant to limit the scope of the present disclosure. Abbreviations used herein have their common meaning in the fields of chemistry and biology.

[0040] "Nanopore" refers to a pore, channel, or passageway formed or otherwise provided in a membrane. The membrane can be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane made of a polymeric material. The nanopore can be located adjacent to or in close 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 (nanometers or nm) to about 1000 nm. In some implementations, the nanopore can be a protein.

[0041] "Nucleic acid" refers to a deoxyribonucleotide or ribonucleotide, and a single-stranded It refers to a polymer thereof, either in single stranded or double stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, both synthetic, naturally occurring, and non-naturally occurring. They have similar binding properties as the reference nucleic acid. They are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed groups 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 gene, complementary deoxyribonucleic acid (complementary DNA or cDNA), messenger ribonucleic acid (messenger ribonucleic acid or mRNA), oligonucleotide, and polynucleotide.

[0042] The term "nucleotide" can be understood to refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as 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 hybridized to a complementary base), unless the context clearly dictates otherwise.

[0043] The term "tag" refers to a detectable moiety, which may be an atom or molecule, or a collection of atoms or molecules. A 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 a tag, the tag is called a "tagged nucleotide." A tag can be attached to a nucleotide via a phosphate moiety.

[0044] The term "template" refers to a single-stranded nucleic acid molecule that is copied onto a complementary strand of DNA nucleotides for DNA synthesis. In some cases, the template may refer to the sequence of DNA that is copied during the synthesis of mRNA.

[0045] The term "primer" refers to a short nucleic acid sequence that provides a starting point for DNA synthesis. An enzyme that catalyzes DNA synthesis, such as DNA polymerase, can add new nucleotides to the primer for DNA replication.

[0046] The term "polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides. The term encompasses both the entire polypeptide and the domain that has polymerase activity. DNA polymerases are well known to those of skill in the art and include, but are not limited to, DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima, or modified versions thereof. These include both DNA-dependent polymerases, such as reverse transcriptase, and RNA-dependent polymerases. At least five families of DNA-dependent DNA polymerases are known. The most well-known are Family A, Family B, and Family C. There is little or no sequence similarity between these various families. Most of the Family A polymerases are polymerases that are not related to the RNA-dependent DNA polymerases. Family B polymerases are single-chain proteins that can contain multiple enzymatic 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 auxiliary elements. Family C polymerases are typically multisubunit proteins with polymerization and 3' to 5' exonuclease activity. Three types of DNA polymerases have been found in Escherichia coli (E. coli): 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. One family A polymerase, polymerase γ, 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, and bacterial RNA polymerases, as well as phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

[0047] The term "light phase" generally refers to a period during which the tag of the tagged nucleotide is forced into the nanopore by the electric field applied through the AC signal. The term "dark phase" generally refers to a period during which the tag of the tagged nucleotide is forced out of the nanopore by the electric field applied through the AC signal. An AC cycle can include a light phase and a dark phase. In different embodiments, the polarity of the voltage signal applied to the nanopore cell can be different to put the nanopore cell into the light phase (or dark phase).

[0048] The term "signal value" refers to the value of a sequencing signal output from a sequencing cell. According to certain embodiments, the sequencing signal is an electrical signal measured at a point in the circuitry of one or more sequencing cells and / or output from it. 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 the voltage and / or current and / or may represent an indirect measurement. For example, the signal value can be the measured period of time it takes for the voltage or current to reach a specified value. The signal value can represent any measurable quantity that correlates with the resistivity of the nanopore and from which the resistivity and / or conductance of the nanopore (threaded and / or unthreaded) can be derived. As another example, the signal value can correspond to the intensity of light from, for example, a fluorophore attached to a nucleotide added to a nucleic acid using a polymerase.

[0049] The term "osmolality", also known as osmolality, refers to a unit of measure for solute concentration. Osmolality measures the number of osmoles of solute particles per unit volume of solution. An osmole is a unit of measure for the number of moles of solute that contribute to the osmotic pressure of a solution. Osmolality allows for the measurement of the osmotic pressure of a solution and the determination of how a solvent diffuses across a semipermeable membrane (osmosis) that separates two solutions with different osmolar concentrations.

[0050] The term "osmolyte" refers to any soluble compound that, when dissolved in a solution, increases the osmolality of the solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] According to certain embodiments, the techniques and systems disclosed below relate 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 a pore into the membrane reduces the likelihood of the insertion of additional pores into the membrane. This therefore makes the pore insertion more self-limiting and reduces or eliminates the need for active feedback during the insertion step.

[0052] Examples of nanopore systems, circuits, and sequencing operations are described first. This is followed by an example technique for replacing the nanopore in a DNA sequencing cell. Embodiments of the invention can be implemented in many ways. These methods include processes, systems, and computer program products embodied on a computer-readable storage medium and / or a processor, such as a processor configured to execute instructions stored on and / or provided by a memory coupled to the processor.

[0053] I. Nanopore-Based Sequencing Chips FIG. 1 is a top 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 control circuitry integrated onto the 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 group can receive a different sample for characterization. Each of the nanopore cells can be used to sequence nucleic acids. 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 interface with other circuitry, such as a computer processor.

[0054] In some embodiments, the nanopore sensor chip 100 includes multiple chips in the same package, such as forming a multi-chip module (MCM) or a system-in-package (SiP). The chips can include, for example, memory, processors, field-programmable gate arrays (FPGAs), application-specific integrated circuits (ASICs), data converters, high-speed I / O interfaces, etc.

[0055] In some embodiments, the nanopore sensor chip 100 is connected (e.g., docked) to a nanochip workstation 120. The nanochip workstation 120 can include various components for performing (e.g., automatically performing) various embodiments of the processes disclosed below. These processes can include, for example, an analyte delivery mechanism, such as a pipette, for delivering a lipid suspension or other membrane structure suspension, an analyte solution, and / or other liquids, suspensions, or solids. The nanochip workstation components can further include a robotic arm, one or more computer processors, and / or memory. A plurality of polynucleotides can be detected on an array 140 of nanopore cells 150. In some embodiments, each of the nanopore cells 150 is individually addressable.

[0056] 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 size and / or chemical structure are attached to different nucleotides in the nucleic acid molecule to be sequenced. In some embodiments, complementary strands may be synthesized to the template of the nucleic acid molecule to be sequenced by hybridizing nucleotides tagged with different polymers to the template. In some implementations, both the nucleic acid molecule and the attached tag are translocated through the nanopore. An ionic current passing through the nanopore can indicate which nucleotides are within the nanopore due to the particular size and / or structure of the tag attached to that nucleotide. In some implementations, only the tag is transferred into the nanopore. Different tags within the nanopore can also be detected in many different ways.

[0057] A. Nanopore Sequencing Cell Structure 2 illustrates an embodiment of an exemplary nanopore cell 200 in a nanopore sensor chip, such as the nanopore cell 150 in the nanopore sensor chip 100 of FIG. 1, which may be used to evaluate properties of polynucleotides or polypeptides. The nanopore cell 200 may include a well 205 formed by dielectric layers 201 and 204, a membrane, such as a lipid bilayer 214, formed on the well 205, and a sample chamber 215 on and separated from the well 205 by the lipid bilayer 214. The well 205 may include a quantity of electrolyte 206. The sample chamber 215 may hold a bulk electrolyte 208 that includes a nanopore, such as soluble protein nanopore transmembrane molecular complexes (PNTMCs), and an analyte of interest (e.g., a nucleic acid molecule to be sequenced).

[0058] The nanopore cell 200 can include a working electrode 202 at the bottom of a well 205 and a counter electrode 210 disposed in a sample chamber 215. A 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 initiated by the voltage signal. Thus, a nanopore 216 is formed in the lipid bilayer 214. The individual membranes (e.g., lipid bilayer 214 or other membrane structures) in the array can be neither chemically nor electrically connected to each other. Thus, each of the nanopore cells in the array can be an independent sequencing machine. It generates data specific to a single polymer molecule associated with a nanopore that acts on an analyte of interest and modulates an ionic current through an otherwise impermeable lipid bilayer.

[0059] Additional embodiments of systems and methods for pore insertion are described below in Section III. In particular, these systems and methods describe self-limiting pore insertion that effectively achieves single pore insertion in the membrane of a cell.

[0060] As shown in FIG. 2, the nanopore cell 200 may be formed on a substrate 230, such as a silicon substrate. A dielectric layer 201 may be formed on the substrate 230. Dielectric materials used to form the dielectric layer 201 may include, for example, glass, oxide, nitride, and the like. An electrical circuit 222 for controlling electrical stimuli and processing signals detected from the nanopore cell 200 may be formed on the substrate 230 and / or in the dielectric layer 201. For example, multiple patterned metal layers (e.g., metal 1 through metal 6) may be formed in the dielectric layer 201. And, multiple active devices (e.g., transistors) may be fabricated on the substrate 230. In some embodiments, a signal source 228 is included as part of the electrical circuit 222. The electrical circuit 222 may include, for example, amplifiers, integrators, analog-to-digital converters, noise filters, feedback control logic, and / or various other components. The electrical circuit 222 may further be connected to a processor 224, which is connected to a memory 226. Here, the processor 224 can analyze the sequencing data and determine the sequences of the sequenced polymer molecules in the array.

[0061] The working electrode 202 may be formed on the dielectric layer 201 and may form at least a portion 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 may be made of a metal or other material that is resistant to corrosion and oxidation, such as, for example, platinum, gold, titanium nitride, and graphite. For example, the working electrode 202 may be a platinum electrode that is electroplated with platinum. In another example, the working electrode 202 may be a titanium nitride (TiN) fabrication electrode. The working electrode 202 may be porous, which may therefore increase its surface area and provide a capacitance associated with the working electrode 202. Because the working electrode of a nanopore cell may be independent of the working electrode of another nanopore cell, the working electrode may be referred to as the cell electrode in this disclosure.

[0062] A dielectric layer 204 may 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 may include, for example, glass, oxide, silicon mononitride (SiN), polyimide, or other suitable hydrophobic insulating materials. The top surface of the dielectric layer 204 may be silanized. The silanization may form a hydrophobic layer 220 on the top surface of the dielectric layer 204. In some embodiments, the hydrophobic layer 220 has a thickness of about 1.5 nanometers (nm).

[0063] A well 205 formed by the walls of the dielectric layer 204 contains a mass of electrolyte 206 above the working electrode 202. The mass of electrolyte 206 may be buffered and may include 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 (CaCl 2 ), strontium chloride (SrCl 2 ), manganese chloride (MnCl 2 ), and magnesium chloride (MgCl 2 In some embodiments, the bulk electrolyte 206 has a thickness of about 3 microns (μm).

[0064] 2, a membrane may be formed on top of the dielectric layer 204. The membrane spans the well 205. In some embodiments, the membrane includes a lipid monolayer 218 formed on top of a hydrophobic layer 220. When the membrane reaches the opening of the well 205, the lipid monolayer 208 can transition to a lipid bilayer 214 that spans the opening of the well 205. The lipid bilayer can include or consist of lipids, such as phospholipids. The lipids may be selected, for example, from diphytanoyl-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine, 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methylester (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 phospholipid derivatives (e.g., mPEG phospholipids, polyglycerin phospholipids, functional phospholipids, terminally active phospholipids), diphytanoyl phospholipids (e.g., DPhPC, DOP). hPC, DPhPE, and DOPhPE). In some embodiments, the bilayer may be formed using, for example, the following non-lipid based materials: amphiphilic block copolymers (e.g., poly(butadiene)-block-poly(ethylene oxide), PEG diblock copolymers, PEG triblock copolymers, PPG triblock copolymers, and poloxamers) and other amphiphilic copolymers, which may be non-ionic or ionic. In some embodiments, the bilayer may be formed from a combination of lipid-based and non-lipid based materials. In some embodiments, the bilayer material may be provided in a solvent phase that includes one or more organic solvents, such as an alkane (e.g., decane, tridecane, hexadecane, etc.) and / or one or more silicone oils (e.g., AR-20).

[0065] As shown, lipid bilayer 214 is embedded with a single nanopore 216, formed, for example, by a single PNTMC. As described above, nanopore 216 can be formed by inserting a single PNTMC into lipid bilayer 214 by electroporation. Nanopore 216 allows for the insertion of at least a portion of an analyte of interest and / or a small ion (e.g., Na + , K + , Ca 2+ , C.I. - ) may be large enough to pass between the two sides of the lipid bilayer 214.

[0066] The sample chamber 215 resides above the lipid bilayer 214 and can hold a solution of analytes to be characterized. The solution is an aqueous solution containing bulk electrolyte 208, which can be buffered to an optimal ion concentration and 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 include 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 (CaCl 2 ), strontium chloride (SrCl 2 ), manganese chloride (MnCl 2 ), and magnesium chloride (MgCl 2 ).

[0067] The counter electrode (CE) 210 can be an electrochemical potential sensor. In some embodiments, the counter electrode 210 is shared among multiple nanopore cells and can therefore be referred to as a common electrode. In some cases, the common potential and common electrode can be common to all nanopore cells, or at least to 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 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 electrical characteristics (e.g., resistance, capacitance, and ionic current flow) of the lipid bilayer 214. In some embodiments, the nanopore cell 200 can also include a reference electrode 212.

[0068] In some embodiments, various checks are performed during the creation of the nanopore cell as part of the calibration. Once the nanopore cell is created, further calibration steps may be performed, for example, to identify a nanopore cell (e.g., one nanopore in a cell) that is working as desired. Such calibration checks may include physical checks, voltage calibration, open channel calibration, and identification of cells with a single nanopore.

[0069] B. Nanopore sequencing cell detection signal The nanopore cells in the nanopore sensor chip, such as the nanopore cell 150 in the nanopore sensor chip 100, are made by single molecule nanopore-based synthesis (Nano-SBS) techniques. This allows for parallel sequencing using sequencing of multiple sites.

[0070] 3 shows an embodiment of a nanopore cell 300 for 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 in the sample chamber of the nanopore cell 300. By way of example, the template 332 can be circular or linear. A nucleic acid primer can be hybridized to a site on the template 332, to which four different polymer-tagged nucleotides 338 can be added.

[0071] In some embodiments, an enzyme (e.g., a polymerase 334, such as a DNA polymerase) is associated with the nanopore 316 for use in synthesizing a complementary strand to the template 332. For example, the polymerase 334 may be attached to the nanopore 316 by a covalent bond. The polymerase 334 may catalyze the incorporation of nucleotides 338 onto a primer using a single-stranded nucleic acid molecule as a template. The nucleotides 338 may include a tag species ("tag") that uses a nucleotide that is one of four different types: A, T, G, or C. Once the tagged nucleotide is properly complexed with the polymerase 334, the tag may be drawn (e.g., loaded) into the nanopore by an electrical force, such as a force generated in the presence of an electric field generated by a voltage applied across the lipid bilayer 314 and / or the nanopore 316. The end of the tag may be positioned within the barrel of the nanopore 316. A tag held within the barrel of the nanopore 316 can generate a unique ion sequestration signal 340 due to the distinct chemical structure and / or size of the tag, and thus the added base to which the tag is attached can be electronically identified.

[0072] As used herein, a "loaded" or "threaded" tag is disposed within and / or remains near the nanopore for a substantial period of time, such as 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 instances, the probability that the loaded tag will pass through (and / or be detected by) the nanopore after being released following a nucleotide incorporation event is suitably high, such as 90% to 99%.

[0073] In some embodiments, before the polymerase 334 is 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 the four types of tagged nucleotides described above. The polymerase can then undergo isomerization and phosphoryl transfer reactions to incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule.

[0074] In some cases, some of the tagged nucleotides (complementary bases) may not match the current position of the nucleic acid molecule (template). Tagged nucleotides that are not base-paired to the nucleic acid molecule can also pass through the nanopore. These unpaired nucleotides can be rejected by the polymerase within a time scale that is shorter than the time scale that correctly paired nucleotides remain associated with the polymerase. Tags directed to unpaired nucleotides can pass through the nanopore quickly and can be detected over a short period of time (e.g., less than 10 ms). On the other hand, tags directed to paired nucleotides can be loaded into the nanopore and detected over a long period of time (e.g., less than 10 ms). The unpaired nucleotide can be detected over a period of time (at least 10 ms) that is determined by a downstream processor based at least in part on the time that the nucleotide is detected in the nanopore.

[0075] The conductance (or equivalently, resistance) of the nanopore containing the loaded (threaded) tag is measured via a signal value (e.g., voltage or current passing through the nanopore). Thus, the identification of the tag type, and therefore the nucleotide at the current position, is provided. 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, operating the nanopore sensor for a long period of time using a direct current can have other undesirable effects that can change the composition of the electrodes, unbalance the ion concentration across the nanopore, and adversely affect the life of the nanopore cell. Applying an alternating current (AC) waveform can reduce electron transfer, avoid these undesirable effects, and have certain advantages, as described below. The nucleic acid sequencing methods utilizing tagged nucleotides described herein are fully compatible with applied AC voltages. Thus, to achieve these advantages, AC waveforms can be used.

[0076] Being able to recharge the electrodes during the AC detection cycle may be advantageous when sacrificial electrodes are used, which are electrodes that change their molecular characteristics in a current-transfer reaction (e.g., electrodes that include silver) or electrodes that change their molecular characteristics in a current-transfer reaction. When a DC signal is used, the electrodes may wear out during the detection cycle. Recharging can prevent the electrodes from reaching a wear limit, such as being completely worn out, which may be a problem when the electrodes are small (e.g., when the electrodes are small enough to provide an array of electrodes with at least 500 electrodes per square millimeter). Electrode life extends with the width of the electrode in some cases, i.e., electrode life depends at least in part on the width of the electrode.

[0077] Suitable conditions for measuring the ionic current through a nanopore are known to those skilled in the art. Examples thereof are provided below. The measurement may be performed with a voltage applied across the membrane and the pore. In some embodiments, the voltage used is in the range of -400mV to +400mV. The voltage used is preferably in a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. The voltage used may more preferably be in the range of 100mV to 240mV, most preferably in the range of 160mV to 240mV. Increasing the applied potential can increase the nanopore's discrimination between different nucleotides. Nucleic acid sequencing using AC waveforms and tagged nucleotides is described in U.S. Patent Publication No. 2014 / 0134616, filed November 6, 2013, and entitled "Nucleic Acid Sequencing Using Tags," the entire contents of which are incorporated herein by reference. In addition to the tagged nucleotides described in U.S. Patent Application Publication No. 2014 / 0134616, sequencing can be performed using nucleotide analogs with fewer sugar or acyclic moieties, such as (S)-glycerol nucleoside triphosphates (gNTPs) of the five common nucleobases: adenine, cytosine, guanine, uracil, and thymine (Horhota et al., Organic Letters, 8:5345-5347

[2006] ).

[0078] C. Electrical circuitry of the nanopore sequencing cell 4 illustrates an embodiment of an electrical circuit 400 within a nanopore cell, such as nanopore cell 400, which may include portions of electrical circuit 222 of FIG. 2. As described above, in some embodiments, electrical circuit 400 includes a counter electrode 410, which may be shared among multiple nanopore cells or all of the nanopore cells in a nanopore sensor chip, and thus may also be referred to as a common electrode. The common electrode is coupled to a voltage source V LIQ 420 may be configured to apply a common potential to a bulk electrolyte (e.g., bulk electrolyte 208) in contact with a lipid bilayer (e.g., lipid bilayer 214) in the nanopore cell. In some embodiments, an AC non-Faraday mode is utilized, where an AC signal (e.g., a square wave) is used to apply a voltage V LIQ is modulated, and the modulated voltage V LIQ is applied to the bulk electrolyte in contact with the lipid bilayer in the nanopore cell. LIQ is a square wave with 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) may be modeled by a large capacitor (not shown), for example, 100 μF or more.

[0079] 4 also shows an electrical model 422 that represents the electrical properties of a working electrode 402 (e.g., working electrode 202) and a lipid bilayer (e.g., lipid bilayer 214). The electrical model 422 includes a capacitor 426 (C 二分子層 ) and resistor 428 (R 細孔 ), which can vary based on the presence of a particular tag in the nanopore. The electrical model 422 also includes the double layer capacitance (C 二重層 ) and also includes a capacitor 424 that represents the electrical properties of working electrode 402 and well 205. Working electrode 402 can be configured to apply a distinct potential that is independent of the working electrodes in other nanopore cells.

[0080] 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 lipids are deposited to form a lipid bilayer, the impedance between these two electrodes may be very low because the well of the nanopore cell is not sealed. Therefore, to avoid a short circuit situation, the pass device 406 can be kept open. After the lipid solvent is deposited in the nanopore cell and the well of the nanopore cell is sealed, the pass device 406 can be closed.

[0081] The circuit 400 includes on-chip integrating capacitors 408(n cap The integration capacitor 408 may be pre-charged by using a reset signal 403 that closes the switch 401, and the integration capacitor 408 is connected to a voltage source V PRE 405. In some embodiments, the voltage source V PRE 405 provides a particular reference voltage, such as 900 mV. When switch 401 is closed, integrating capacitor 408 is connected to a voltage source V PRE The capacitor 405 may be precharged to a reference voltage level.

[0082] Once precharging of the integrating capacitor 408 is complete, a reset signal 403 can be used to open the switch 401 and reset the integrating capacitor 408 to the voltage source V PRE At this point, the voltage source V LIQ Depending on the level of the voltage source V, the potential of the counter electrode 410 may be at a higher level than that of the working electrode 402 (and the integrating capacitor 408) or vice versa. LIQ During the positive phase of the square wave from the voltage source V (e.g., the light or dark phase of the 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. LIQDuring the negative phase of the square wave from the AC voltage source signal (e.g., the dark or light phase of the AC voltage source signal cycle), the potential of the counter electrode 410 is at a lower level than the potential of the working electrode 402. Thus, in some embodiments, the integrating capacitor 408 is Between them, a voltage source V PRE From the pre-charged voltage level of 405, it can be further charged to a higher level and discharged to a lower level during the dark period by the potential difference between the counter electrode 410 and the working electrode 402. In other embodiments, charging and discharging occur during the dark and light periods, respectively.

[0083] The integration capacitor 408 may be charged or discharged over a period of time depending on the sampling rate of the analog-to-digital converter (ADC) 435, which may be greater than 1 kHz, 5 kHz, 10 kHz, 100 kHz, or higher than these values. For example, at a sampling rate of 1 kHz, the integration capacitor 408 may be charged / discharged over a period of about 1 ms. Following this, a voltage level may be sampled and converted by the ADC 435 at the end of the integration period. A particular voltage level corresponds to a particular tag species in the nanopore and therefore to the nucleotide at the current position on the template.

[0084] After being sampled by ADC 435, integrating capacitor 408 may again be precharged by using reset signal 403 to close switch 401 so that integrating capacitor 408 is again coupled to voltage source V PRE 405. The steps of pre-charging the integrating capacitor 408, waiting a period of time for the integrating capacitor 408 to charge or discharge, and sampling and converting the integrating capacitor voltage level by the ADC 435 may be repeated for each cycle through the sequencing process.

[0085] 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 assembling the ADC output data from the array of nanopore cells into various data frames. In some embodiments, the digital processor 430 performs further 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.

[0086] Thus, a voltage signal applied across the nanopore can be used to detect a particular state of the nanopore. One of the possible states of the nanopore is the open channel state when tagged polyphosphate is clear of the nanopore barrel. This is also referred to below as the unthreaded nanopore state. The other four possible states of the nanopore correspond to states when one of the four different types of tagged polyphosphate nucleotides (A, T, G, or C) is retained within the nanopore barrel. Yet another possible state of the nanopore is when the lipid bilayer is ruptured.

[0087] When the voltage level on the integrating capacitor 408 is measured after a period of time, different states of the nanopore may result in different voltage level measurements. This is because the rate of voltage decay (falling due to discharging or rising due to charging) on ​​the integrating capacitor 408 (i.e., the slope of the slope of the voltage on the integrating capacitor 408 versus time plot) is proportional to the nanopore resistance (e.g., resistor R 細孔428) is dependent on the resistance of the nanopore. Notably, since in different states the resistance associated with the nanopore is different due to the distinct chemical structures of the molecules (tags), different corresponding rates of voltage decay may be observed, which may be used to identify different states of the nanopore. The voltage decay curve may be an exponential curve with an RC time constant τ=RC, where R is the time constant of the nanopore (i.e., R 細孔 is the resistance associated with resistor 428. At the same time as R, the membrane (i.e., C 二分子層 The decay curve may not exactly match the exponential curve, depending on the detailed implementation of the bilayer. However, the decay curve may be similar to the exponential curve and be monotonic, thus allowing detection of the tag.

[0088] 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, with the tag inside the barrel of the nanopore, the resistance associated with the nanopore may be in the range of 200 MOhm to 40 GOhm. In other embodiments, the integrating capacitor 408 is omitted because the voltage to the ADC 435 still changes due to voltage decay in the electrical model 422.

[0089] The rate of decay of the voltage on the integrating 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 period of time. For example, the voltage on the integrating capacitor 408 can first be measured by the ADC 435 at time t1. Then, the voltage is measured again by the ADC 435 at time t2. The steeper the slope of the voltage on the integrating capacitor 408 versus time curve, the larger the voltage difference. The steeper the slope of the voltage curve, the smaller the voltage difference. Thus, the voltage difference can be used as a metric to determine the rate of decay of the voltage on the integrating capacitor 408, and therefore the state of the nanopore cell.

[0090] In other embodiments, the rate of voltage decay is determined by measuring the period of time required for a selected amount of voltage decay. For example, the time required for a voltage to drop or rise from a first voltage level V1 to a second voltage level V2 can be measured. If the slope of the voltage versus time curve is steeper, the time required is shorter. If the slope of the voltage versus time curve is less steep, the time required is longer. Thus, the measured required time is proportional to the time required for the integration capacitor n cap This can be used as a metric to determine the rate of decay of the voltage on 408, and therefore the state of the nanopore cell. Those skilled in the art will appreciate the various circuits that can be used to measure the resistance of the nanopore, including signal value measurement techniques, such as, for example, measuring voltage or current.

[0091] In some embodiments, the electrical circuit 400 includes a pass device (e.g., pass device 406) and an additional capacitor (e.g., integrating capacitor 408(n cap )), which therefore facilitates a reduction in the size of the nanopore-based sequencing chip. Due to the thin nature of the membrane (lipid bilayer), the membrane (e.g., capacitor 426 (C 二分子層The capacitance associated with capacitor 426 may be sufficient to create the necessary RC time constant without requiring additional on-chip capacitance. Thus, capacitor 426 may be used as an integrating capacitor, and the voltage signal V PRE and then the voltage signal V LIQ The elimination of additional capacitors and pass devices in the electrical circuitry that would otherwise be fabricated on-chip can greatly reduce the footprint of a single nanopore cell in a nanopore sequencing chip, thus facilitating the scaling of nanopore sequencing chips to include many more cells (e.g., to have millions of cells in a nanopore sequencing chip).

[0092] D. Data Sampling in Nanopore Cells To perform nucleic acid sequencing, an integrating capacitor (e.g., integrating capacitor 408(n cap ) or capacitor 426(C 二分子層 The voltage level of the nucleotide sequence is sampled by an ADC (e.g., ADC435) while a tagged nucleotide is added to the nucleic acid. For example, if the applied voltage is V LIQ V PRE If it is lower, an electric field across the nanopore, applied through the counter and working electrodes, can force the nucleotide tag into the barrel of the nanopore.

[0093] 1. Insertion An insertion event is when a tagged nucleotide is attached to a template (e.g., a piece of nucleic acid) and the tag moves in and out of the barrel of a nanopore. This movement can occur multiple times during an insertion event. If the tag is within the barrel of the nanopore, the resistance of the nanopore can be greater and the current that can flow through the nanopore is smaller.

[0094] During sequencing, the tag may not be inside the nanopore (called the open channel state) depending on the AC cycle. Here, the current is maximum due to the smaller resistance of the nanopore. When the tag is pulled into the nanopore barrel, the nanopore is in the bright mode. When the tag is pushed out of the nanopore barrel, the nanopore is in the dark mode.

[0095] 2. Light and dark periods During an AC cycle, the voltage on the integrating capacitor may be sampled multiple times by the ADC. For example, in one embodiment, an AC voltage signal is applied across the system, for example at about 100 Hz. The acquisition rate of the ADC may be about 2000 Hz per cell. Thus, about 20 data points (voltage measurements) may be acquired per AC cycle (cycle of the AC waveform). A number of data points corresponding to one cycle of the AC waveform may be referred to as a set. A set of data points for an AC cycle may, for example, correspond to a light mode (period) when the tag is pressed into the barrel of the nanopore, V LIQ But, V PRE If the V is lower than the V, the subset may be obtained. Another subset may be obtained, for example, LIQ But, V PRE The higher case may correspond to the dark mode (period) when the tag is pushed out of the barrel of the nanopore by the applied electric field.

[0096] 3. Measurement voltage For each of the data points, when switch 401 is opened, e.g., V LIQ But, V PRE If it is higher, V PRE From V LIQ or as a rise to V LIQ But, V PRE If lower, V PRE From V LIQ As a descent to V LIQ As a result of charging / discharging by the integrating capacitor (e.g., integrating capacitor 408(n cap ) or capacitor 426(C 二分子層The voltage at the working electrode changes and decays. The final voltage value is V LIQ The rate of change of the voltage level on the integrating capacitor may be governed by the value of the resistance of the bilayer that may contain the nanopore and thus the molecule (e.g., the tag of the tagged nucleotide) in the nanopore. The voltage level may be measured at a predetermined time after switch 401 is opened.

[0097] The switch 401 can be operated at the rate of data acquisition. The switch 401 can be closed for a relatively short period between two acquisitions of data, typically immediately after the measurement by the ADC. LIQ This allows for the acquisition of multiple data points during each sub-period (light or dark) of the AC cycle. If switch 401 remains open, the voltage level on the integrating capacitor, and therefore the output value of the ADC, is fully decayed and remains in that state. Instead, if switch 401 is closed, the integrating capacitor is again precharged (V PRE Switch 401 thus enables the acquisition of multiple data points over each subperiod (bright or dark) of the AC cycle. Such multiple measurements are not required for the large number of measurements (which may be averaged, for example) using a fixed ADC. Multiple measurements can allow for higher resolution (8 to 14 bits, typically achieved by scanning multiple times). Multiple measurements can also provide kinetic information about the molecule threaded within the nanopore. Timing information can allow for the determination of the period over which the threading occurs. This can also be used to help determine whether the sequence of multiple nucleotides added to a nucleic acid strand has been determined.

[0098] FIG. 5 shows example data points acquired from a nanopore cell during the light and dark periods of an AC cycle. The changes in the data points are highlighted in FIG. 5 for illustrative purposes. The voltage applied to the working electrode or integrating capacitor (V PRE) is at a constant level, e.g., 900 mV. The voltage signal 510 (V LIQ ) is an AC signal depicted as a square wave, where the duty cycle can be any suitable value up to 50%, such as, for example, about 40%.

[0099] During the light period 520, the voltage signal 510 (V LIQ ) is the voltage V applied to the working electrode PRE The electric field caused by different voltage levels applied at the working and counter electrodes (e.g., by the flow of charges and / or ions on the tag) can force the tag into the barrel of the nanopore. When switch 401 is opened, the voltage at the node in front of the ADC (e.g., at the integrating capacitor) drops. After a voltage data point is acquired (e.g., after a specified period of time), switch 401 can be closed and the voltage at the measurement node rises, V PRE We return again to . This process of measuring multiple voltage data points can be repeated. In this manner, multiple data points can be obtained during the light period.

[0100] As shown in Figure 5, V LIQ The first data point 522 (also called the first point delta (FPD)) in the light period after the change in the sign of the signal may be lower than the subsequent data point 524. This may be because there is no tag in the nanopore (open channel), and therefore the resistance here is low and the discharge rate is high. In some examples, the first data point 522 is V, as shown in FIG. LIQ The signal can exceed a level that may be caused by the capacitance of the bilayer connecting the signal to an on-chip capacitor. Data point 524 may be acquired after a threading event occurs, i.e., after a tag is forced into the barrel of the nanopore, where the resistance of the nanopore, and therefore the rate of discharge of the integrating capacitor, depends on the particular type of tag that is forced into the barrel of the nanopore ..., as described below.二重層 The charge built up at 424 can be reduced slightly for each measurement.

[0101] During the dark period 530, the voltage signal 510 (V LIQ ) is the voltage applied to the working electrode (V PRE ) such that any tags are pushed out of the barrel of the nanopore. When switch 401 is opened, a voltage signal 510 (V LIQ ) voltage level is V PRE After a voltage data point is taken (e.g., after a specified period of time), switch 401 can be closed, causing the voltage at the measurement node to drop and V PRE 5. This process of measuring multiple voltage data points can be repeated. Thus, multiple data points can be acquired during the dark period, including the first point delta 532 and subsequent data points 534. As described above, during the dark period, any nucleotide tags are pushed out of the nanopore, and thus, in addition to use in normalization, minimal information about any nucleotide tags is acquired.

[0102] FIG. 5 also illustrates the voltage signal 510 (V LIQ ) is the voltage applied to the working electrode (V PRE ), no threading event occurs (open channel). Thus, the resistance of the nanopore is low and the rate of discharge of the integrating capacitor is high. As a result, the first data point 542 and subsequent data points 5 The data points obtained, including 44, indicate a low voltage level.

[0103] One might expect that the voltage measured during the light or dark periods would be roughly the same for each measurement of constant resistance of the nanopore (e.g., made during the light mode of a given AC cycle while one tag is within the nanopore). However, this is due to the fact that a charge (C 二重層) builds up. This build-up of charge can lengthen the time constant of the nanopore cell. As a result, the voltage levels can be shifted, thus reducing the measured values ​​for each of the data points in a cycle. Thus, as shown in FIG. 5, within a cycle, the data points may vary somewhat from one data point to another.

[0104] Further details regarding the measurements can be found, for example, in U.S. Patent Publication No. 2016 / 0178577, entitled "Nanopore-Based Sequencing With Varying Voltage Stimulus"; No. 2016 / 0178554, entitled "Non-Destructive Bilayer Monitoring Using Measurement Of Bilayer Response To Electrical Stimulus," U.S. Patent Publication 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 entire disclosures of which are incorporated herein by reference for all purposes.

[0105] 4. Normalization and Base Calling For each of the available nanopore cells of the nanopore sensor chip, a production mode for sequencing nucleic acids may be performed. The ADC output data acquired during sequencing may be normalized to provide higher accuracy. The normalization accounts for offset effects such as cycle shape, gain drift, charge injection offset, and baseline shift. In some implementations, the signal values ​​of the light cycle corresponding to the insertion event may be smoothed so that a single signal value (e.g., average value) is acquired for the cycle. Alternatively, adjustments may be made to the measured signal to reduce intracycle decay (one type of cycle shape effect). Gain drift typically extends across the entire signal and changes on the order of 100s to 1,000s of seconds. As an example, gain drift may be triggered by changes in the solution (pore resistance) or bilayer capacitance. Baseline shifts occur with time scales up to 100ms and are related to the voltage offset at the working electrode. The baseline shift may be driven by a change in the effective rectification ratio from the insertion as a result of the need to maintain charge balance in the sequencing cell from light to dark.

[0106] After normalization, each of the embodiments can determine a cluster of voltages for the inserted channel, where each cluster corresponds to a different tag type and therefore 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 a cutoff voltage for distinguishing between different nucleotides (bases).

[0107] III. Self-Limiting Pore Insertion After a pore is inserted into the membrane of a cell, the voltage across the membrane begins to drop rapidly due to the relatively high conductance of the pore. The voltage drop across the membrane reduces the driving force for further pore insertion in the membrane.

[0108] FIG. 6 illustrates an embodiment of a circuit diagram 600 for a nanopore sensor cell, highlighting some of the various voltages and sensor cell components that may be involved in the systems and methods described herein. Examples of these include: a voltage (V app ) 602, the voltage across the bilayer (V bly )604, working electrode (C 二重層 ) 608 and an integrating capacitor (N CAP ) 610 and the voltage used for precharging (V pre ) 606, and the voltage applied to the counter electrode (V liq )612.

[0109] Described herein are methods and systems that utilize this property to insert protein pores and control single pore insertion without active feedback during the insertion step. In some embodiments of the pore insertion method, an AC-connected voltage is applied via a capacitive working electrode. This voltage is maintained across the membrane due to the low conductance of the membrane without pores. In some embodiments, the voltage can be applied to the entire array of cells regardless of the current state of pore insertion. In some embodiments, the voltage can be applied to the cells that have membranes. The applied voltage waveform can be stepped up as a ramp, multiple expansion steps, or other shapes designed to lower the probability of additional protein pore insertion while also reducing the risk of damaging the membrane. This can be achieved by limiting the voltage application transients by using small voltage steps, a gentle rate of voltage increase in the voltage ramp, etc.

[0110] For example, in some embodiments, the pore insertion voltage (V app) may be applied as a stepped voltage waveform 700 starting at 0 mV and rising in 100 mV increments every 5 seconds to a maximum voltage of 600 mV. In some embodiments, the initial voltage may be about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mV. In some embodiments, the step increase may 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 of the steps 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, each of the steps can have a variable duration. For example, in some embodiments, some or all of the steps at the lower voltage can have a longer duration than those at the higher voltage. 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 may be predetermined, such as the initial starting voltage, the magnitude of the voltage step increase, the duration of each of the steps, and / or the maximum voltage.

[0111] In some embodiments, as shown in Figure 8A, the pore insertion voltage may be applied as a ramped voltage waveform 800 starting at 0 mV and rising at a rate of 1 V per minute to a maximum voltage of 600 mV. In some embodiments, the initial voltage may 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, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.8, 3.9, 3.1, 3.1, 3.2, 3.3, 3.4, 3.5, 3.7, 3.8, 3.1, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.1, 3.1, 3.2, 3.3, 3.4, 3.5, 3.7, 3.8, 3.1, 3.1, 3.2, 3.3, 3.4 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 may be predetermined, such as the initial starting voltage, the rate of voltage rise, and / or the maximum voltage.

[0112] In some embodiments, one or more components of the pore insertion voltage waveform may 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 forms a seal across the cell. In some embodiments, these measurements may be taken before the voltage waveform is applied, such that the waveform is completely determined before it is applied. This differs from active feedback based methods that use measurements taken during stimulation to modify one or more stimulation parameters. Because the poration methods described herein are self-limiting, it is not necessary to utilize active poration methods that involve measuring changes in electrical or physical properties of the system or components of the system that result from inserting a pore into the membrane, and then adjusting the poration voltage accordingly to prevent the insertion of a second pore into the membrane.

[0113] In some embodiments, the methods described herein can be applied to an array of sensors with capacitive electrodes at the base of a microwell with a suspended membrane and a counter electrode on the opposite side of the membrane. These sensors can be used to detect the presence of a pore after application of the insertion drive voltage has been removed from all cells. Although it is possible to detect the presence of a pore during application of the voltage, this is not necessary with this method. A pore can be inserted without feedback to the application of a voltage on any individual sensor in the array or in the collection.

[0114] This method effectively scans the voltage required to overcome the pore insertion activation barrier, which may vary between individual membranes within an array, between narrow or wide regions on an array, or from an array from one device to another from a second device. In addition, poration voltages may vary between pore mutants and between membrane compositions and structures, 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 robust enough to work effectively with a large number of different types of pore arrays, or with the same type of pore array with a certain amount of variability.

[0115] In addition, by sweeping from a low voltage to a high voltage, the membrane may be more likely to have pores inserted before the bilayer reaches a critical voltage level that damages the membrane. In addition, as shown in Figures 9A and 9B, once a pore is inserted, the pore can dissipate the increased voltage across the membrane, thus reducing both the risk of damage to the membrane and the possibility of further pore insertion when the voltage is further increased after the pore is inserted. As long as the magnitude of the voltage step or the rate of increase of the voltage ramp is not too large, the pore can effectively dissipate the excess voltage increase across the membrane, thus reducing the risk of membrane damage and reducing the possibility 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 ramp to shorten the time to complete the poration step.

[0116] In some embodiments, the upper limit of the voltage waveform can be determined by comparing the kinetics and / or probability of pore insertion as a function of voltage and time to the kinetics and / or probability of membrane damage as a function of voltage and time. 10A shows a plot of the number of pore insertions at 1000 s and 1000 s at 1000 s. FIG. 10B shows a plot of the number of deactivations / shorts, which typically result from membrane destruction and damage, as a function of voltage. From these two plots, an optimal maximum voltage can be determined that balances high pore insertions and low deactivations / shorts.

[0117] In some embodiments, the concentration of pores in the solution during the pore insertion step is selected to be 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 insertion of pores into the membrane without application of a voltage across the membrane to assist 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 may reduce the possibility of multiple pores being inserted into a single membrane.

[0118] In some embodiments, leakage currents can cause a rise in voltage in one or more cells in the array after the membrane is placed over the cell. These trapped charges can vary in their magnitude over time and from cell to cell. This makes it difficult to apply a uniform voltage across all of the cell membranes when inducing poration. For example, when trapped charges are present in cells in an array in varying amounts, it is difficult to apply a uniform voltage (V app ) to all of the cells may result in the cells experiencing different amounts of effective voltage during the poration step. This may result in a high level of variability in the number of cells with single pore insertions and / or in some cells the voltage may be applied in excessive amounts, which may cause damage to the membrane. Using a stepped or ramped voltage waveform can solve these problems.

[0119] In some embodiments, the formation of the membrane over the cell opening is accomplished by flowing a solvent and membrane material, such as a lipid or block copolymer, over the cell opening. The membrane can then be thinned to a bilayer by applying a voltage across the membrane, for example, if a lipid is used, as further described in US Patent Publication No. 20170283867A1, and / or by manipulating an osmolarity imbalance across the membrane, as further described in International Patent Publication No. WO2018001925, the entire contents of each of which are incorporated herein by reference for all purposes. As described below, a thinned membrane is a membrane that is thinned sufficiently (i.e., e.g., the thickness is less than the length of the pore) so that a pore 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 (i.e., e.g., the thickness is greater than the length of the pore) to allow for the insertion of a pore. In some embodiments, the formation of a thinned membrane (i.e., lipid bilayer) on the cells in the array can be completed before starting 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, for example, by using the same voltage waveform, such as any of the voltage waveforms described herein for both the thinning and poration processes. A complex of pores can be flowed onto the membrane during the combined thinning and poration process. In some embodiments, the combined thinning and poration process can be applied after the membrane material has already been dispensed onto the cells and an unthinned membrane is formed across the cells in the array by applying a voltage during the dispensing of the membrane material. The initial formation of an unthinned membrane can result in non-uniform charge trapping. Additionally, a non-uniform osmotic pressure can be achieved across the membrane during the combined thinning and poration process. Combining the thinning and poration steps can substantially reduce the time it takes to prepare an array of pore sensors, thus improving the throughput of the sensor array system.

[0120] The methods described herein offer many advantages, including improving the rate of successful single pore insertion, reducing the rate of multiple pore insertion, and reducing the likelihood of membrane damage.

[0121] IV. AC Modulation of Voltage Waveform In some embodiments, as shown in FIG. 7, the pore insertion waveform 700 may be a voltage waveform with AC modulation. As shown, the pore insertion waveform 702 is stepped. The AC modulation 702 component may be superimposed on top of the voltage waveform 700 to provide a rapid voltage fluctuation at each of the stepped voltages. The voltage fluctuation or change allows electrical measurements to be taken while the pore insertion waveform 700 is applied during the electroporation step during pore insertion. These electrical measurements may be used to check the integrity of the membrane (i.e., detect membrane defects such as short conditions), check the membrane leakiness (i.e., membrane resistance and / or conductance), and check the insertion of the pore, and generally to monitor the progress of the electroporation step. In some embodiments, measurements may not be taken while the voltage waveform is applied. In these embodiments, measurements are generally taken before, after, or between applications of the voltage waveform. Superimposing an AC modulation component onto the voltage waveform allows for the simultaneous application of the voltage waveform and the taking of measurements.

[0122] In some embodiments, other voltage waveforms are also superimposed with AC modulation components. For example, a membrane formation waveform may be AC ​​modulated such that electrical measurements may be taken while the membrane formation waveform is being applied during the step of forming a membrane on a well. These electrical measurements may be used to check whether the well is covered with membrane formation material (i.e., to check for short conditions), to check the integrity of the membrane (i.e., to detect membrane defects such as short conditions), to check the leakiness of the membrane (i.e., the membrane resistance and / or conductance), whether the membrane is of a suitable thickness for pore insertion, and generally to monitor the progress of the membrane formation step.

[0123] Another voltage waveform that can be AC ​​modulated in some embodiments is a translocation voltage waveform that can be used to translocate molecules through a pore.

[0124] In some embodiments, the amplitude of the AC modulation component may be minimized to reduce its effect on the main function of the voltage waveform (i.e., membrane formation or pore insertion) while allowing accurate measurements to be obtained. A relatively large amplitude AC modulation component may cause higher than expected transient voltages to significantly affect the membrane during the membrane formation step and / or pore insertion step. This may cause, for example, membrane failure 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 may be spread out with the amplitude of the voltage waveform being modulated.

[0125] In some embodiments, the frequency of the AC modulation component may be minimized while still allowing accurate measurements. In some embodiments, the frequency of the AC modulation may be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 times the sampling maximum frequency. In some embodiments, the frequency may be 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.

[0126] Figure 8B shows an embodiment of the self-limiting poration waveform shown in Figure 8A with an AC modulation component 802 added to make a measurement. In this embodiment, the amplitude of the AC modulation component is 100 mV, which is shown on the graph as the thickness of the line.

[0127] Any other voltage waveform may have an AC modulation component superimposed on it so that measurements may be taken while the voltage waveform is being applied.

[0128] V. Computer Systems Any of the computer systems described herein can utilize any suitable number of subsystems, many of which may be any number. An example of such a subsystem is shown in FIG. 11, in computer system 1110. In some embodiments, the computer system includes a single computing device, where the subsystems may be components of that computing device. In other embodiments, the computer system includes multiple computing devices, each of which is a subsystem having internal components. Computer systems can include desktop and laptop computers, tablets, mobile phones, and other mobile devices.

[0129] 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, storage device(s) 1179, and a monitor 1176 connected to a display adapter 1182. Peripheral and input / output (I / O) devices connected to an I / O controller 1171 may 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, the I / O port 1177 or an external interface 1181 (e.g., Ethernet®, Wi-Fi®, etc.) may 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 a system bus 1180 allows a central processor 1173 to communicate with each of the subsystems and control the execution of instructions from a system memory 1172 or storage device(s) 1179 (e.g., fixed disk such as a hard drive or optical disk), as well as the exchange of information between the subsystems. The system memory 1172 and / or the storage device(s) 1179 may embody a computer-readable medium. Another subsystem is a data collection device 1175, such as a camera, microphone, accelerometer, or other sensor. Any of the data described herein may be output from one component to another and may be output to a user.

[0130] A computer system may include multiple identical components or subsystems connected together, for example, by an external interface 1181, by an internal interface, or through a removable storage device that may be connected and disconnected from one component to another. In some embodiments, computer systems, subsystems, or devices communicate over a network. In such an example, one computer may be considered a client and another computer may be considered a server. Each of them may be part of the same computer system. A client Each of the client and server may include multiple systems, subsystems, or components.

[0131] Various aspects of the embodiments may be implemented in the form of control logic using hardware circuits (e.g., APSICs or FPGAs) and / or using computer software, typically with modular or integrated programmable processors. As used herein, a processor may include a single core processor, a multi-core processor on the same integrated chip, or multiple processing units on a single circuit board, or networked, as well as dedicated hardware. Based on the disclosure and teachings provided herein, those skilled in the art will know and understand other ways and / or methods of implementing each of the embodiments of the present invention using hardware and combinations of hardware and software.

[0132] Any of the software components or functions described in this application may be implemented as software code executed by a processor using any suitable computer language, such as, for example, Java, C, C++, C#, Objective C, Swift, or a scripting language, such as, for example, Perl or Python, using conventional or object-oriented techniques. The software code may 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 may include random access memory (RAM), read only memory (ROM), magnetic media such as a hard drive or a floppy disk, or optical media such as a compact disk (CD) or a digital versatile disk (DVD), flash memory, etc. The computer-readable medium may be any combination of such storage or transmission devices.

[0133] Such a program may also be encoded and transmitted using a carrier signal adapted for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet. Thus, a computer-readable medium may be created using a data signal encoded with such a program. A computer-readable medium encoded with program code may be packaged with a compatible device or provided separately from other devices (e.g., downloaded via the Internet). Any such computer-readable medium may be provided on or within a single computer product (e.g., a hard drive, CD, or an entire computer system), or may reside on or within different computer products within a system or network. A computer system may include a monitor, printer, or other suitable display for providing any of the results described herein to a user.

[0134] Any of the methods described herein may be performed completely or partially using a computer system including one or more processors that may be configured to perform the steps described above. Thus, each of the embodiments may be directed to a computer system configured to perform any of the steps of the methods described herein, potentially using different components performing each of the steps or each of the groups of steps. Although the steps of the methods herein are presented as sequenced steps, they may be performed simultaneously or at different times or in different orders. Additionally, each of these step portions may be used with each of the other step portions from other methods. Also, all of one step or each of its portions may be optional. Additionally, any of the steps of any of these methods may be performed using modules, units, circuits, or other means of systems for performing these steps.

[0135] The specific details of the particular embodiments may be combined in any suitable manner without departing from the spirit and scope of the embodiments of the invention, however, other embodiments of the invention may be directed to specific embodiments relating to each individual aspect or specific combinations of those individual aspects.

[0136] Although the previous embodiments have been described in some detail for purposes of clarity of understanding, the present invention is not limited to those details provided. There are many alternative ways to implement the present invention. The disclosed embodiments are for purposes of illustration and description, not limitation. The above descriptions of exemplary embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms described. Many modifications and variations are possible in light of the above teachings.

[0137] When a feature or element is said herein to be "on" another feature or element, this can be directly on the other feature or element, or there may be intervening features and / or elements between them. In contrast, when a feature or element is said to be "directly on" another feature or element, this means that there are no intervening features or elements between them. When a feature or element is said to be "connected," "attached," or "coupled" to another feature or element, it will also be understood that this can be directly connected, attached, or connected to the other feature or element, or there may be intervening features or elements between them. In contrast, when a feature or element is said to be "directly connected," "directly attached," or "directly coupled" to another feature or element, this means that there are no intervening features or elements between them. Although described or illustrated with respect to one embodiment, the features and elements described or illustrated may be applicable to other embodiments. It will also be understood by those skilled in the art that when referring to a structure or feature located "adjacent" another feature, this may include overlapping or underlying portions of the adjacent feature.

[0138] 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. It will be further understood that when the terms "comprises" and / or "comprising" are used herein, they specify the presence of the features, steps, operations, elements, and / or components set forth herein, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the terms "and" and / or "or" include any and all combinations of one or more of the associated listed items. They may be abbreviated as " / ".

[0139] Spatially relative terms such as "under," "below," "lower," "over," and "upper" may be used herein to describe the relationship of one element or feature to another element(s) or feature(s) as illustrated in the drawings for ease of description. It will be understood that these spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the drawings. For example, the top and bottom of a device in the drawings may be different. When reversed, an element described as "under" or "beneath" another element or feature will now be "over" that other element or feature. Thus, the exemplary term "under" can include both over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and interpreted accordingly by the spatially relative descriptors used herein. Similarly, the terms "upwardly," "downwardly," "vertical," "horizontal," etc. are used herein for descriptive purposes only, unless specifically indicated otherwise.

[0140] Although the terms "first" and "second" may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element described below could be referred to as a second feature / element, and similarly, a second feature / element described below could be referred to as a first feature / element, without departing from the teachings of the present invention.

[0141] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and its variations, such as "comprises" and "comprising," mean that various components may be employed together in the methods and clauses (e.g., configurations and apparatuses, including devices and methods). For example, the term "comprising" is understood to imply the inclusion of any element or step described herein, but not the exclusion of any other element or step.

[0142] As used herein, including as used in each instance, and unless expressly specified otherwise, all numerical values ​​in the specification and claims may be read as if preceded by the words "about" or "approximately," even if each of them is not expressly stated. The phrases "about" or "approximately" may be used in describing a magnitude and / or location to indicate that the described value and / or location is within a reasonably expected range of values ​​and / or locations. For example, a numerical value may have a value of + / -0.1% of the indicated value (or range of values), + / -1% of the indicated value (or range of values), + / -2% of the indicated value (or range of values), + / -5% of the indicated value (or range of values), + / -10% of the indicated value (or range of values), etc. Any numerical value given 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, it is also disclosed as "about 10." Any numerical range recited herein is intended to include all subranges therebetween. When a value is disclosed as "less than or equal to" that value, it is also understood that "greater than or equal to" that value, and possible ranges between the values, as appropriately understood by one of ordinary skill in the art, are also disclosed. For example, when a value "X" is disclosed, this can be understood as "less than or equal to X," as well as "greater than or equal to X." (e.g., X is a numerical value). It is also understood that throughout this application data is provided in a number of different formats, and that this data represents endpoints as well as starting points and ranges for any combination of the data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that numbers greater than, greater than, less than, less than, less than, and equal to 10 and 15 are considered to be disclosed, as well as numbers between 10 and 15. It is also understood that every unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0143] Although various illustrative embodiments have been described above, any number of modifications may 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 described are performed may often be changed in alternative embodiments. In other alternative embodiments, one or more method steps may be skipped altogether. Optional features of the various device and system embodiments may be included in some embodiments and not included in other embodiments. Thus, 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.

[0144] The examples and figures included herein show, for purposes of illustration, not limitation, specific embodiments in which the present subject matter may be practiced. As noted above, other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such embodiments of the present subject matter, if more than one of them are actually disclosed, may be referred to herein by the term "invention," either individually or collectively, for convenience only, without any intention of independently limiting the scope of the present application to any single invention or inventive concept. Thus, although specific embodiments are shown and described herein, any arrangements calculated to achieve the same purpose may be substituted for the specific embodiments shown herein. The present disclosure is intended to cover any and all adaptations or variations of the various embodiments. Combinations of the above embodiments with other embodiments not specifically described herein will be apparent to one of ordinary skill in the art upon reviewing the preceding description.

[0145] All patents, patent applications, publications, and descriptions discussed herein are hereby incorporated by reference in their entirety for all purposes. None are admitted to be prior art.

Claims

1. 1. A method of forming an array of nanopore sensor cells, comprising: 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 a power source electrically connected thereto; applying a voltage waveform across the membrane of the cell, the voltage waveform beginning at a first voltage and increasing over a period of time to a second voltage; inserting the nanopore into the membrane during the step of applying the voltage waveform; The method includes:

2. 2. The method of claim 1, wherein the first voltage is between about 0 and 100 mV and the second voltage is between about 100 to 2000 mV.

3. The method of claim 1 , wherein the working electrode is a capacitive electrode.

4. 4. The method of claim 1, wherein the voltage waveform includes a plurality of incremental steps between the first voltage and the second voltage.

5. The method of claim 4 , wherein the plurality of incremental steps are increments of about 1 to 100 mV.

6. The method of claim 4 , wherein the plurality of incremental steps are increments of about 1 to 25 mV.

7. 7. The method of claim 4, wherein each incremental step has a duration of between about 0.1 and 60 seconds.

8. The method of claim 7 , wherein the duration of the incremental steps is variable.

9. The method of claim 7 , wherein the duration of the incremental steps at a lower voltage is longer than the duration of the incremental steps at a higher voltage.

10. The method of claim 7 , wherein the period of the incremental steps is constant.

11. 4. The method of claim 1, wherein the voltage waveform includes a ramp between the first voltage and the second voltage.

12. 12. The method of claim 11, wherein the ramp is between about 0.1 and 2.0 volts per minute.

13. The method of claim 12 , wherein the ramp has a constant slope.

14. The method of claim 12 , wherein the ramp has a variable slope.

15. The method of claim 12 , wherein the ramp has a slope at a lower voltage that is less than a slope at a higher voltage.

16. The method of claim 1 , wherein applying the voltage waveform is applied to a non-thinned film.

17. 17. The method of claim 16, further comprising thinning the unthinned film with the applied voltage waveform.

18. The method of claim 1 , wherein the power supply is AC connected to the working electrode.

19. 2. The method of claim 1 , wherein the voltage waveform includes an AC modulation component configured such that electrical measurements can be taken through the working electrode while the voltage waveform is applied across the membrane of the cell.

20. 20. The method of claim 19, wherein the AC modulation component has an amplitude of less than 100 mV.

21. 20. The method of claim 19, wherein the AC modulation component has a frequency between 10 Hz and 1000 Hz.

22. 1. A system for molecular sequencing 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, the working electrodes being powered by an AC connected power source; A counter electrode; a power source electrically connected to each of the working electrodes; A controller, a controller programmed to apply a voltage waveform to the cell using the working and counter electrodes, the voltage waveform beginning at a first voltage and increasing over a period of time to a second voltage; A system including:

23. 23. The system of claim 22, wherein the working electrode is a capacitive electrode.

24. 23. The system of claim 22, wherein the voltage waveform includes a plurality of incremental steps between the first voltage and the second voltage.

25. 23. The system of claim 22, wherein the voltage waveform includes a ramp between the first voltage and the second voltage.

26. 23. The system of claim 22, wherein the controller is further programmed to apply the voltage waveform to an unthinned membrane.

27. 23. The system of claim 22, wherein the power source is AC connected to each of the working electrodes.

28. 1. A method of forming a membrane covered cell, comprising the steps of: flowing a membrane-forming material over a cell, the cell having a working electrode, the working electrode being powered by a power source electrically connected thereto; disposing a layer of a membrane-forming material over the cell; applying a voltage waveform across the layer of film-forming material using the working electrode and a counter electrode on an opposite side of the layer of film-forming material, the voltage waveform including an AC modulation component, the AC modulation component configured such that electrical measurements can be taken across the working electrode while the voltage waveform is being applied across the layer of film-forming material. applying a voltage waveform across the layer of film-forming material using the working electrode and a counter electrode on an opposite side of the layer of film-forming material; thinning the layer of membrane-forming material into a membrane, the membrane configured to receive a nanopore; The method includes:

29. 30. The method of claim 28, wherein the AC modulation component has an amplitude of less than 100 mV.

30. 30. The method of claim 28, wherein the AC modulation component has a frequency between 10 Hz and 1000 Hz.

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