System and method for inserting self-restrictive protein pores in membranes

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 on December 11, 2018, the entire content of which is hereby incorporated by reference for all purposes.

[0002] Incorporation by Reference All publications and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference and was set forth herein in its entirety.

Background Art

[0003] Nanopore - based sequencing chips are analytical tools that can be used for deoxyribonucleic acid (DNA) sequencing. These devices can incorporate a very large number of sensor cells configured as an array. For example, a sequencing chip can include an array of one million cells, such as a 1000 - row by 1000 - column cell array. Each cell of the array can include a membrane and a protein pore having a pore size on the order of 1 nanometer in inner diameter. Such nanopores have been shown to be effective in the rapid sequencing of nucleotides.

[0004] When a potential is applied across a nanopore immersed in a conductive fluid, a small ion current due to the conduction of ions across the nanopore can exist. The size of the current is susceptible to the pore size and the type of molecule placed within 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 becomes possible. As a method of measuring the resistance of the molecule, a voltage or other signal in a circuit including the nanopore can be measured (e.g., by an integrating capacitor). Thus, it becomes possible to detect what molecule is in the nanopore.

[0005] For the array determination chip to function properly, generally, only one pore should be inserted in the membrane for a given cell. When multiple pores are inserted into a single membrane, the interpretation of the electrical signature generated by nucleotides that have passed through multiple pores simultaneously becomes much more difficult.

[0006] The application of a voltage across the membrane during the pore insertion step may perhaps facilitate the pore insertion process by reducing the stability of the membrane and making the pore itself more easily inserted into the membrane. However, the application of an excessive voltage across the membrane can cause significant damage to the membrane. As a result, the cell becomes unusable.

[0007] Therefore, it is preferable to provide a system and method for reliably inserting a single pore into a membrane while reducing the risk of excessive damage to the membrane. SUMMARY OF THE INVENTION

[0008] Various embodiments provide techniques and systems related to the insertion of a single pore into a membrane in a cell of a nanopore-based array determination chip. In some embodiments, the insertion of the pore into the membrane reduces the likelihood of further pore insertion into the membrane.

[0009] Other embodiments are directed to systems and computer-readable media associated with the methods described below. readable media.

[0010] In some embodiments, a method is provided for forming an array of nanopore sensor cells. This method includes introducing nanopores as close as possible 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 supply; applying a voltage waveform across the membrane of the cell, the voltage waveform starting at a first voltage and increasing over a period of time to a second voltage; and inserting nanopores into the membrane during the step of applying the voltage waveform.

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

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

[0013] In some embodiments, the voltage waveform includes a plurality of gradual steps between the first voltage and the second voltage.

[0014] In some embodiments, the multiple gradual steps progress in increments of approximately 1 to 100 mV.

[0015] In some embodiments, multiple gradual steps progress by approximately 1 to 25 mV.

[0016] In some embodiments, each progressive step has a duration between approximately 0.1 and 60 seconds.

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

[0018] In some embodiments, the duration of the gradual step at lower voltages is longer than the duration of the gradual step at higher voltages.

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

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

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

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

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

[0024] In some embodiments, the slope has a smaller slope at lower voltages than the slope at higher voltages.

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

[0026] In some embodiments, this method further includes thinning an unthinned film using an applied voltage waveform.

[0027] In some embodiments, a system for determining the arrangement of molecules is provided. The system includes an array of cells on a substrate, each cell having a working electrode and an opening configured to be sealed by a film, the working electrode being powered by an alternating current (AC) connected power supply; a counter electrode; a power supply, AC connected to each working electrode; and a controller, programmed to supply a voltage waveform to the cells using the working electrode and the counter electrode, starting at a first voltage and increasing over a period of time up to a second voltage.

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

[0029] In some embodiments, the voltage waveform includes a plurality of gradual steps between the first voltage and the second voltage.

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

[0031] In some embodiments, the controller is further programmed to supply a voltage waveform to the film that has not been thinned.

[0032] In some embodiments, a method is provided for forming an array of nanopore sensor cells. This method may include introducing nanopores as close to the cell as possible, wherein the cell has a working electrode and a membrane sealing the cell, and the working electrode is powered by an electrically connected power supply; applying a voltage waveform across the membrane of the cell, wherein the voltage waveform starts at a first voltage and increases over a period of time to a second voltage, and the voltage waveform includes an alternating current (AC) modulated component, the AC modulated component is configured such that electrical measurements can be obtained through the working electrode while the voltage waveform is applied across the membrane of the cell; and inserting nanopores 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 is provided for forming a film-covered cell. This method may include: flowing a film-forming material onto a cell, the cell having a working electrode, the working electrode being powered by an electrically connected power source; arranging a layer of film-forming material onto the cell; applying a voltage waveform across the layer of film-forming material using the working electrode and a counter electrode on the opposite side of the layer of film-forming material, the voltage waveform including an alternating current (AC) modulated component, the AC modulated component being configured such that electrical measurements can be obtained through the working electrode while the voltage waveform is being applied across the layer of film-forming material; and thinning the layer of film-forming material into a film, the film being configured to accept nanopores.

[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] For a good understanding of the characteristics and advantages of the embodiments of the present invention, please refer to the following detailed description and accompanying drawings. It can be obtained by illuminating it.

[0037] Novel features of the present invention are described in particular in the following claims. A good understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description and accompanying drawings, which illustrate exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 is a top view of an embodiment of a nanopore sensor chip having an array of nanopore cells. [Figure 2]Figure 2 shows an embodiment of a nanopore cell in a nanopore sensor chip that may be used to evaluate the properties of polynucleotides or polypeptides. [Figure 3] Figure 3 shows an embodiment of a nanopore cell in which nucleotide sequencing is performed using nanopore-based, synthetic sequencing (Nano-sequencing-by-synthesi or Nano-SBS) technology. [Figure 4] Figure 4 shows an embodiment of an electrical circuit in a nanopore cell. [Figure 5] Figure 5 shows an example of data points acquired from a nanopore cell during the light and dark phases of an AC cycle. [Figure 6] Figure 6 shows an embodiment of the circuit diagram of a nanopore sensor cell. [Figure 7] Figure 7 shows a stepped voltage waveform that can be used to facilitate pore insertion. [Figure 8] A and B show sloped voltage waveforms that can be used to facilitate pore insertion. [Figure 9] A and B demonstrate that, in some embodiments, once a pore is inserted, the pore can dissipate the increased voltage across the membrane itself, thus reducing both the risk of damage to the membrane and the possibility of further pore insertion when the voltage increases further after the pore has been inserted. [Figure 10] A plots the number of pore insertions in the array with respect to voltage and time, and B plots the number of deactivations / shorts resulting from film breakdown with respect to voltage and time. [Figure 11] Figure 11 shows a computer system relating to a particular aspect of this disclosure.

[0039] term Unless otherwise defined, technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Methods, devices, and materials similar to or equivalent to those described herein may be used in the practice of the disclosed art. The following terms are provided to facilitate understanding of certain terms used frequently and are not intended to limit the scope of this disclosure. Abbreviations used herein have their common meanings in the fields of chemistry and biology.

[0040] "Nanopores" refer to pores, channels, or passages formed within or otherwise provided within a membrane. The membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane made of a polymer material. Nanopores may be located adjacent to or near sensing circuits, such as complementary metal oxide semiconductor (CMOS) or field-effect transistor (FET) circuits, or electrodes connected to such sensing circuits. In some examples, nanopores have characteristic widths or diameters on the order of about 0.1 nanometers (nm) to about 1000 nm. In some implementations, nanopores may be proteins.

[0041] "Nucleic acids" refer to deoxyribonucleotides, or ribonucleotides, and single-stranded nucleic acids. This term refers to polymers of either nucleotides or double-stranded nucleotides. The term encompasses known nucleotide analogs, or nucleic acids containing modified skeletal chain residues or chains. These include synthetic, naturally occurring, and non-naturally occurring substances. They possess similar binding properties to the reference nucleic acid. They are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, specific nucleic acid sequences also implicitly include their conservedly modified variants (e.g., degenerate codon substitutions), complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed group and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid can be used interchangeably with genes, complementary deoxyribonucleic acid (complementary DNA or cDNA), messenger ribonucleic acid (mRNA), oligonucleotides, and polynucleotides.

[0042] In addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, the term "nucleotide" may be understood to refer to their associated structural variants, including functionally equivalent derivatives and analogues, in the context in which nucleotides are used (such as hybridization with complementary bases), unless otherwise explicitly indicated by that context.

[0043] The term "tag" refers to a detectable portion that may be an atom or molecule, or an aggregate of atoms or molecules. A tag can provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive or capacitive) signature. This signature may be detectable with the help of nanopores. Typically, when a nucleotide is attached to a tag, the tag is called a "tagged nucleotide." Tags may be attached to nucleotides via phosphate moieties.

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

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

[0046] The term "polymerase" refers to an enzyme that synthesizes polynucleotides directed toward a template. This term encompasses both the entire polypeptide and the domain possessing polymerase activity. DNA polymerases are well known to those skilled in the art and include, but are not limited to, DNA polymerases cleaved from or derived from Pyrococcus friosus, Thermococcus litralis, and Thermotoga maritima, or their modified versions. These include both DNA-dependent polymerases and RNA-dependent polymerases, such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known. The most well-known are families A, B, and C. Similarities in sequencing among these various families are minimal or nonexistent. Most family A polymerases are polymerases. DNA polymerases are single-chain proteins that can include multiple enzymatic functions, including exonuclease activity from 3′ to 5′ and exonuclease activity from 5′ to 3′. Family B polymerases typically have a single catalytic domain with polymerase and exonuclease activity from 3′ to 5′, as well as cofactors. Family C polymerases are typically multi-subunit proteins with polymerization and exonuclease activity from 3′ to 5′. In Escherichia coli (or E. coli), three types of DNA polymerases have been found: DNA polymerase I (family A), DNA polymerase II (family B), and DNA polymerase III (family C). In eukaryotic cells, three different family B polymerases, DNA polymerases α, δ, and ε, are involved in nuclear replication. Polymerase γ, one of the family A polymerases, is used for mitochondrial DNA replication. Other types of DNA polymerases include phage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, as well as bacterial RNA polymerases, and phage and viral polymerases. RNA polymerases can be DNA-dependent or RNA-dependent.

[0047] The term "light phase" generally refers to the period during which the tags of tagged nucleotides are pushed into the nanopores by an electric field applied via an AC signal. The term "dark phase" generally refers to the period during which the tags of tagged nucleotides are pushed out of the nanopores by an electric field applied via an AC signal. An AC cycle may include both a light phase and a dark phase. In different embodiments, the polarity of the voltage signal applied to the nanopore cell can be varied to bring the nanopore cell into a light phase (or dark phase).

[0048] The term "signal value" refers to the value of the sequencing signal output from a sequencing cell. According to a particular embodiment, the sequencing signal is an electrical signal measured at and / or output from a point in the circuit of one or more sequencing cells. For example, the signal value is (or represents) voltage or current. The signal value may represent the result of a direct measurement of voltage and / or current, and / or an indirect measurement. For example, the signal value may be the measured period of time it takes for the voltage or current to reach a specified value. The signal value may be related to the resistivity of nanopores and may represent any measurable quantity from which the resistivity and / or conductance of (penetrated and / or unpenetrated) nanopores can be derived. In another example, the signal value may correspond to the intensity of light from a phosphor attached to a nucleotide added to a nucleic acid using polymerase, for example.

[0049] The term "osmolarity," also known as osmotic pressure concentration, refers to the unit of measurement for solute concentration. Osmolarity measures the number of osmoles of solute particles per unit volume of solution. An osmole is the unit of measurement for the number of moles of solute that contribute to the osmotic pressure of a solution. Osmolarity enables the measurement of the osmotic pressure of a solution and the determination of how a solvent disperses across a semipermeable membrane (osmotic action) separating two solutions with different osmotic pressures.

[0050] The term "osmolite" refers to any soluble compound that, when dissolved in a solution, increases the osmolality of that solution. [Modes for carrying out the invention]

[0051] In certain embodiments, the technologies 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 a membrane reduces the possibility of further insertion of pores into the membrane. This, in turn, makes the pore insertion more self-limiting, reducing or eliminating the need for active feedback during the insertion step.

[0052] Examples of nanopore systems, circuits, and sequencing operations are described first. Subsequently, examples of techniques for replacing nanopores in DNA sequencing cells are described. Embodiments of the present invention can be carried out in many ways. These methods include processes, systems, and computer program products embodied on a computer-readable storage medium, and / or processors, for example, a processor configured to execute instructions stored on and / or provided by memory connected to the processor.

[0053] I. Nanopore-based sequencing chips Figure 1 is a top view of an embodiment of a nanopore sensor chip 100 having an array 140 of nanopore cells 150. Each nanopore cell 150 includes a control circuit integrated on the silicon substrate of the nanopore sensor chip 100. In some embodiments, side walls 136 are included in the array 140 to separate each group of nanopore cells 150, so that each group can receive different samples for characterization. Each nanopore cell may be used to determine the sequence of 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 number of pins 110 that interact with other circuits, such as a computer processor.

[0054] In some embodiments, the nanopore sensor chip 100 includes multiple chips in the same package, for example, forming a multi-chip module (MCM) or a system-in-package (SiP). The chips may include, for example, memory, a processor, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a data converter, a high-speed I / O interface, and the like.

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

[0056] II. Nanopore Alignment Determination Cell The nanopore cells 150 in the nanopore sensor chip 100 can be implemented in many different ways. For example, in some embodiments, tags of different sizes and / or chemical structures are attached to different nucleotides in the nucleic acid molecule to be sequenced. In some embodiments, complementary strands may be synthesized on the nucleic acid molecule to be sequenced by hybridizing nucleotides tagged with different polymers with a template. In some implementations, both the nucleic acid molecule and the attached tags move through the nanopores. Ionic currents passing through the nanopores can indicate nucleotides within the nanopores, depending on the specific size and / or structure of the tag attached to that nucleotide. In some implementations, only the tags are moved into the nanopores. Different tags within the nanopores can also be detected in many different ways.

[0057] A. Determining the nanopore arrangement of cell structures Figure 2 shows an exemplary embodiment of a nanopore cell 200 in a nanopore sensor chip, such as the nanopore cell 150 in the nanopore sensor chip 100 of Figure 1. This can be used to evaluate the properties of polynucleotides or polypeptides. The nanopore cell 200 may include a well 205 formed of dielectric layers 201 and 204, a membrane such as a lipid bilayer 214 formed on the well 205, and a sample chamber 215 on the lipid bilayer 214, separated from the well 205 by the lipid bilayer 214. The well 205 may contain a large amount of electrolyte 206. The sample chamber 215 can hold a nanopore-containing bulk electrolyte 208, such as soluble protein nanopore transmembrane molecular complexes (PNTMCs), and the analyte of interest (e.g., a nucleic acid molecule to be sequenced).

[0058] The nanopore cell 200 may include a working electrode 202 located at the bottom of a well 205 and a counter electrode 210 positioned within 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 a lipid bilayer 214 by an electroporation process initiated by a voltage signal. Thus, a nanopore 216 is formed within the lipid bilayer 214. Individual membranes in the array (e.g., lipid bilayer 214 or other membrane structures) may be unconnected to each other chemically or electrically. Thus, each nanopore cell in the array can be an independent sequencing machine. This generates data specific to a single polymer molecule associated with a nanopore that acts on the analyte and modulates an ionic current through an otherwise impermeable lipid bilayer.

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

[0060] As shown in Figure 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, oxides, nitrides, etc. An electrical circuit 222 for controlling electrical stimulation and processing signals detected from the nanopore cell 200 may be formed on the substrate 230 and / or within the dielectric layer 201. For example, multiple patterned metal layers (e.g., metal 1 to metal 6) may be formed within the dielectric layer 201. 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, etc. The electrical circuit 222 may be further connected to a processor 224 connected to a memory 226. Here, the processor 224 can analyze the sequencing data and determine the sequence of polymer molecules that have been sequenced 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 metallic electrode. For non-Faraday conductivity, the working electrode 202 may be made of a metal or other material resistant to corrosion and oxidation, such as platinum, gold, titanium nitride, and graphite. For example, the working electrode 202 may be a platinum electrode 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. This, therefore, increases its surface area and gives the capacitance associated with the working electrode 202. The working electrode of a nanopore cell may be independent of the working electrode of another nanopore cell; therefore, the working electrode may be referred to as the cell electrode in this disclosure.

[0062] A dielectric layer 204 may be formed on top of the dielectric layer 201. The dielectric layer 204 forms a wall surrounding the well 205. The dielectric material used to form the dielectric layer 204 may include, for example, glass, oxide, silicon mononitride (or SiN), polyimide, or other suitable hydrophobic insulating materials. The upper surface of the dielectric layer 204 may be silane-treated. Silane treatment can form a hydrophobic layer 220 on top of the upper surface of the dielectric layer 204. In some embodiments, the hydrophobic layer 220 has a thickness of about 1.5 nanometers (nm).

[0063] The well 205 formed by the wall of the dielectric layer 204 contains a large amount of electrolyte 206 on the working electrode 202. The large amount 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 (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2). In some embodiments, the large amount of electrolyte 206 has a thickness of about 3 microns (μm).

[0064] Furthermore, as shown in Figure 2, a film may be formed on the dielectric layer 204. This film extends across the well 205. In some embodiments, this film includes a lipid monolayer 218 formed on the hydrophobic layer 220. When this film reaches the opening of the well 205, the lipid monolayer 208 can transition into a lipid bilayer 214 that extends across the opening of the well 205. The lipid bilayer may contain or consist of lipids such as phospholipids. Lipids are selected from, for example, diphytanyl-phosphatidylcholine (DPhPC), 1,2-diphytanyl-sn-glycero-3-phosphocholine, 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidyl-methyl ester (DOPME), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanyl-sn-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy( [Polyethylene glycol)-350], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, GM1 ganglioside, lysophosphatidylcholine (LPC), or any combination thereof. Other phospholipid derivatives may also be used.For example, phosphatidic acid derivatives (e.g., DMPA, DDPA, DSPA), phosphatidylcholine derivatives (e.g., DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol derivatives (e.g., DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine derivatives (e.g., DMPE, DPPE, DSPE, DOPE), phosphatidylserine derivatives (e.g., DOPS), PEG phospholipid derivatives (e.g., mPEG phospholipids, polyglycerol 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 copolymer, PEG triblock copolymer, PPG triblock copolymer, and poloxamer), and other amphiphilic copolymers, which may be nonionic or ionic. In some embodiments, the bilayer may be formed from a combination of a lipid-based material and a non-lipid-based material. In some embodiments, the bilayer material may be supplied in a solvent phase containing one or more organic solvents, such as alkanes (e.g., decane, tridecane, hexadecane, etc.) and / or one or more silicone oils (e.g., AR-20).

[0065] As shown here, the lipid bilayer 214 has a single nanopore 216 embedded in it, for example, formed by a single PNTMC. As described above, the nanopore 216 can be formed by inserting a single PNTMC into the lipid bilayer 214 by electroporation. The nanopore 216 contains at least one site of the target analyte and / or small ions (e.g., Na + , K + Ca 2+ , CI - ) can be large enough to pass between the two sides of the lipid bilayer 214.

[0066] The sample chamber 215 rests on the lipid bilayer 214 and can hold a solution of the analyte to be characterized. The solution is an aqueous solution containing the bulk electrolyte 208, which can be buffered to an optimal ion concentration and maintained at an optimal pH to keep the nanopores 216 open. The nanopores 216 traverse the lipid bilayer 214 and provide a single pathway for ion flow from the bulk electrolyte 208 to the working electrode 202. In addition to the nanopores (e.g., PNTMC) and the analyte, the bulk electrolyte 208 may further contain one or more of the following: lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2).

[0067] The counter electrode (CE) 210 may be an electrochemical potential sensor. In some embodiments, the counter electrode 210 is shared among multiple nanopore cells and may therefore be called a common electrode. In some cases, the common potential and common electrode may be common to all nanopore cells, or at least to all nanopore cells within a particular grouping. The common electrode may be configured to apply the common potential to a bulk electrolyte 208 in contact with the nanopores 216. The counter electrode 210 and the working electrode 202 may be connected to a signal source 228 to provide an electrical stimulus (e.g., a voltage bias) across the lipid bilayer 214 and may be used to sense the electrical characteristics of the lipid bilayer 214 (e.g., resistance, capacitance, and ion current flow). In some embodiments, the nanopore cell 200 may also include a reference electrode 212.

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

[0069] B. Detection signal of nanopore arrangement determination cell Nanopore cells in nanopore sensor chips, such as the nanopore cell 150 in nanopore sensor chip 100, are single-molecule nanopore cells synthesized using nano-SBS (Nano-SBS) technology. This enables parallel sequencing using sequence determination.

[0070] Figure 3 shows an embodiment of a nanoporous 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 target analyte) and a primer can be introduced into the bulk electrolyte 308 within the sample chamber of the nanoporous cell 300. For example, the template 332 may be circular or linear. The nucleic acid primer can be hybridized to a portion of the template 332. Herein, nucleotides 338 tagged with four different polymers may be added.

[0071] In some embodiments, an enzyme (e.g., polymerase 334, such as DNA polymerase) is associated with the nanopore 316 for use in synthesizing a complementary strand onto a template 332. For example, polymerase 334 may be attached to the nanopore 316 by covalent bond. Polymerase 334 can act as a catalyst in the integration of nucleotide 338 onto a primer, using a single-stranded nucleic acid molecule as a template. Nucleotide 338 may include a tag species ("tag") using a nucleotide that is one of four distinct types: A, T, G, or C. Once the tagged nucleotide is properly complexed with polymerase 334, the tag may be drawn into (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 ends 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-blocking signal 340 due to its distinct chemical structure and / or size. Thus, the added base to which the tag is attached is electronically identified.

[0072] As used herein, a “loaded” or “threaded” tag is positioned within and / or near a nanopore for a considerable 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 examples, the probability that a loaded tag will pass through (and / or be detected by) a nanopore after being released following a nucleotide incorporation event is preferably high, such as 90% to 99%.

[0073] In some embodiments, the conductance of the nanopore 316 is high, such as about 300 picosiemens (300 pS), before polymerase 334 is connected to the nanopore 316. Once the tag is loaded into the nanopore, the distinct chemical structure and / or size of the tag generates a unique conductance signal (e.g., signal 340). For example, the conductance of the nanopore may be about 60 pS, 80 pS, 100 pS, or 120 pS. Each of these corresponds to one of the four types of tagged nucleotides described above. The polymerase can then incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule through isomerization and phosphate transfer reactions.

[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 with the nucleic acid molecule can also pass through the nanopores. These unpaired nucleotides can be rejected by polymerase within a shorter timescale than the timescale at which properly paired nucleotides remain associated with the polymerase. Tags directed at unpaired nucleotides can pass rapidly through the nanopores and may be detected over short periods (e.g., less than 10 ms). Tags directed at paired nucleotides, on the other hand, can be loaded into the nanopores and detected over longer periods (e.g., It can be detected over a period of at least 10 ms. Therefore, unpaired nucleotides can be identified by a downstream processor based on at least a portion of the time the nucleotide is detected within the nanopore.

[0075] The conductance (or equivalent to resistance) of the nanopore containing the loaded (inserted) tag is measured via a signal value (e.g., voltage or current passing through the nanopore). This provides identification of the tag species, and therefore the nucleotide at its current position. In some embodiments, a direct current (DC) signal is applied to the nanopore cell (e.g., so that the direction in which the tag moves through the nanopore is not reversed). However, using DC to operate the nanopore sensor over long periods can have other undesirable effects, such as altering the electrode composition, creating an imbalance in ion concentration across the nanopore, and adversely affecting the lifespan 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 have a good affinity with applied AC voltages. Therefore, AC waveforms can be used to achieve these advantages.

[0076] The ability to recharge electrodes during an AC detection cycle may be preferable when sacrificial electrodes are used, which are electrodes whose molecular characteristics change during the current-passing reaction (e.g., electrodes containing silver), or electrodes whose molecular characteristics change during the current-passing reaction. When a DC signal is used, the electrodes may wear out during the detection cycle. Recharging can prevent the electrodes from reaching their wear limit, such as complete wear, which can be problematic 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 is extended in some cases with the width of the electrodes; that is, electrode life depends at least partially on the width of the electrodes.

[0077] Suitable conditions for measuring ion currents passing through nanopores are known to those skilled in the art. Examples of these are provided below. Measurements can be performed using a voltage applied across the membrane and pores. In some embodiments, the voltage used is in the range of -400mV to +400mV. The voltage used is preferably in the 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, and most preferably in the range of 160mV to 240mV. By increasing the applied potential, the ability of nanopores to distinguish between different nucleotides can be enhanced. Nucleic acid sequencing using AC waveforms and tagged nucleotides is described in U.S. Patent Publication No. 2014 / 0134616, filed November 6, 2013, entitled “Nucleic Acid Sequencing Using Tags.” The entirety of that publication is incorporated herein by reference. In addition to tagged nucleotides as described in U.S. Patent Application Publication No. 2014 / 0134616, sequencing may be performed using nucleotide analogs with fewer sugar or acyclic moieties, such as (S)-glycerol nucleoside triphosphates (gNTPs) of five common nucleic acid bases: adenine, cytosine, guanine, uracil, and thymine (Horhota et al., *Organic Letters*, 8:5345-5347

[2006] ).

[0078] C. Electrical circuit of nanopore arrangement determination cell Figure 4 shows an embodiment of an electrical circuit 400 within a nanopore cell, such as nanopore cell 400 (which may include each part of electrical circuit 222 in FIG. 2). As described above, in some embodiments, the electrical circuit 400 may be shared among a plurality of nanopore cells or all nanopore cells in a nanopore sensor chip, and thus includes a counter electrode 410, which may also be referred to as a common electrode. The common electrode is configured to apply a common potential to a bulk electrolyte (e.g., bulk electrolyte 208) in contact with a lipid bilayer (e.g., lipid bilayer 214) in the nanopore cell by connecting to a voltage source V LIQ 420. In some embodiments, an AC non-Faradaic mode is utilized, and an AC signal (e.g., a rectangular wave) is used to modulate the voltage V LIQ , and this modulated voltage V LIQ is applied to the bulk electrolyte in contact with the lipid bilayer in the nanopore cell. In some embodiments, V LIQ is a rectangular wave having a magnitude of ±200 to 250 mV and a frequency, for example, between 25 and 400 Hz. The bulk electrolyte between the counter electrode 410 and the lipid bilayer (e.g., lipid bilayer 214) may be modeled by, for example, a large capacitor (not shown) of 100 μF or more.

[0079] Figure 4 also shows an electrical model 422 representing the electrical properties of the working electrode 402 (e.g., working electrode 202) and the lipid bilayer (e.g., lipid bilayer 214). The electrical model 422 includes a capacitor 426 (C 二分子層 ) that models the capacitance associated with the lipid bilayer and a resistor 428 (R 細孔 ) that models the variable resistance associated with the nanopore. These can change based on the presence of specific tags in the nanopore. The electrical model 422 also has a double-layer capacitance (C 二重層 ) and includes a capacitor 424 that represents the electrical properties between the working electrode 402 and the well 205. The working electrode 402 may be configured to apply a distinct potential 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 the voltage stimulation applied across the lipid bilayer in the nanoporous cell. Before the lipids deposit to form the lipid bilayer, the impedance between these two electrodes may be very low because the wells of the nanoporous cell are not sealed. Therefore, to avoid a short circuit, the pass device 406 may be kept open. After the lipid solvent has deposited in the nanoporous cell and the wells of the nanoporous cell are sealed, the pass device 406 may be closed.

[0081] Circuit 400 is an on-chip integrating capacitor 408(n cap ) may further include. The integrating capacitor 408 may be pre-charged by using the reset signal 403 which closes the switch 401, and the integrating capacitor 408 is connected to a voltage source V PRE It will be connected to 405. In some embodiments, the voltage source V PRE 405 provides a specific reference voltage of a magnitude such as 900mV. When switch 401 is closed, the integrating capacitor 408 controls the voltage source V PRE It can be pre-charged to a reference voltage level of 405.

[0082] Once the pre-charging of the integrating capacitor 408 is complete, a reset signal 403 may be used to open the switch 401, and the voltage source V of the integrating capacitor 408 may be used. PRE The connection from 405 is cut off. At this point, the voltage source V LIQ Depending on the level of the voltage source V, the potential of the counter electrode 410 can be higher than that of the working electrode 402 (and the integrating capacitor 408). The reverse is also true. For example, the voltage source V LIQ During the positive phase of the square wave from the voltage source (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 (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. Therefore, in some embodiments, the integrating capacitor 408 is in the light phase. In between, a voltage source V PRE From the pre-charged voltage level of 405, it can be further charged to a higher level, and during the dark period, it can be discharged to a lower level due to the potential difference between the counter electrode 410 and the working electrode 402. In other embodiments, charging and discharging occur during the dark period and the light period, respectively.

[0083] The integrating capacitor 408 can 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 higher than 1 kHz, 5 kHz, 10 kHz, or 100 kHz. For example, at a sampling rate of 1 kHz, the integrating capacitor 408 can be charged / discharged over a period of approximately 1 ms. Subsequently, a voltage level is sampled, which can be 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 a nucleotide at its current position on the template.

[0084] After sampling by the ADC435, the integrating capacitor 408 can be pre-charged again by using the reset signal 403 which closes the switch 401, and the integrating capacitor 408 can be pre-charged again by the voltage source V PRE The connection to 405 is established. The steps of pre-charging the integrating capacitor 408, waiting for a certain period of time for the integrating capacitor 408 to charge or discharge, and sampling and converting the voltage level of the integrating capacitor by the ADC 435 may be repeated in each cycle through the array determination process.

[0085] The digital processor 430 can process the ADC output data for purposes such as normalization, data buffering, data filtering, data compression, data reduction, event extraction, or assembly of ADC output data from an 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] Therefore, a voltage signal applied across the nanopore can be used to detect specific states of the nanopore. One possible state of the nanopore is the open-channel state, when the tagged polyphosphate is removed from the barrel of the nanopore. This is also referred to below as the uninserted nanopore state. The other four possible states of the nanopore each correspond to the state when one of four different types of tagged polyphosphate nucleotides (A, T, G, or C) is retained within the barrel of the nanopore. Yet another possible state of the nanopore is when the lipid bilayer is broken.

[0087] When the voltage level on the integrating capacitor 408 is measured after a certain period of time, different states of the nanopores can result in different voltage level measurements. This is because the rate of voltage decay (decrease due to discharge or increase due to charging) on ​​the integrating capacitor 408 (i.e., the slope of the voltage gradient on the integrating capacitor 408 relative to the time plot) is affected by the nanopore resistance (e.g., resistor R). 細孔This is because it depends on the resistance of 428. In particular, in different states, the resistance associated with the nanopore differs due to the distinct chemical structure of the molecule (tag), so different, corresponding rates of voltage decay may be observed, which can 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 nanopore (i.e., R 細孔 This is the resistor associated with resistor 428). C is, R simultaneously with the membrane (that is, C 二分子層 This is the capacitance associated with capacitor 426). The time constant of the nanopore cell can be, for example, about 200 to 500 ms. The decay curve may not exactly match the exponential curve due to the detailed implementation of the bilayer. However, the decay curve is similar to the exponential curve, monotonic, and therefore can enable tag detection.

[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 electrical model 422.

[0089] The rate of voltage decay over the integrating capacitor 408 can be determined by different methods. As described above, the rate of voltage decay can be determined by measuring the voltage decay over a certain period of time. For example, the voltage over the integrating capacitor 408 can first be measured by the ADC 435 at time t1. Subsequently, the voltage is measured again by the ADC 435 at time t2. If the slope of the voltage over the integrating capacitor 408 with respect to the time curve is steeper, the voltage difference is larger. If the slope of the voltage curve is not steeper, the voltage difference is smaller. Therefore, the voltage difference can be used as a criterion for determining the rate of voltage decay over the integrating capacitor 408, and thus the state of the nanopore cell.

[0090] In other embodiments, the rate of voltage decay is determined by measuring the time required for a selected amount of voltage decay. For example, the time required for the voltage to drop, or the time required for the voltage to rise from a first voltage level V1 to a second voltage level V2, can be measured. If the voltage slope is steeper with respect to the time curve, the required time is shorter. If the voltage slope is not steeper with respect to the time curve, the required time is longer. Thus, the measured required time is equal to the integrating capacitor n cap The rate of voltage attenuation on 408 can be used as a criterion for determining the state of the nanopore cells. Those skilled in the art will understand the various circuits that can be used to measure the resistance of nanopores, including signal value measurement techniques such as voltage or current measurement.

[0091] In some embodiments, the electrical circuit 400 includes an on-chip pass device (e.g., pass device 406) and an additional capacitor (e.g., an integrating capacitor 408(n cap )) and does not include. This therefore facilitates a reduction in the size of nanopore-based sequencing chips. Due to the thinness of the film (lipid bilayer), the film (e.g., capacitor 426(C)) does not contain. 二分子層The capacitance associated with )) can be sufficient on its own to create the required RC time constant without requiring additional on-chip capacitance. Thus, capacitor 426 can be used as an integrating capacitor, and the voltage signal V PRE It can be pre-charged by, and then the voltage signal V LIQ It can be discharged or charged by this. In electrical circuits, the elimination of additional capacitors and pass devices, which are otherwise fabricated on-chip, can greatly reduce the mounting area of ​​a single nanopore cell in a nanopore sequencing chip. This, therefore, facilitates scaling of the nanopore sequencing chip to include more cells (for example, 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 (for example, integrating capacitor 408(n) cap ) or capacitor 426(C 二分子層 The voltage level of the tagged nucleotides is sampled by an ADC (e.g., ADC435) while the nucleic acid is being added. It can be converted. For example, if the applied voltage is V LIQ ga V PRE If the field is low enough, the nucleotide tag can be pushed into the barrel of the nanopore by the electric field applied through the counter electrode and the working electrode across the nanopore.

[0093] 1. Insertion An insertion event occurs 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 inside the barrel of a nanopore, the resistance of the nanopore may be greater, and the current that can flow through the nanopore may be smaller.

[0094] During sequencing, the tags may not be inside the nanopores depending on the AC cycle (this is called the open-channel state). Here, the current is maximum because the resistance of the nanopores is lower. When the tags are drawn into the barrel of the nanopore, the nanopore enters the light mode. When the tags are pushed out of the barrel of the nanopore, the nanopore enters the dark mode.

[0095] 2. Light and dark periods During an AC cycle, the voltage across the integrating capacitor can 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 ADC acquisition rate may be about 2000 Hz per cell. Thus, about 20 data points (voltage measurements) can be acquired for each AC cycle (cycle of the AC waveform). Multiple data points corresponding to one cycle of the AC waveform can be called a set. One set of data points for an AC cycle can correspond to, for example, the light mode (period) when a tag is pressed into the barrel of nanopores, V LIQ However, V PRE A subset may be obtained if it is lower. Another subset is, for example, V LIQ However, V PRE At higher levels, the dark mode (period) can be accommodated when the tag is pushed out of the nanopore barrel by the applied electric field.

[0096] 3. Measurement voltage For each data point, when switch 401 is opened, for example, V LIQ However, V PRE If higher, V PRE From V LIQ As an increase to, or V LIQ However, V PRE If it is lower, V PRE From V LIQ As a descent to V LIQ As a result of charging / discharging, an integrating capacitor (e.g., integrating capacitor 408(n cap ) or capacitor 426(C 二分子層The voltage at )) changes and attenuates. Due to the charging of the working electrode, the final voltage value is V LIQ It may deviate from this. The rate of change of the voltage level on the integrating capacitor can be controlled by the value of the resistance of the bilayer, which may contain nanopores and therefore may contain molecules (e.g., tags of tagged nucleotides) in the nanopores. The voltage level can be measured at a predetermined time after switch 401 is opened.

[0097] Switch 401 can be operated at the rate of data acquisition. Switch 401 can be closed during the relatively short period between two data acquisitions, typically immediately after measurement by the ADC. The switch is V LIQ This allows 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 sufficiently attenuated and remains in that state. Alternatively, if switch 401 is closed, the integrating capacitor is pre-charged again (V PRE Up to this point, the system is ready for another measurement. Thus, switch 401 allows for the acquisition of multiple data points across each sub-period (bright or dark) of the AC cycle. Such multiple measurements can be performed using a fixed ADC (for example, they may be averaged, due to the large number of measurements). This allows for higher resolutions (8-bit to 14-bit). Multiple measurements can also provide motion information about molecules inserted into nanopores. Timing information can enable the determination of the time period over which insertion occurs. This can also be used to help determine whether the sequence of multiple nucleotides added to a nucleic acid chain has been determined.

[0098] Figure 5 shows an example of data points acquired from a nanopore cell during the light and dark phases of an AC cycle. In Figure 5, changes in data points are highlighted for illustrative purposes. Voltage (V) applied to the working electrode or integrating capacitor. PREThe voltage signal applied to the counter electrode of the nanopore cell is 510 (V LIQ ) is an AC signal represented as a square wave. The duty cycle here can be any preferred value, such as 50% or less, for example, about 40%.

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

[0100] As shown in Figure 5, V LIQ The first data point 522 (also called the first point delta (FPD)) during the light phase after the change in the signal sign 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 Figure 5. LIQ The level can be exceeded. This can be caused by the capacitance of the bilayer connecting the signal to the on-chip capacitor. Data point 524 can be obtained after the insertion event occurs, i.e., after the tag has been pushed into the barrel of the nanopore. Here, the resistance of the nanopore, and therefore the discharge rate of the integrating capacitor, depends on the specific type of tag pushed into the barrel of the nanopore. Data point 524 is C as described below.二重層 The accumulated charge at 424 can slightly reduce each measurement.

[0101] During the dark period 530, the voltage signal 510 (V) applied to the counter electrode LIQ ) is the voltage (V) applied to the working electrode. PRE At a higher voltage than ), each tag is pushed out of the nanopore barrel. When switch 401 is opened, the voltage signal 510(V LIQ The voltage level of ) is V PRE Because it is higher, the voltage at the measurement node rises. After the voltage data point is acquired (for example, after a specified period), switch 401 may be closed, the voltage at the measurement node drops, V PRE Returning to the previous step, this process of measuring multiple voltage data points can be repeated. Thus, multiple data points, including the first point delta 532 and the subsequent data point 534, can be acquired during the dark period. As described above, during the dark period, any nucleotide tags are extruded from the nanopores, and thus minimal information about any nucleotide tag is acquired, in addition to its use in normalization.

[0102] Figure 5 also shows the voltage signal 510(V) applied to the counter electrode during the light period 540. LIQ ) is the voltage (V) applied to the working electrode. PRE This also shows that even at lower values, no insertion event occurs (open channel). Therefore, the resistance of the nanopores is low and the discharge rate of the integrating capacitor is high. As a result, the first data point 542 and the subsequent data point 5 The acquired data points, including 44, indicate low voltage levels.

[0103] The voltage measured during the light or dark phase may be expected to be approximately the same for each measurement of the constant resistance of the nanopore (for example, performed during the light mode of a given AC cycle while one tag is inside the nanopore). However, this is because the charge (C) in the double-layer capacitor 424 is different. 二重層This may not be the case when the charges are stacked. This stacked charge can lengthen the time constant of the nanopore cell. As a result, the voltage level may be shifted, and therefore, the measured value for each data point in a given cycle will be smaller. Thus, as shown in Figure 5, within a cycle, the data points may shift somewhat from one data point to another.

[0104] Further details regarding the measurement can be found, for example, in U.S. Patent Publication No. 2016 / 0178577, titled "Nanopore-Based Sequencing With Varying Voltage Stimulus". U.S. Patent Publication No. 2016 / 0178554, entitled "Varying Voltage Stimulus," U.S. Patent Application No. 15 / 085,700, entitled "Non-Destructive Bilayer Monitoring Using Measurement Of Bilayer Response To Electrical Stimulus," and U.S. Patent Application No. 15 / 085,713, entitled "Electrical Enhancement Of Bilayer Formation." The entirety of these disclosures is incorporated herein by reference for all purposes.

[0105] 4. Normalization and base calling For each of the usable nanopore cells of the nanopore sensor chip, a production mode for nucleic acid sequencing can be performed. The ADC output data acquired during sequencing can be normalized to provide higher accuracy. Normalization consists of offset effects such as cycle shape, gain drift, charge injection offset, and baseline shift. In some implementations, the signal values ​​of the light-period cycle corresponding to insertion events may be flattened so that a single signal value (e.g., mean) is acquired for each cycle. Alternatively, adjustments may be made to the measured signal to reduce intra-cycle decay (one type of cycle shape effect). Gain drift generally spreads across the entire signal and changes on the order of seconds, from 100s to 1,000s. For example, gain drift can be triggered by changes in solution (pore resistance) or changes in bilayer capacitance. Baseline shift occurs with a timescale of up to 100ms and relates to the voltage offset at the working electrode. Baseline shifts can be driven by changes in the effective rectification ratio from insertion, resulting from the need to maintain charge equilibrium in the sequencing cell from light to dark.

[0106] After normalization, each embodiment can determine a cluster of voltages for an 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. In another example, the clusters can be used to determine a cutoff voltage for distinguishing different nucleotides (bases) from each other.

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

[0108] Figure 6 shows an embodiment of circuit diagram 600 for a nanopore sensor cell. Here, some of the various voltages and components of the sensor cell that may be related to the system and method described herein are highlighted. Examples of these include: the voltage (V) applied between the working electrode and the counter electrode. app )602, voltage across two layers (V bly )604, working electrode (C 二重層 )608 and integrating capacitor (N CAP )610 The voltage used for pre-charging (V pre )606, and the voltage applied to the counter electrode (V liq )612.

[0109] Described herein are methods and systems that utilize the property of inserting protein pores and controlling single pore insertion without active feedback during the insertion step. In some embodiments of this 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 may be applied across an entire array of cells, regardless of the current state of pore insertion. In some embodiments, the voltage may be applied to cells with membranes. The applied voltage waveform may be progressively increased as a slope, as multiple expansion steps, or as other shapes designed to reduce the probability of additional protein pore insertion while also reducing the risk of membrane damage. This can be achieved by limiting the voltage application transients by using small voltage steps, a gradual rate of voltage rise in the voltage slope, etc.

[0110] For example, in some embodiments, as shown in Figure 7, the pore insertion voltage (V appThe voltage can be applied as a stepped voltage waveform 700, starting at 0mV and increasing by 100mV every 5 seconds up to a maximum voltage of 600mV. In some embodiments, the initial voltage may be about 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100mV. 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 300mV. In some embodiments, the duration of each step may be approximately 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 step may have a variable duration. For example, in some embodiments, some or all of the steps at lower voltages may have a longer duration than those of the steps at higher voltages. In some embodiments, the maximum voltage is approximately 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 step, and / or the maximum voltage.

[0111] In some embodiments, as shown in Figure 8A, the pore insertion voltage may be applied as a sloped voltage waveform 800, starting at 0mV and rising at a rate of 1V per minute to a maximum voltage of 600mV. In some embodiments, the initial voltage may be approximately 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100mV. In some embodiments, the rate of voltage rise may be approximately 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. These are 0.8, 1.9, and 2.0V. In some embodiments, the maximum voltage is approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000mV. 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 elements of the pore insertion voltage waveform may be determined based on measured electrical and / or physical properties of the cell components, such as membrane sealing resistance, which is the resistance across the membrane after the membrane has formed a seal across the cell. In some embodiments, these measurements may be acquired before the voltage waveform is applied, and the waveform is determined before application. This differs from active feedback-based methods that use measurements acquired during stimulation to modify one or more stimulation parameters. Because the electroporation methods described herein are self-limiting, there is no need to utilize active electroporation methods that involve measuring changes in the electrical or physical properties of the system, or components of the system, resulting from the insertion of pores into the membrane, and subsequently adjusting the electroporation voltage accordingly to prevent the insertion of a second pore into the membrane.

[0113] In some embodiments, the method described herein may be applied to an array of sensors having a capacitive electrode at the base of a microwell with a suspension membrane, and a counter electrode on the opposite side of the membrane. These sensors may be used to detect the presence of pores after the insertion drive voltage has been removed from all cells. While it is possible to detect the presence of pores while the voltage is applied, this method does not require it. Pores can be inserted without feedback to the application of voltage on any individual sensor in the array or in the assembly.

[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 between an array from one device and another from a second device. In addition, the electroporation voltage 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 across a narrow to wide range, a single voltage waveform can be made robust enough to function effectively across a large number of different types of pore arrays, or across the same type of pore array with a certain degree of variability.

[0115] In addition, sweeping from low to high voltages increases the likelihood that pores will be inserted into the membrane before the bilayer reaches a critical voltage level that damages the membrane. Furthermore, as shown in Figures 9A and 9B, once pores are inserted, they can dissipate the increased voltage across the membrane, thus reducing both the risk of membrane damage and the likelihood of further pore insertion when the voltage increases further after pore insertion. As long as the magnitude of the voltage step or the rate of increase in the voltage gradient is not too large, the pores can effectively dissipate the excess voltage increase across the membrane, thus reducing the risk of membrane damage and the likelihood of further pore insertion. On the other hand, it is desirable to increase the magnitude of the voltage step or the rate of increase in the voltage gradient in order to shorten the time required to complete the electroporation step.

[0116] In some embodiments, the upper limit of the voltage waveform can be determined by comparing the dynamics and / or probability of pore insertion, depending on the voltage and time, with the dynamics and / or probability of membrane damage, also depending on the voltage and time. For example, Figure 10A shows the array depending on the voltage. Figure 10B shows a plot of the number of pore insertions. Figure 10B typically shows a plot of the number of deactivations / shorts resulting from film breakdown and damage, depending on the voltage. From these two plots, a balanced, optimal maximum voltage with a high number of pore insertions and a low number of deactivations / shorts can be determined.

[0117] In some embodiments, the concentration of pores in the solution during the pore insertion step is selected to be sufficiently low to reduce passive insertion of pores into the membrane, while still sufficiently high to allow voltage-assisted insertion of pores into the membrane. Passive insertion of pores refers to insertion of pores into the membrane without the application of voltage across the membrane for assistance in pore insertion. In some embodiments, the percentage of pores inserted through passive insertion is less than 50%, 40%, 30%, 20%, or 10%. The percentage of pores inserted through voltage-assisted insertion is at least 50%, 60%, 70%, 80%, or 90%. Reducing the rate of passive pore insertion can reduce the likelihood of multiple pores being inserted into a single membrane.

[0118] In some embodiments, leakage current can cause a voltage increase in one or more cells in the array after the film has been placed on the cell. This confined charge can vary in magnitude from cell to cell over time. This makes it difficult to apply a uniform voltage across the entire film of the cell when inducing electroporation. For example, when confined charges are present in varying amounts in the cells of the array, it is difficult to apply a uniform voltage (V app Applying the voltage to all cells means that during the electroporation step, the cells may receive different amounts of effective voltage. This can lead to a high level of variability in the number of cells with single-pore insertions, and / or, in some cells, an excessive amount of voltage may be applied. This can cause damage to the membrane. Using a stepped or sloped voltage waveform can solve these problems.

[0119] In some embodiments, the formation of a membrane over a cell opening is achieved by flowing a solvent and membrane material, such as a lipid or block copolymer, over the cell opening. Subsequently, for example, if a lipid is used, the membrane can be thinned to a bilayer by applying a voltage across the membrane, as further described in U.S. Patent Publication No. 20170283867A1, and / or by manipulating an imbalance of osmolal concentrations across the membrane, as further described in International Patent Publication No. WO2018001925. The entirety of each of these is incorporated herein by reference for all purposes. As described below, a thinned membrane is one that is thinned sufficiently (i.e., for example, its thickness is less than the length of the pore) so that pores can be inserted into the membrane. A non-thinned membrane, on the other hand, is one that is too thick to allow pore insertion (i.e., its thickness is greater than the length of the pore). In some embodiments, the formation of a thinned membrane (i.e., a lipid bilayer) on the cells in the array can be completed before initiating the electroporation process and before inserting pores into the membrane. In other embodiments, the membrane thinning process can be combined with the pore insertion process by using the same voltage waveform, for example, one of the voltage waveforms described herein, for both the thinning and electroporation processes. The pore complex can be flowed onto the membrane between the combined thinning and electroporation processes. In some embodiments, the combined thinning and electroporation process can be applied after the membrane material has already been distributed onto the cells and an unthinned membrane has been formed across the cells in the array by applying a voltage during the distribution of the membrane material. The formation of the initial unthinned membrane can lead to non-uniform charge confinement. In addition, non-uniform osmotic pressure can be achieved across the membrane during the combined thinning and electroporation process. Combining the thinning step with the electroporation step can substantially reduce the time required to prepare the array of pore sensors. This thus improves the throughput of the sensor array system.

[0120] The method described herein offers several advantages, including improving the rate of good single-pore insertions, reducing the rate of multiple-pore insertions, and lowering the likelihood of membrane damage.

[0121] IV. AC Modulation of Voltage Waveforms In some embodiments, as shown in Figure 7, the pore insertion waveform 700 may be a voltage waveform with AC modulation. As shown here, the pore insertion waveform 702 is stepwise. The AC modulation component 702 may be superimposed on top of the voltage waveform 700 to provide rapid voltage fluctuations at each of the stepwise voltages. The voltage fluctuations or changes allow electrical measurements to be taken during the electroporation step, while the pore insertion waveform 700 is applied. These electrical measurements may be used to check the integrity of the film (i.e., to detect film defects such as short circuits), to check the film's leakability (i.e., film resistance and / or conductance), and to check pore insertion, and may generally be used 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 a voltage waveform enables simultaneous application of the voltage waveform and acquisition of measurement values.

[0122] In some embodiments, an AC modulation component is also superimposed on other voltage waveforms. For example, a film-forming waveform may be AC-modulated so that electrical measurements can be obtained while the film-forming waveform is applied during the step of forming a film on a well. These electrical measurements may be used to check whether the well is covered with the film-forming material (i.e., to check for short circuits), to check the integrity of the film (i.e., to detect film defects such as short circuits), to check the leakability of the film (i.e., the resistance and / or conductance of the film), to check whether the film is of a suitable thickness for pore insertion, and generally to monitor the progress of the film-forming step.

[0123] In some embodiments, another voltage waveform that can be AC ​​modulated is a translocation voltage waveform that can be used to translocate molecules through pores.

[0124] In some embodiments, the amplitude of the AC modulation component may be minimized to reduce its influence on the primary function of the voltage waveform (i.e., film formation or pore insertion) while still allowing for accurate measurements. A relatively large amplitude AC modulation component may result in higher-than-expected transient voltages that significantly affect the film during the film formation and / or pore insertion steps. This can, for example, cause film defects or multiporation. Therefore, 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 modulated voltage waveform. In other embodiments, the amplitude of the AC modulation component may broaden along with the amplitude of the modulated voltage waveform.

[0125] In some embodiments, the frequency of the AC modulation component can be minimized while still allowing for accurate measurement. In some embodiments, the frequency of AC modulation may be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1 times the maximum sampling 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, or 600. The frequencies are 700, 800, 900, or 1000 Hz. In some embodiments, the frequencies are 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, or less than 10 Hz. In some embodiments, the frequencies are 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 electroporation waveform shown in Figure 8A, to which an AC modulation component 802 is added for measurement. In this embodiment, the amplitude of the AC modulation component is 100mV, 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 can be taken while that voltage waveform is being applied.

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

[0129] The subsystems shown in Figure 11 are interconnected via the system bus 1180. Additional subsystems are shown, including a printer 1174, a keyboard 1178, a storage device (one or more) 1179, and a monitor 1176 connected to a display adapter 1182. Peripheral devices and input / output (I / O) devices connected to the I / O controller 1171 may be connected to the computer system by any of the many means known to those skilled in the art, such as I / O ports 1177 (e.g., Universal Serial Bus (USB), FireWire®). For example, I / O port 1177 or 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 the system bus 1180 allows the central processor 1173 to communicate with each subsystem and control the execution of multiple instructions from the system memory 1172 or storage device(s) 1179 (e.g., a fixed disk such as a hard drive, or an optical disk), as well as the exchange of information between subsystems. The system memory 1172 and / or storage device(s) 1179 can embody computer-readable media. Another subsystem is a data acquisition 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 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 via removable storage devices that can be connected to and detached from one component to another. In some embodiments, the computer system, subsystems, or devices communicate over a network. In such examples, 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. Each of the two components, the system and the server, may include multiple systems, subsystems, or components.

[0131] Various embodiments of the invention may be implemented in the form of control logic using computer software, using hardware circuits (e.g., APSICs or FPGAs) and / or, generally, modular or integrated programmable processors. As used herein, the 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 on dedicated hardware. Based on the disclosures and teachings provided herein, those skilled in the art will know and understand other forms and / or methods of implementing each embodiment of the 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 preferred computer language, such as Java®, C, C++, C#, Objective C, or Swift, or using a scripting language, such as Perl or Python, that uses prior art 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. Preferred non-temporary computer-readable media may include random access memory (RAM), read-only memory (ROM), magnetic media such as hard drives or floppy disks, optical media such as compact disks (CDs) or digital versatile disks (DVDs), flash memory, etc. The computer-readable medium may be any combination of such storage or transmission devices.

[0133] Such programs may also be transmitted using carrier signals that are encoded and adapted for transmission over wired, optical, and / or wireless networks conforming to various protocols, including the Internet. Therefore, computer-readable media may be created using data signals encoded with such programs. Computer-readable media encoded with program code may be packaged using compatible devices or provided separately from other devices (e.g., downloaded via the Internet). Any such computer-readable media 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 the user with any of the results described herein.

[0134] Any of the methods described herein may be carried out entirely or in part using a computer system comprising one or more processors that can be configured to perform the steps described herein. Thus, each embodiment may be directed to a computer system configured to perform any of the steps of the methods described herein using different components that potentially perform each of the steps or each of the groups of steps. The steps of the methods herein are presented as sequential steps, but may be performed simultaneously, at different times, or in different orders. In addition, each of the parts of these steps may be used in conjunction with each of the parts of other steps from other methods. Furthermore, all of a step, or each of its parts, may be optional. In addition, any of the steps of these methods may be carried out using a module, unit, circuit, or other means of a system for performing these steps.

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

[0136] While the earlier embodiments have been described in some detail for the purpose of clarifying understanding, the present invention is not limited to those details provided. There are many alternative ways to carry out the present invention. The disclosed embodiments are for illustrative purposes only and are not limiting. The above description of exemplary embodiments of the present invention is presented for illustrative and explanatory purposes only. They are not intended to be comprehensive or to limit the present invention to the exact form described. Based on the above teachings, many modifications and variations are possible.

[0137] When we say that one feature or element is "on" another feature or element, this could mean that it is directly on that other feature or element, or that there may be features and / or elements intervening between them. In contrast, when we say that one feature or element is "directly on" another feature or element, this means that there are no features or elements intervening between them. When we say that one feature or element is "connected," "attached," or "coupled" to another feature or element, this can also mean that it is directly connected, attached, or coupled to that other feature or element, or that there may be features or elements intervening between them. In contrast, when we say that one feature or element is "directly connected," "directly attached," or "directly coupled" to another feature or element, this means that there are no features or elements intervening between them. Although described or shown in relation to one embodiment, the features and elements described or shown can be applied to other embodiments. When referring to a structure or feature positioned "adjacent to" another feature, it will also be understood by those skilled in the art that there may be parts that overlap with or lie beneath that adjacent feature.

[0138] The terminology used herein is intended solely to describe specific embodiments and is not intended to limit 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 otherwise explicitly indicates otherwise. Where the terms “comprises” and / or “comprising” are used herein, it will be further understood that they indicate the presence of the features, steps, operations, elements, and / or components described 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 / or” include any and all combinations of one or more related, enumerated items. These may be abbreviated as “ / ”.

[0139] Spatially relative terms such as "under," "below," "lower," "over," and "upper" are used, and to facilitate explanation, the relationship between one element or feature and another element(s) or feature(s)(s)(s) may be described here, as shown in the drawings. It will be understood that these spatially relative terms are intended to include different orientations of the device in use or operation, in addition to the orientation shown in the drawings. For example, the top and bottom of the device in the drawing are In the reverse case, an element described as “under” or “beneath” another element or feature becomes “over” that other element or feature. Therefore, the exemplary term “under” may include both “over” and “under.” Devices may be oriented in other directions (rotated 90 degrees or otherwise) and may be interpreted accordingly by the spatially relative descriptors used herein. Similarly, terms such as “upwardly,” “downwardly,” “vertical,” and “horizontal” are used herein for descriptive purposes only, unless otherwise specifically indicated.

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

[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,” means that various components may be adopted together in this Method and each of its provisions (e.g., configurations and apparatus including devices and methods). For example, the term “comprising” is understood to imply that it includes any element or step described herein, but does not imply that it excludes any other element or step.

[0142] In this specification and in the claims, as used herein, all numerical values ​​may be read as if preceded by the words “about” or “approximately,” even if not explicitly stated, including as used in each example. The phrase “about” or “approximately” may be used when describing a magnitude and / or location to indicate that the value and / or location described is within the reasonably expected range of the value and / or location. For example, a numerical value may have values ​​such as + / -0.1% of the value (or range of value), + / -1% of the value (or range of value), + / -2% of the value (or range of value), + / -5% of the value (or range of value), + / -10% of the value (or range of value), and so on. Any numerical value given herein should also be understood to include its approximate or round value unless the context otherwise indicates otherwise. For example, when the value "10" is disclosed, it is also disclosed as "about 10". Any numerical range listed herein is intended to include all partial ranges belonging to them. When a value is disclosed as "less than or equal to" its value, it should also be understood, as will be appropriately understood by those skilled in the art, that "greater than or equal to" and the possible range between values ​​are also disclosed. For example, when the value "X" is disclosed, it is understood to include "less than or equal to" X, and similarly, "greater than or equal to" X. It is also disclosed as "equal to" (for example, X is a number). Throughout this application, it is understood that data is provided in many different formats, and this data represents a range for any combination of end points, start points, and data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that numbers greater than 10 and 15, greater than or equal to, less than, less than or equal to, and equal to these are considered to be disclosed, as are the numbers between 10 and 15. It is understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0143] While various illustrative embodiments have been described above, many of these embodiments may be modified without departing from the scope of the invention as defined 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 together. Any features of the various device and system embodiments may be included in some embodiments and not in others. Therefore, the above description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as defined in the claims.

[0144] The examples and figures included herein are not limiting, but illustrate specific embodiments from which the subject matter may be put into practice. As stated above, other embodiments may be used and derived from them, so as to be structural and logical substitutions and modifications may be made without departing from the scope of this disclosure. If more than one such embodiment of the subject matter of the present invention is actually disclosed, they may be referred to herein by the term “invention” individually or collectively, for convenience only, without any intention of voluntarily limiting the scope of this application to any single invention or concept relating to the invention. Thus, while specific embodiments are illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown herein. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments and other embodiments not specifically described herein will become apparent to those skilled in the art by considering the foregoing description.

[0145] All patents, patent applications, publications, and descriptions described herein are incorporated herein by reference in their entirety for all purposes. There is no prior art to speak of. The claims as they were at the time of filing are shown below. [Claim 1] A method for forming an array of nanopore sensor cells, The method involves introducing nanopores as close to the cell as possible, wherein the cell comprises a working electrode and a membrane sealing the cell, and the working electrode is powered by an electrically connected power source. Applying a voltage waveform across the film of the cell, wherein the voltage waveform starts at a first voltage and increases over a period of time up to a second voltage, and applying the voltage waveform across the film of the cell. During the step of applying the voltage waveform, the nanopores are inserted into the film, A method that includes this. [Claim 2] The method according to claim 1, wherein the first voltage is between approximately 0 and 100 mV, and the second voltage is between approximately 100 and 2000 mV. [Claim 3] The method according to claim 1, wherein the working electrode is a capacitive electrode. [Claim 4] The method according to any one of claims 1 to 3, wherein the voltage waveform includes a plurality of gradual steps between the first voltage and the second voltage. [Claim 5] The method according to claim 4, wherein the plurality of gradual steps advance by approximately 1 to 100 mV. [Claim 6] The method according to claim 4, wherein the plurality of gradual steps advance by approximately 1 to 25 mV. [Claim 7] The method according to any one of claims 4 to 6, wherein each incremental step has a duration between approximately 0.1 and 60 seconds. [Claim 8] The method according to claim 7, wherein the duration of the gradual steps is variable. [Claim 9] The method according to claim 7, wherein the duration of the gradual step at a lower voltage is longer than the duration of the gradual step at a higher voltage. [Claim 10] The method according to claim 7, wherein the duration of the gradual steps is constant. [Claim 11] The method according to any one of claims 1 to 3, wherein the voltage waveform includes a slope between the first voltage and the second voltage. [Claim 12] The method according to claim 11, wherein the slope is between approximately 0.1 and 2.0 V per minute. [Claim 13] The method according to claim 12, wherein the incline has a constant slope. [Claim 14] The method according to claim 12, wherein the incline has a variable slope. [Claim 15] The method according to claim 12, wherein the slope has a slope at lower voltages that is smaller than the slope at higher voltages. [Claim 16] The method according to any one of claims 1 to 3, wherein the application of the voltage waveform is applied to a film that has not been thinned. [Claim 17] The method according to claim 16, further comprising thinning the film that has not been thinned using the applied voltage waveform. [Claim 18] The method according to claim 1, wherein the power supply is AC-connected to the working electrode. [Claim 19] The method according to claim 1, wherein the voltage waveform includes an AC modulation component, and the AC modulation component is configured such that an electrical measurement can be obtained through the working electrode while the voltage waveform is applied across the film of the cell. [Claim 20] The method according to claim 19, wherein the AC modulation component has an amplitude of less than 100 mV. [Claim 21] The method according to claim 19, wherein the AC modulation component has a frequency between 10 Hz and 1000 Hz. [Claim 22] A system for determining the sequence of molecules, An array of cells on a substrate, each of which has a working electrode and an opening configured to be sealed by a film, wherein the working electrode is powered by an AC-connected power supply, Counter electrode and, A power supply, which is electrically connected to each of the working electrodes, It is a controller, A controller programmed to supply a voltage waveform to the cell, using the working electrode and the counter electrode, which starts at a first voltage and increases over a period of time up to a second voltage. A system that includes this. [Claim 23] The system according to claim 22, wherein the working electrode is a capacitive electrode. [Claim 24] The system according to claim 22, wherein the voltage waveform includes a plurality of gradual steps between the first voltage and the second voltage. [Claim 25] The system according to claim 22, wherein the voltage waveform includes a slope between the first voltage and the second voltage. [Claim 26] The system according to claim 22, wherein the controller is further programmed to supply the voltage waveform to a film that has not been thinned. [Claim 27] The system according to claim 22, wherein the power supply is AC-connected to each of the working electrodes. [Claim 28] A method for forming cells covered with a membrane, The process involves flowing a film-forming material onto a cell, wherein the cell has a working electrode, and the working electrode is powered by an electrically connected power source. Placing a layer of film-forming material on the cell, Applying a voltage waveform across the layers of the film-forming material using the working electrode and a counter electrode on the opposite side of the layers of the film-forming material, wherein the voltage waveform includes an AC modulation component, and the AC modulation component is configured such that an electrical measurement can be obtained through the working electrode while the voltage waveform is being applied across the layers of the film-forming material, The process involves thinning the layer of the film-forming material into a film, wherein the film is configured to accept nanopores, and the process involves thinning the layer of the film-forming material into a film. A method that includes this. [Claim 29] The method according to claim 28, wherein the AC modulation component has an amplitude of less than 100 mV. [Claim 30] The method according to claim 28, wherein the AC modulation component has a frequency between 10 Hz and 1000 Hz.

Claims

1. A method for forming an array of nanopore sensor chips, A step of introducing nanopores as close as possible to each cell in a cell array, wherein each cell has a working electrode and a membrane sealing the cell, and each working electrode is configured to be powered by a common power source, A step of applying a predetermined voltage waveform across the membrane of each cell, wherein the voltage waveform starts at a first voltage and increases over a period of time to a second predetermined voltage, and the same predetermined voltage waveform is applied to each cell, and During the step of applying the predetermined voltage waveform, the step of inserting the nanopores into the film, Includes, The voltage waveform includes a plurality of gradual steps between the first voltage and the second voltage. The voltage waveform includes an AC modulation component, and the AC modulation component is configured such that an electrical measurement can be obtained through the working electrode while the voltage waveform is applied across the cell membrane. The aforementioned method.

2. The method according to claim 1, wherein the AC modulation component has an amplitude of less than 100 mV.

3. The method according to claim 1, wherein the AC modulation component has a frequency between 10 Hz and 1000 Hz.

4. The method according to claim 1, wherein the electrical measurement is used to detect the insertion of nanopores.

5. The method according to claim 1, wherein the insertion of nanopores is not detected while a predetermined voltage waveform is applied.

6. The method according to claim 5, wherein after applying a predetermined voltage waveform, it is detected whether nanopores have been inserted into the membrane of each cell.

7. The method according to claim 1, wherein the nanopores are present in a solution of a selected concentration such that voltage-assisted nanopore insertion accounts for at least 50% of the nanopores inserted into the membrane in the cell array, and passive insertion accounts for less than 50% of the nanopores inserted into the membrane in the cell array.

8. The method according to claim 1, wherein the nanopores are present in a solution of a selected concentration such that voltage-assisted nanopore insertion accounts for at least 70% of the nanopores inserted into the membrane in the cell array, and passive insertion accounts for less than 30% of the nanopores inserted into the membrane in the cell array.

9. The method according to claim 1, wherein the nanopores are present in a solution of a selected concentration such that voltage-assisted nanopore insertion accounts for at least 90% of the nanopores inserted into the membrane in the cell array, and passive insertion accounts for less than 10% of the nanopores inserted into the membrane in the cell array.

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