Compositions and methods for reducing Prussian blue formation during nanopore sequencing

Abstract

The present application discloses electrochemical cells, nanopore devices, and related buffer compositions useful for nanopore-based nucleic acid sequencing. Methods for using the electrochemical cells, devices, and compositions in nanopore-based nucleic acid sequencing methods, such as nanopore sequencing-by-expansion (Nano-SBX) and nanopore sequencing-by-synthesis (Nano-SBS) methods, are also disclosed.

Description

[Technical Field]

[0001] field This application relates to an electrochemical cell and associated buffer composition that reduces the harmful formation of Prussian blue in nanopore-based assays, as well as a method for using the composition in nanopore-based nucleic acid detection techniques such as nanopore sequencing. [Background technology]

[0002] background Nanopore-based nucleic acid sequencing is a widely studied and compelling approach. In one approach, single-stranded polynucleotide sequences are detected by changes in ionic current as the polynucleotides move through nanopores embedded in a lipid bilayer that separates the two sides of an electrochemical cell. During the movement of the polynucleotides, partial occlusion of the nanopore opening alters the flow of ions over time, resulting in changes in current that can be measured by the cell.

[0003] Sequencing by Expansion ("SBX") is a nanopore-based nucleic acid sequencing method that uses a biochemical process to transfer the DNA sequence to a measurable polymer molecule called "Xpandomer." See, for example, U.S. Patent No. 7,939,259 entitled "High Throughput Nucleic Acid Sequencing by Expansion" and PCT International Publication No. 2020236526 entitled "Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing." In the SBX process, the target nucleic acid sequence is encoded along a mainchain Xpandomer sequence having a reporter construct approximately 10 nm apart, designed to provide a highly signal-to-noise, well-distinguished response signal during nanopore transfer. The enhanced signal-to-noise provided by the distinct response signals provides a significant increase in the sequence reading efficiency and accuracy of the Xpandomer compared to the native nucleic acid molecule.

[0004] Nanopore-based sequencing-by-synthesis ("SBS") uses a polymerase (or other chain elongation enzyme) covalently bound to a nanopore to synthesize a DNA strand (i.e., a copy strand) complementary to a target sequence template. Nanopores embedded in a membrane within an electrochemical cell are used to simultaneously detect the identity of each nucleotide monomer as it is added to its growth strand. See, for example, U.S. Patent Application Publications 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0368710, and 2018 / 0057870, and International Publication 2019 / 166457. Each added nucleotide monomer is detected by monitoring the signal resulting from changes in ion flow through the nanopore as the tag portion bound to each added nucleotide monomer enters the nanopore and alters the ion flow. For optimal performance, the tag portion must be present in the nanopore for a sufficient time to provide a detectable, identifiable, and reproducible signal related to changes in ion flow through the nanopore, allowing the specific nucleotide associated with the tag to be clearly distinguished from other tagged nucleotides in the SBS solution (relative to baseline "open current" flow).

[0005] However, nanopore-based sequencing is burdensome because it must resolve small current-signal differences immersed in significant background noise within microvolume electrochemical cells. This measurement challenge is complicated by small changes in materials and parameters affecting the electrochemical cell, including but not limited to electrode materials, electrochemical cell materials, electrochemical solution buffer salts, and redox-active reagents, as well as pH, voltage, temperature, and viscosity.

[0006] Therefore, there is still a need for electrochemical cell designs and materials, buffer and redox reagent compositions, and methods for using them, in order to reduce or prevent the formation of harmful byproducts (e.g., Prussian blue precipitate) that interfere with accurate electrochemical signal detection in nanopore-based nucleic acid sequencing devices, systems, and methods. [Overview of the project]

[0007] overview This disclosure generally relates to electrochemical cells and associated buffer compositions used in cells that reduce the harmful formation of Prussian blue or related precipitates during nanopore-based assays, as well as methods for using the cells and compositions for nanopore-based nucleic acid detection techniques such as nanopore sequencing. This summary is intended to introduce the subject matter of this disclosure but does not exhaust all embodiments, combinations, or variations contemplated and described herein. Further embodiments are contemplated and described by the detailed description, drawings, and claims disclosure.

[0008] In at least one embodiment, the present disclosure provides an electrochemical cell comprising (a) a nanopore embedded in a membrane, the membrane separating the cell into cis and trans chambers operably connected by the nanopore, the cis and trans chambers each comprising an electrode, a solution containing ferrocyanide ions, ferricyanide ions, and a buffer composition, and (b) an inlet port and an outlet port operably connected to the cell, the inlet port and the outlet port comprising a metal.

[0009] In at least one embodiment of the electrochemical cell of this disclosure, the metal is polarized to a potential of approximately 0.3V relative to an Ag / AgCl reference electrode in a buffer solution of 1M NH4Cl, 800mM urea, 100mM HEPES, pH 7.4, or in a buffer solution of 1M NH4Cl, 800mM urea, 100mM MES at pH 6.2-6.8. -3 mA / cm 2The following current densities are observed. In at least one embodiment, the metal contains less than 10% iron, less than 8% iron, less than 6% iron, or less than 5% iron. In at least one embodiment, the metal is a nickel alloy. In at least one embodiment, the metal is a nickel alloy selected from C276, C22, 625, and MP35N.

[0010] In at least one embodiment of the electrochemical cell of the present disclosure, the cell contains a solution volume between about 0.1 femtoliters and 1000 femtoliters.

[0011] In at least one embodiment of the electrochemical cell of the present disclosure, the total concentrations of ferrocyanide ions and ferricyanide ions are between approximately 25 mM and 250 mM.

[0012] In at least one embodiment of the electrochemical cell of this disclosure, the buffer composition contains ammonium acetate at a concentration of about 0 mM to about 1500 mM. In at least one embodiment, the buffer composition contains ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the buffer composition contains ammonium acetate at a concentration of about 0 mM to about 1500 mM and ammonium chloride at a concentration of less than about 2000 mM.

[0013] In at least one embodiment of the electrochemical cell of the present disclosure, by applying alternating current (AC) in the voltage range of 450mV to 1200mV to the cell, no visible formation of Prussian blue occurs within the cell for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, or at least 10 hours.

[0014] In at least one embodiment, the present disclosure provides a method for sequencing a target nucleic acid, the method is (a) An electrochemical cell comprising: (i) a nanopore embedded in a membrane, the membrane separating the cell into a cis and a trans chamber operably connected by the nanopore, the cis and trans chambers each containing an electrode, a solution containing ferrocyanide ions and ferricyanide ions, and a buffer composition, and the nanopore; and (ii) an inlet port and an outlet port containing a metal operably connected to the cell. (b) Adding a molecule derived from a target nucleic acid to the cis chamber. (c) Applying a voltage to the cell to move at least a portion of the molecule through the nanopore. (d) Detecting a change in the flow of voltage within the cell as the molecule moves through the nanopore, the change in the flow of voltage indicating the sequence of the target nucleic acid. Comprising.

[0015] In at least one embodiment of the sequencing method of the present disclosure, the metal polarizes to a potential of about 0.3 V with respect to an Ag / AgCl reference electrode in a buffer solution of 1M NH4Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or in a buffer solution of 1M NH4Cl, 800 mM urea, 100 mM MES at pH 6.2 - 6.8 and exhibits a current density of 10 -3 mA / cm 2 or less. In at least one embodiment, the metal contains less than 10% iron, less than 8% iron, less than 6% iron, or less than 5% iron. In at least one embodiment, the metal is a nickel alloy. In at least one embodiment, the metal is a nickel alloy selected from C276, C22, 625, and MP35N.

[0016] In at least one embodiment of the sequencing of the present disclosure, the cell confines a solution volume between about 0.1 femtoliters and 1000 femtoliters.

[0017] In at least one embodiment of the sequencing method of the present disclosure, the total concentration of ferrocyanide ions and ferricyanide ions is between about 25 mM and 250 mM.

[0018] In at least one embodiment of the sequencing method of the present disclosure, the buffer composition contains ammonium acetate at a concentration of about 0 mM to about 1500 mM. In at least one embodiment, the buffer composition contains ammonium chloride at a concentration less than 2000 mM. In at least one embodiment, the buffer composition contains ammonium acetate at a concentration of about 0 mM to about 1500 mM and ammonium chloride at a concentration less than 2000 mM. In at least one embodiment, the concentration of ammonium acetate is 0 mM and the concentration of ammonium chloride is about 600 mM.

[0019] In at least one embodiment of the sequencing method of the present disclosure, by applying an alternating current (AC) to the cell within a voltage range of 450 mV to 1200 mV, visible formation of Prussian blue does not occur within the cell for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, or at least 10 hours.

[0020] In at least one embodiment of the sequencing method of the present disclosure, the applied voltage includes a baseline voltage, and optionally, the baseline voltage is about 55 mV to about 95 mV. In at least one embodiment, the applied voltage includes a pulse voltage, and optionally, the pulse voltage is about 320 mV to about 550 mV. In at least one embodiment, the pulse voltage has a duration of about 5 μs to about 10 μs. In at least one embodiment, the time between pulse voltages is about 0.5 ms to about 1.7 ms. In at least one embodiment, the applied voltage provides an alternating current, and optionally, the alternating current has a periodicity of about 0.4 seconds to about 6 seconds.

[0021] In at least one embodiment of the sequencing method of the present disclosure, the molecule derived from the target nucleic acid includes an Xpandomer. In at least one embodiment, the Xpandomer includes a plurality of XNTP subunits coupled with sequences corresponding to all or part of the continuous nucleotide sequence of the target nucleic acid, wherein each XNTP subunit of the chain includes a reporter construct, nucleic acid base residues and selectively cleavable bonds, and cleavage of the selectively cleavable bonds results in an Xpandomer longer than the plurality of XNTP subunits of the chain.

[0022] In at least one embodiment of the sequencing method of the present disclosure, the method further comprises selectively cleaving cleavable bonds to obtain an Xpandomer.

[0023] In at least one embodiment of the sequencing method of the present disclosure, the Xpandomer includes a reporter construct for analyzing genetic information in a sequence corresponding to all or part of a sequence of nucleotides of a target nucleic acid. In at least one embodiment, detecting a change in voltage includes detecting a change in voltage caused by the reporter construct of the Xpandomer moving a nanopore.

[0024] In at least one embodiment of the sequencing method of the present disclosure, a reporter construct for analyzing genetic information includes a reporter code and a migration control element, the migration control element providing sterically hindered migration control, pausing the migration of the Xpandomer as it passes through a nanopore subjected to a baseline voltage, the migration control element engaging with the reporter code within the opening of the nanopore, the reporter code being sensed by the nanopore. [Brief explanation of the drawing]

[0025] A better understanding of the novel features and advantages of this disclosure will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of this disclosure are utilized, and to the accompanying drawings (also referred to herein as "Figure" and "FIG.").

[0026] [Figure 1] This shows one embodiment of a cell 100 in a nanopore-based sequencing chip. [Figure 2] This document describes one embodiment of a cell 200 that performs nucleotide sequencing using Nano-SBS technology. [Figure 3] This shows one embodiment of a cell in which nucleotide sequencing is to be performed using a preloaded tag. [Figure 4] This document illustrates one embodiment of process 400 for nucleic acid sequencing using preloaded tags. [Figure 5] This shows one embodiment of the circuit 500 within a nanopore-based sequencing chip cell. [Figure 6] One embodiment of the circuit 600 within the cell of a nanopore-based sequencing chip is shown, in which the voltage applied to the nanopore can be configured to vary over a period of time during which the nanopore is in a specific detectable state. [Figure 7] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 8] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 9] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 10] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 11] This is a schematic diagram showing further details of one embodiment of XNTP. [Figure 12] This is a schematic diagram showing one embodiment of Xpandomer passing through biological nanopores. [Modes for carrying out the invention]

[0027] Detailed explanation This disclosure provides an electrochemical cell and associated buffer compositions for use in cells. The electrochemical cell is useful for carrying out nanopore-based methods for assaying nucleic acids, including nanopore-based sequencing. The features of the electrochemical cell and associated buffer compositions result in a remarkable advantage: reduced formation of harmful precipitates during the use of the cell in nanopore-based assays. Harmful precipitates include Prussian blue, which can precipitate as a solid in redox reactions involving ferricyanide and ferrocyanide ions, as well as metallic iron found in components of the electrochemical cell such as inlet and outlet ports. The formation of such precipitates can rapidly clog ports and / or nanopores, thereby significantly reducing the accuracy and efficiency of any nanopore-based assay. This disclosure also provides methods for using the electrochemical cell and compositions for nanopore-based nucleic acid detection techniques such as nanopore-based sequencing-by-synthesis (Nano-SBS) and nanopore-based sequencing by expansion.

[0028] In this specification and the accompanying claims, the singular forms "a" and "an" include multiple references unless the context explicitly indicates otherwise. Thus, for example, a reference to "protein" includes multiple proteins, and a reference to "compound" refers to multiple compounds. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" is interchangeable and not intended to be limiting. Where the description of various embodiments uses the term "comprising," it should be further understood that in some particular examples the embodiment could be described alternatively using the terms "essentially consisting of" or "consisting of."

[0029] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intervening integer of the value and each tenth of each intervening integer of the value are understood to be included in the invention, unless the context explicitly indicates otherwise, between the upper and lower limits of that range, and any other listed or intervening values ​​within that stated range. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in the invention, subject to any specifically excluded limitations within the stated range. If a stated range includes one or both limits, the range excluding (i) one or (ii) both of the included limits is also included in the invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0030] In general, the nomenclature used herein and the techniques and procedures described herein are well understood and commonly used by those skilled in the art, including, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2012 (hereinafter, "Sambrook"); and the general techniques and methodologies described in Current Protocols in Molecular Biology, edited by FMAusubel et al. (hereinafter, "Ausubel"), which was first published in book form in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., with regular supplements until 2011, and is now available as an online journal, Volumes 00-130 (1987-2020), and published in the Wiley Online Library by Wiley & Sons, Inc.

[0031] All publications, patents, patent applications, and other documents referenced in this disclosure are incorporated herein by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, or other document is individually indicated as being incorporated herein by reference for all purposes.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. It should be understood that the terms used herein are intended to describe only specific embodiments and are not intended to limit them. For the purposes of interpreting this disclosure, the following definitions of terms apply, and where appropriate, singular terms are also included in their plural forms and vice versa.

[0033] Nanopore-based devices for detecting nucleic acids

[0034] Nanopore-based devices for detecting nucleic acids have been developed for rapid sequencing, and various designs and uses are known in the art. See, for example, U.S. Patents 9,494,554, 9,567,630, 9,557,294, and 9,605,309, respectively, which are incorporated herein by reference. These devices generally include an electrochemical cell having a chamber containing nanopores embedded in a membrane. The membrane acts to separate the cell chamber into two sub-chambers called the cis and trans sides of the cell, each containing electrodes. The membrane may be an organic membrane such as a lipid bilayer, or a synthetic membrane made of a polymer material that does not exist naturally. The pores of the nanopores act as channels (or passages) within the membrane between the cis and trans sides of the cell. Depending on the nanopores used, the pores may have a width or diameter ranging from about 1 angstrom to about 10,000 angstroms. Nanopores may be naturally occurring pore-forming proteins such as α-hemolysin derived from Staphylococcus aureus (S. aureus), or non-naturally occurring variants or variants of wild-type pore-forming proteins. A range of naturally occurring and non-naturally occurring nanopores with various pore sizes and properties is known in the art. See, for example, U.S. Patents 1,0351908, 1,0934582, and 1,0227645.

[0035] Within an electrochemical cell, nanopores embedded in a membrane are positioned in close proximity to electrodes coupled to a sensing circuit, such as a complementary metal-oxide-semiconductor (CMOS) or field-effect transistor (FET) circuit. When a potential is applied across the nanopores (through the electrodes) immersed in a conductive fluid, a small current can be observed due to the flow of ions through the nanopores. This ion flow is sensitive to the pore size, and therefore, molecules entering the pores affect the ion flow and voltage measured through this sensor circuit.

[0036] Electrochemical cells for nanopore-based sequencing of nucleic acids are typically used in a large-scale parallel configuration, where thousands of such cells are configured as an array within a single device, often referred to as a chip (or biochip). Typically, a nanopore-based sequencing chip device may incorporate an array of more than one million electrochemical cells, containing 1,000 rows × 1,000 columns of such cells (see, for example, a chip manufactured by Roche Sequencing Solutions, Santa Clara, California, USA). Methods for manufacturing and using such nanopore array microchips can also be found in U.S. Patent Applications Publications 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0368710, and 2018 / 0057870, as well as International Publication 2019 / 166457, which are incorporated herein by reference, respectively. Each well in the array is fabricated using a standard CMOS process with surface modifications that allow constant contact between the bioreagent and the conductive salt. Each well can support a phospholipid bilayer membrane embedded with a nanopore polymerase conjugate. The electrodes in each well are individually addressable via a computer interface. All reagents used are introduced into a simple flow cell above the array microchip using a computer-controlled syringe pump. The chip supports analog-to-digital conversion and reports electrical measurements independently from all electrodes at a rate exceeding 1000 points / second. Nanopore measurements are performed asynchronously at least once every 1 millisecond (msec) on each of the 8M addressable nanopore-containing membranes in the array and can be recorded on the interfaced computer. Further description of exemplary electrochemical cells useful for nanopore-based nucleic acid assays such as sequencing, including chambers and electrode materials, buffers, sensing circuits, array devices, and their use in various applications, is provided below.

[0037] Figure 1 shows an exemplary embodiment of an electrochemical cell 100 found in an array of such cells useful for nanopore-based sequencing. A membrane 102 is formed on the surface of the cell. In some embodiments, the membrane 102 is a lipid bilayer. A bulk electrolyte solution 114 containing nanopores or "protein nanopore transmembrane complexes" (PNTMCs) 104 is placed directly on this surface within the cell, and a single PNTMC 104 is inserted into the membrane 102 using electroporation. The individual nanopore-embedded membranes within each cell of the array are not chemically or electrically connected to one another. Thus, each cell is an independent sequencing machine, generating data specific to a single polymer molecule associated with the PNTMC.

[0038] Continuing to refer to Figure 1, the thin film of electrolyte solution 108 is isolated from the bulk electrolyte solution 114 by the ion-impermeable membrane 102. Thus, the nanopore-embedded membrane separates the electrochemical cell 100 into two chambers: a cis chamber containing the bulk electrolyte solution 114 and a trans chamber containing the thin film of electrolyte solution 108. The thin film of electrolyte solution 108 in the trans chamber is in contact with the metallic working electrode 110 connected to the analog measurement circuit 112. The bulk electrolyte solution in the cis chamber is in contact with the counter electrode 116 and the reference electrode 117.

[0039] The pores in PNTMC 104 provide channels through membrane 102 that allow analytes (e.g., nucleic acids) and ions to flow, resulting in modulation of the ionic current across the impermeable bilayer. Thus, the pores in PNTMC 104 provide the sole pathway for the ionic current to flow from the metallic working electrode 110 to the bulk electrolyte solution 114 in contact with the counter electrode 116. The electrochemical cell also includes a reference electrode 117 to further enhance its sensitivity as an electrochemical potential sensor.

[0040] A thin film of electrolyte constitutes a femtoliter volume of solution within a trans chamber that is in direct contact with the working electrode. This small amount of electrolyte in contact with the electrode of the electrochemical cell must receive repetitive voltage pulses to sense the ion flow through the nanopore. The need to maintain the ion flow through the nanopore using these repetitive electrochemical measurements with electrodes in a small amount of electrolyte, and using repetitive measurements corresponding to individual molecular parts moving through the pore, requires a highly sensitive sensor system. This electrochemical measurement system is highly sensitive to any precipitate or aggregated molecular entities that could clog the pore or otherwise interfere with the ion flow between the working and counter electrodes. Therefore, the electrolyte salt, electrochemical cell material, and electrochemical measurement conditions should be carefully controlled to prevent or reduce harmful precipitate formation or electrode wear in the system.

[0041] The electrolyte solution 108 may contain one 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 film of the electrolyte solution has a thickness of about 3 microns (μm). The thickness of the film of the electrolyte solution may be in the range of 0 to 5 microns.

[0042] In addition to the salts described above, the electrolyte membrane may contain redox-active ions such as ferrocyanide ions and ferricyanide ions. These redox ions are used in certain nanopore-based sequencing methods performed in electrochemical cells. For example, Nano-SBX measurements performed in the cells of this disclosure may contain ferricyanide and ferrocyanide ions. Such redox-active ions may undergo irreversible reactions depending on their concentration and the specific electrochemical conditions at the working and counter electrodes. Indeed, as described elsewhere in this specification, the irreversible formation of Prussian blue, a precipitate in electrochemical cells, is highly detrimental to any nanopore-based measurements in the cell. Therefore, the concentrations of redox-active ions such as ferricyanide and ferrocyanide should be carefully controlled. In at least one embodiment of this disclosure, the ferricyanide and ferrocyanide ions in the solution within the electrochemical cell should be maintained at a total concentration between approximately 25 mM and 250 mM.

[0043] It has also been found that the salt used in the electrolyte solution of an electrochemical cell can affect the ability of the cell to be used for highly sensitive nanopore-based nucleic acid measurements. For example, many nanopore-based sequencing protocols use ammonium chloride as a buffer salt in the electrolyte solution provided in the cell chamber. It has been found that controlling the concentration of ammonium chloride by substituting it with ammonium acetate as the salt in the electrolyte solution during nanopore-based nucleic acid sequencing methods such as Nano-SBX can help reduce or prevent the formation of redox precipitates such as Prussian blue, which are highly detrimental to the accuracy and sensitivity of the electrochemical cell as described above. Therefore, in some embodiments of the electrochemical cell of this disclosure, the buffer composition used in the cell contains ammonium acetate at a concentration of about 0 mM to about 1500 mM and / or ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the buffer composition contains ammonium acetate at a concentration of about 0 mM to 1500 mM and ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the concentration of ammonium acetate is 0 mM and the concentration of ammonium chloride is about 600 mM.

[0044] Dielectric materials are typically used to form an oxide layer 106 that defines the trans chamber of the cell. Useful dielectric materials include glass, oxides, and silicon mononitride (SiN). In some embodiments, the upper surface of the dielectric oxide layer 106 in contact with the bulk electrolyte 114 can be silanized. Silanization forms a hydrophobic layer on top of the dielectric layer. In some embodiments, this hydrophobic layer has a thickness of about 1.5 nanometers (nm). Alternatively, a hydrophobic dielectric material such as hafnium oxide can be used to form the upper part of the dielectric layer.

[0045] As shown in Figure 1, the membrane is a lipid bilayer at least partially formed on the dielectric oxide layer 106 and spans a well containing a thin film of electrolyte 108. For example, the membrane can be formed on a hydrophobic layer on the dielectric oxide layer, and when the membrane reaches the opening of the well, the lipid monolayer migrates into a lipid bilayer that extends across the opening of the well. The hydrophobic layer can facilitate the formation of a lipid monolayer on the dielectric layer and the migration from the lipid monolayer to a lipid bilayer.

[0046] The bulk electrolyte 114 may further comprise one of 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). Therefore, in some embodiments of the electrochemical cells of this disclosure, the buffer composition of the bulk electrolyte used in the cell comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM and / or ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the buffer composition comprises ammonium acetate at a concentration of about 0 mM to 1500 mM and ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the concentration of ammonium acetate is 0 mM and the concentration of ammonium chloride is about 600 mM. Similarly, in some embodiments, the ferricyanide and ferrocyanide ions in the bulk electrolyte solution of the electrochemical cell should be maintained at a total concentration between approximately 25 mM and 250 mM.

[0047] As mentioned above regarding electrolyte thin films, the composition of the salts used in the bulk electrolyte (e.g., ammonium chloride versus ammonium acetate) and the redox-active ions can affect the accuracy and sensitivity of nanopore-based measurements performed using electrochemical cells. Depending on the type of electrochemical measurement being performed, particularly the potential applied to the electrode in contact with the solution, it is possible to adjust the composition of the solution to reduce or prevent the formation of harmful precipitates.

[0048] To perform the necessary insertion and removal of solutions, the electrochemical cells of this disclosure include inlet and outlet ports operably connected to the cell's chamber or reservoir. Although the schematic diagram of the electrochemical cell in Figure 1 does not show the inlet and outlet ports, the incorporation of such ports into electrochemical cells is known in the art. The inlet and outlet ports allow for the insertion of reagents necessary for nanopore-based nucleic acid detection, including materials for fabricating membranes with embedded nanopores, nucleotide synthesis reagents, and electrolytes and buffers. Typically, the inlet and outlet ports include metal tubing attached to the cell to provide a fluid connection from the outside to the cell's chamber, which can be used to insert or remove solutions. Thus, at least a portion of the inlet and outlet port tubing is in contact with the electrochemical solution in the cell chamber. Therefore, the composition of the ports must be electrochemically inert at voltages used to induce ion flow through the nanopores and to perform perceptual measurements of changes in ion flow as molecular portions enter and / or move through the nanopores. Stainless steel tubing has been used to provide metal tubing for inlet and outlet ports, although steel includes iron. One of the remarkable advantages of the electrochemical cell of this disclosure is that the inlet and outlet ports contain metal with less than 10% iron, thereby reducing or preventing the harmful formation of precipitates in the cell, such as Prussian blue. Although not intended to be bound by theory or mechanism, corrosion of stainless steel components of the electrochemical cell (e.g., ports) is caused by potassium ferricyanide (K3[Fe(CN6)3]), a strong oxidizing agent, and chloride ions Cl, which induce pitting corrosion. - The presence of Fe accelerates the process from stainless steel (and other iron-containing metals) to Fe 3+ It is thought to cause the release of ions. Fe 3+ The ion is a ferrocyanide ion ([Fe(CN)6]) present in the solution. 4-) can react to form the precipitate Prussian blue (Fe4[Fe(CN)6]3). The solutions used in electrochemical cells for nanopore sequencing typically require the presence of ferrocyanide or ferricyanide ions, and stainless steel components (e.g., inlet and outlet ports) in contact with the solution are thought to result in the formation of the precipitate Prussian blue under the electrochemical measurement conditions typically used in cells for nanopore-based sequencing. When solid precipitates form within the electrochemical cell, clogging occurs and typically the cell rapidly fails during the course of nanopore-based measurements.

[0049] Iron-containing metals such as stainless steel have been determined to have problems with Prussian blue formation. More specifically, Prussian blue or other metal precipitate formation occurs when the metal is polarized to a potential of about 0.3 V relative to an Ag / AgCl reference electrode in a buffer of 1M NH4Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or in a buffer of 1M NH4Cl, 800 mM urea, 100 mM MES at pH 6.2 - 6.8 -3 mA / cm 2 It has been found that it can be reduced or prevented by using metal inlet and outlet ports that exhibit the following current densities.

[0050] Thus, in at least one embodiment of the electrochemical cell of the present disclosure, the inlet and outlet ports of the cell are 10 when polarized to a potential of about 0.3 V relative to an Ag / AgCl reference electrode in a buffer solution of 1M NH4Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or in a buffer solution of 1M NH4Cl, 800 mM urea, 100 mM MES at pH 6.2 - 6.8 -3 mA / cm 2The ports are made from metals exhibiting the following current densities. As mentioned above, generally, the metal should not contain iron, and ports made of stainless steel that come into contact with the solution in the cell during use are excluded. Nickel ports, such as those made of iron-free nickel alloys, have been found to provide excellent reduction and / or prevention of Prussian blue formation when the cell is used with solutions containing ferricyanide and ferrocyanide ions. In particular, inlet and outlet ports made from nickel alloys selected from C276, C22, 625, and MP35N provide excellent nanopore-based sequencing measurements and lifetimes.

[0051] The electrochemical cell 100 includes a counter electrode (CE) 116 and a reference electrode 117 that functions as an electrochemical potential sensor. In some embodiments, the counter electrode 116 is shared among multiple cells and is therefore also called a common electrode. The common electrode can be configured to apply a common potential to the bulk liquid in contact with nanopores in the multiple cells. The common potential and common electrode are common to all measurement cells.

[0052] In at least one embodiment, a non-Faraday electrochemical cell having a titanium nitride (TiN) working electrode has been found to be advantageous for nanopore-based sequencing of nucleic acids. The cell has a general structure similar to the cell in Figure 1, but includes a TiN working electrode that exhibits increased electrochemical capacity. The electrochemical cell further includes a conductive or metallic layer connecting the working electrode of the cell to the circuit of the array in which the cell is part.

[0053] Therefore, in at least one embodiment, the metallic working electrode 110 is a titanium nitride (TiN) metal electrode with increased electrochemical capacitance. The electrochemical capacitance associated with the metallic working electrode 110 can be increased by maximizing the specific surface area of ​​the electrode. The specific surface area of ​​the metallic working electrode 110 is expressed in units of mass (e.g., m²). 2 / kg) or volume units (e.g., m 2 / m 3 or m -1 ) or base area units (e.g., m 2 / m2 This is the total surface area of ​​the electrode per unit area. A larger surface area increases the electrochemical capacitance of the metal working electrode, allowing more ions to move at the same applied potential before the capacitor is charged. The surface area of ​​the working electrode 110 can be increased by making the TiN electrode "sponge-like" or porous. A TiN sponge allows for immersion in the electrolyte and creates a large effective surface area in contact with the electrolyte.

[0054] Capacitance related to film (C membrane ) and capacitance (C) related to the working electrode electrochemical The ratio to ) can be adjusted to achieve optimal overall system performance. System performance improvements are C electrochemical Maximizing C membrane This can be achieved by reducing C membrane It is tuned to generate the required RC time constant without requiring additional on-chip capacitance, thereby enabling a significant reduction in cell and chip size. The base surface area of ​​the metallic working electrode 110 is greater than or equal to the surface area of ​​the opening of the transside well containing the electrolyte 108, which has walls defined by the oxide layer 106. Thus, the two base surface areas are C membrane and C electrochemical It can be independently optimized to provide a desired ratio between and . By using a spongy, porous TiN working electrode, the electrolyte can diffuse vertically down the uncoated portion of the working electrode through the spaces between the columnar TiN structures, and then horizontally into the coated portion of the working electrode 1102 beneath the dielectric layer 1104. As a result, the effective surface area of ​​the TiN in contact with the electrolyte is maximized, and C electrochemical This is the maximum value.

[0055] Further descriptions of the design of non-Faraday electrochemical cells, TiN working electrodes, and other nanopore-based cell designs useful for the devices, compositions, and methods of this disclosure can be found, for example, in U.S. Patent No. 1,0174371, which is incorporated herein by reference.

[0056] As described elsewhere in this specification, the electrochemical cells of this disclosure can be used as devices for nanopore-based nucleic acid detection and measurement assays, including nucleic acid sequencing. Generally, nanopore-based sequencing of target nucleic acids can be performed using the electrochemical cell of the above design and associated solution compositions comprising electrolytes and buffers that reduce the precipitate formation described above. The present method provides an electrochemical cell comprising (a) an electrochemical cell having (i) nanopores embedded in a membrane, the membrane separating the cell into cis and trans chambers operably connected by the nanopores, the cis and trans chambers each comprising an electrode, a solution containing ferrocyanide ions, ferricyanide ions, and a buffer composition, and (ii) an inlet port and an outlet port containing metal operably connected to the cell; (b) adding molecules derived from a target nucleic acid to the cis chamber; (c) applying a voltage to the cell that causes at least a portion of the molecules to move through the nanopores; and (d) detecting a change in the voltage flow within the cell as the molecules move through the nanopores, wherein the change in the voltage flow indicates the sequence of the target nucleic acid.

[0057] As described elsewhere in this specification, the specific potentials applied, their timing, and duration can significantly affect highly sensitive changes in ion flow conductance through the nanopore in the presence of nucleic acid molecular portions to be detected when performing the sequencing method. Exemplary methods and parameters for applying potentials across nanopore-based electrochemical cell arrays for sequencing are known in the Art and are described, for example, in U.S. Patent No. 11150216 and U.S. Patent No. 11029306, each of which is incorporated herein by reference. The applied potentials, their pulse timing, and duration can also affect the redox chemistry within the cell, potentially leading to the formation of harmful precipitates such as Prussian blue. A remarkable advantage of the electrochemical cells of this disclosure and the associated solutions used therein is that, in some embodiments, the nanopore sequencing method using the electrochemical cells of this disclosure can be performed using alternating current (AC) applied to the cell for 10 hours in a voltage range of 450 mV to 1200 mV, without the visible formation of Prussian blue within the cell.

[0058] In some embodiments of the sequencing method of this disclosure, the applied voltage includes applying a baseline potential to the cells. A typical baseline voltage applied may be in the range of about 55 mV to about 95 mV.

[0059] In some embodiments of the array determination method, the applied voltage may include a pulsed voltage. A pulsed voltage useful for this method may be about 320 mV to about 550 mV. The duration of the pulsed voltage is typically about 5 μs to about 10 μs. In at least one embodiment, the time between pulses is about 0.5 ms to 1.7 ms. In at least one embodiment, the applied voltage supplies alternating current to an electrochemical cell. For example, the resulting alternating current may have a periodicity of about 0.4 seconds to about 6 seconds.

[0060] In at least one embodiment, it is conceivable that a parallel sequencing method can be performed using an array of electrochemical cells of this disclosure. Such parallel nanopore-based sequencing of nucleic acids using a parallel array of electrochemical cells has been used in conjunction with nanopore-based sequencing by synthesis (Nano-SBS) techniques. System compositions and methods for Nano-SBS are known in the art. See, for example, U.S. Patent Applications Publications 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0368710, and 2018 / 0057870A1, and International Publication 2019 / 166457. These known systems, compositions, and methods are conceivable to be used or adapted for use with the electrochemical cells of this disclosure. A description of the use of Nano-SBS in the context of the electrochemical cells and compositions of this disclosure is provided below.

[0061] Figure 2 shows an embodiment of an electrochemical cell 200 used to perform nanopore-based nucleic acid sequencing using Nano-SBS technology. In Nano-SBS technology, a template 202 and primers to be sequenced are introduced into the electrochemical cell 200. Four different tagged nucleotides 208 are added to the bulk aqueous phase to this template-primer complex. When the correctly tagged nucleotides form a complex with polymerase 204, the tails of the tags are positioned within the barrel of the nanopore 206. The tags held within the barrel of the nanopore 206 generate a unique ion-barrier signal 210, thereby electronically identifying the added bases due to the different chemical structures of the tags.

[0062] Figure 3 shows one embodiment of a cell in which nucleotide sequencing is to be performed using a preloaded tag. A nanopore 301 is formed within a membrane 302. An enzyme 303 (e.g., a polymerase such as DNA polymerase) associates with the nanopore. In some cases, the enzyme 303 is covalently bound to the nanopore 301. The polymerase 303 associates with the nucleic acid molecule 304 to be sequenced. The associated nucleic acid molecule 304 may be linear or cyclic. In some embodiments, a nucleic acid primer 305 hybridizes to a portion of the nucleic acid molecule 304. The polymerase 303 uses the single-stranded nucleic acid molecule 304 as a template to catalyze the incorporation of nucleotide 306 into the primer 305. As described above, nucleotide 306 contains a tag species ("tag") 307 that allows it to be distinguished from the other three nucleotides.

[0063] Figure 4 shows one embodiment of process 400 for nucleic acid sequencing using a preloaded tag. In step A, the tagged nucleotide (one of four different types: A, T, G, or C) is not associated with the polymerase. In step B, the tagged nucleotide associates with the polymerase. In step C, the polymerase is close to the nanopore. The tag is drawn into the nanopore by an electric field generated by a voltage applied across the membrane and / or the nanopore. Some of the associated tagged nucleotides do not form base pairs with the nucleic acid molecule. These unpaired nucleotides are typically rejected by the polymerase within a timescale shorter than the timescale in which properly paired nucleotides remain associated with the polymerase. Since the unpaired nucleotides only transiently associate with the polymerase, process 400 shown in Figure 4 typically does not proceed beyond step B.

[0064] Before polymerase docks with the nanopore, the nanopore conductance is approximately 300 picosiemens (300 pS). In step C, the nanopore conductance is approximately 60 pS, 80 pS, 100 pS, or 120 pS, corresponding to one of four types of tagged nucleotides. Polymerase undergoes isomerization and transphosphorylation reactions to incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule. In particular, once the tag is retained within the nanopore, a unique conductance signal (see, e.g., signal 210 in Figure 2) is generated due to the different chemical structures of the tag, thereby electronically identifying the added base. By repeating the cycle (i.e., steps A-E or A-F), sequencing of the nucleic acid molecule becomes possible. In step D, the released tag passes through the nanopore.

[0065] In some cases, as seen in step F of Figure 4, tagged nucleotides that are not integrated into the growing nucleic acid molecule also pass through the nanopore. While unintegrated nucleotides may, in some cases, be detected by the nanopore, this method provides a means for distinguishing between integrated and unintegrated nucleotides based at least partially on the time it takes for the nucleotides to be detected in the nanopore. Tags bound to unintegrated nucleotides pass through the nanopore rapidly and are detected for a short period (e.g., less than 10 ms), while tags bound to integrated nucleotides are loaded into the nanopore and are detected for a longer period (e.g., at least 10 ms).

[0066] Figure 5 shows one embodiment of the circuit 500 within the electrochemical cell of a nanopore-based sequencing chip. As described above, when a molecular portion (e.g., a tag) enters the nanopore 502, a unique conductance signal (see signal 210 in Figure 2, e.g.) is generated due to its different chemical structure and how it fits into the nanopore, thereby electronically identifying the added base. The circuit in Figure 5 maintains a constant voltage across the nanopore 502 while the ionic flow conductance is being measured. In particular, the circuit maintains a constant voltage across the nanopore 502. a or V b Includes an operational amplifier 504 and a pass device 506 that maintain a constant voltage equal to the capacitor n. The ion flow through the nanopore 502 is transmitted to capacitor n. cap It is integrated in 508 and measured by the analog-to-digital (ADC) converter 510. However, circuit 500 only measures the flow of unidirectional current. Furthermore, temperature drift within the operational amplifier 504 can cause the actual voltage applied across the nanopore 502 to vary across different electrochemical cells in the array. The actual voltage applied across the nanopore 502 may drift by tens of millivolts above or below the desired value, thereby causing significant measurement inaccuracies. Reducing the size of the operational amplifier in a large array may introduce other performance issues.

[0067] Figure 6 shows one embodiment of a circuit 600 for use in an electrochemical cell of a nanopore-based sequencing chip, where the voltage applied to the nanopore can be configured to vary over a period of time during which the nanopore is in a particular detectable state. One possible state of the nanopore is an open channel state when a nanomolecular part (e.g., a tag) is present within the barrel of the nanopore. Other possible states of the nanopore correspond to the states when four different types of molecular parts are present within the barrel of the nanopore. Yet another possible state of the nanopore is when the membrane is ruptured. Figure 6 shows a nanopore 602 inserted into a membrane 612, where the nanopore 602 and membrane 612 are positioned between the cell working electrode 614 and the counter electrode 616 so that a voltage is applied across both ends of the nanopore 602. The nanopore 602 is also in contact with the bulk liquid / electrolyte 618. Note that the nanopore 602 and membrane 612 are drawn upside down compared to the nanopore and membrane in Figure 1. Hereinafter, an electrochemical cell means that it includes at least a membrane, a nanopore, a working electrode, and associated circuitry. In some embodiments, the counter electrode is shared among multiple cells (e.g., in an array) and is therefore also called a common electrode. The common electrode may be configured to apply a common potential to the bulk liquid in contact with the nanopore in the measurement cell. The common potential and common electrode are common to all measurement cells. Within each measurement cell there is a metallic working cell electrode, and in contrast to the common electrode, the metallic working cell electrode 614 may be configured to apply a separate potential independent of the working cell electrodes of other measurement cells.

[0068] In addition to the Nano-SBS sequencing described above, in some embodiments, parallel sequencing of nucleic acids can be performed using the electrochemical cell of this disclosure with nanopore-based Sequencing-by-Expansion (Nano-SBX) technology. See, for example, U.S. Patent No. 7,939,259. The SBX technology is based on the polymerization of highly modified non-natural nucleotide analogs called "XNTPs". Typically, SBX uses biochemical polymerization to transfer the DNA template sequence onto a measurable polymer called "Xpandomer". The transferred sequence is encoded along the Xpandomer backbone in a high-signal-versus-noise reporter about 10 nm away, designed for high-signal-versus-noise, high-differentiation response. These differences result in a significant performance improvement in sequence read efficiency and accuracy of Xpandomer compared to natural DNA. A general description of the SBX process is shown in Figures 7, 8, 9, and 10.

[0069] XNTPs are template-dependent enzymatic polymerization and expandable 5'-triphosphate modified unnatural nucleotide analogs. A highly simplified XNTP is shown in Figure 7, which highlights the intrinsic characteristics of these unnatural substrates. XNTP100 has two distinct functional regions: a selectively cleavable phosphoramide bond 110 that links the 5'-α-phosphate 115 to the nucleic acid base 105, and a symmetrically synthesized reporter tether (SSRT) 120 bound within the nucleoside triphosphoramide at a position that allows for controlled expansion by cleavage of the phosphoramide bond. The SSRT contains linkers 125A and 125B separated by the selectively cleavable phosphoramide bond. Each linker is bound to one end of the reporter code 130. XNTP100 is shown in a "constrained configuration" characteristic of the XNTP substrate and the daughter chain product of template-dependent polymerization. The constrained structure of polymerized XNTPs is a precursor to the extended structure, as seen in the Xpandomer product. The transition from the constrained to the extended structure occurs when the PN bond of the phosphoramide in the primary skeleton of the daughter chain is cleaved.

[0070] The synthesis of the Xpandomer polymer is summarized in Figures 8 and 9. As shown in Figure 8, during assembly, the monomer XNTP substrates 145 (XATP, XCTP, XGTP, and XTTP) are polymerized at the elongable ends of the nascent daughter strands 150 by a template-directed polymerization process using a single-strand template 140 as a guide. Generally, this process is initiated from the primer and proceeds in the 5' to 3' direction. Generally, DNA polymerase or other polymerases are used to form the daughter strands, and conditions are selected to obtain complementary copies of the template strand. After the daughter strands are synthesized, the coupled SSRTs form constrained Xpandomer, which further forms the daughter strands. The SSRTs in the daughter strands have a "constrained configuration" of the XNTP substrates. The constrained configuration of the SSRTs is a precursor to the extended configuration, as seen in the Xpandomer product.

[0071] As shown in Figure 9, the transition from the constrained configuration 160 to the extended configuration 165 is due to the cleavage of selectively cleavable phosphoramide bonds (indicated by unshaded ellipses for simplification) within the primary backbone of the daughter strand. In this embodiment, the SSRT includes one or more reporters or reporter codes 130A, 130C, 130G, or 130T that are specific to the nucleic acid bases to which they are linked, thereby encoding the sequence information of the template. In this way, the SSRT provides a means to extend the length of the Xpandomer and reduce the linear density of the sequence information of the parent strand.

[0072] Figure 10 shows the Xpandomer 165 moving from the cis chamber 175 to the trans chamber 185 through the nanopore 180. Upon passing through the nanopore, each reporter code of the linearized Xpandomer generates a distinct and reproducible electronic signal specific to the nucleic acid base to which it is linked. This signal is indicated by the overwrite trace 190.

[0073] Figure 11 shows in more detail a generalized structure of one embodiment of XNTP. XNTP 200 comprises a nucleoside triphosphoramide 210 having linker arm portions 220A and 220B separated by selectively cleavable phosphoramide bond 230. SSRT 275 is linked to the nucleoside triphosphoramide by linking groups 250A and 250B, with the first SSRT terminus linked to a heterocycle 260 (represented here by cytosine, although the heterocycle may be any one of the four standard nucleic acid bases, A, C, G, or T), and the second SSRT terminus linked to an α-phosphate 270 of the nucleic acid base backbone. Those skilled in the art will understand that the final XNTP substrate product can be formed using many suitable coupling chemicals known in the art, for example, that the SSRT conjugate can be achieved by the formation of a triazole linking group.

[0074] In this embodiment, the SSRT275 includes several functional elements, or “features,” such as polymerase-enhancing regions 280A and 280B, reporter codes 285A and 285B, and translation control elements (TCEs) 290A and 290B. In other embodiments, the SSRT may include a single TCE. Each of these features plays a unique function during the migration of the Xpandomer through the nanopore, generating a series of distinctive and reproducible electronic signals. The SSRT275 is designed to control the rate of Xpandomer migration by the TCE via a combination of steric hindrance and / or electrorepulsion, as discussed elsewhere in this specification. Different reporter codes are sized to block ion flow through the nanopore at different measurable levels.

[0075] Specific SSRT polymer sequences can be efficiently synthesized using phosphoramidite chemistry, which is typically used in oligonucleotide synthesis. Reporter codes and other features can be designed by selecting specific phosphoramidite sequences from commercially available and / or proprietary libraries. Such libraries include, but are not limited to, polyethylene glycols having a length of 1 to 12 or more ethylene glycol units, and aliphatic polymers having a length of 1 to 12 or more carbon units. In certain embodiments, the SSRT includes a feature called a "polymerase-enhancing region" at the terminal of the SSRT proximal to the nucleotide triphosphoamide diester. The polymerase-enhancing region may include a positively charged polyamine spacer (e.g., primary, secondary, tertiary, or quaternary amine) or a triamine spacer (three secondary amines separated by three carbons each) that facilitates the uptake of the XNTP structure by nucleic acid polymerase. In certain embodiments, the polymerase-enhancing region comprises two repeating units of spermine, the spermine portion of which is provided by a phosphoramidite monomer having the following structure (as those skilled in the art will recognize, the trifluoroacetamide protecting group is removed at the end of the SSRT synthesis to expose the amine group on spermine). [ka]

[0076] Where used throughout this disclosure, the term “reporter construct” refers to the elements of an SSRT comprising reporter codes, symmetric chemical branching chains, and a migration control element. In certain embodiments, the reporter construct is a polymer comprising a first reporter code, symmetric chemical branching chains carrying a migration control element, and a second reporter code, continuous from a first end to a second end. The term “carrying” refers to a covalent bond between the symmetric branching chain and the migration control element, which results in a favorable orientation of the migration control element relative to the two reporter codes. The symmetric chemical branching chain can be represented by the letter “Y”, with the two reporter codes bonded to the arms of the Y and the migration control element bonded to the stem of the Y. Thus, the two reporter codes are linked inline by the branching chain, while the branching chain carries the migration control element perpendicular to the linear inline SSRT.

[0077] As used throughout this disclosure, the terms “linker A” and “linker B” refer to a polymerase-enhancing region and one or more migration-decelerating features or regions, as well as a region of the SSRT that includes, in certain embodiments, a spacer region containing a polymer such as PEG6, which can be customized to adjust the length of the SSRT traversing within the nanopore.

[0078] In certain embodiments, XNTP may be a compound having the following generalized structure. [ka]

[0079] In one embodiment, for example, when a compound is used to sequence a DNA template, R may be H. In another embodiment, for example, when a compound is used to sequence an RNA template, R may be OH. In certain embodiments, the nucleic acid base is adenine, cytosine, guanine, thymine, uracil, or a nucleic acid base analogue. As those skilled in the art will understand, adenine, cytosine, guanine, thymine, and uracil are naturally occurring nucleic acid bases. As used herein, the term “nucleic acid base analogue” refers to a non-naturally occurring nucleic acid base that can form a Watson-Crick base pair with a complementary nucleic acid base on an adjacent single-stranded nucleic acid template. Exemplary nucleic acid base analogs include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- This list includes, but is not limited to, mannosylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wibutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, 3-nitropyrrole, 8-aza-7-deazaguanine, 8-aza-7-deazynosine, and 8-aza-7-deazaadenine.

[0080] As discussed elsewhere in this specification, the reporter construct is a polymer having a first end and a second end, comprising, continuously from the first end to the second end, a first reporter code, a symmetric chemical branch chain carrying a movement control element, and a second reporter code. This set of features reflects the symmetric structure of the reporter construct (as well as the entire SSRT including the symmetric linkers, linker A and linker B), where the sequences of the two reporter codes are identical and inline linked in opposite directions by the symmetric chemical branch chain. Briefly, synthesis proceeds in the 3' to 5' direction, starting at the 3' end of the TCE. By adding the symmetric branch chain to the 5' end of the TCE, simultaneous polymerization of the first and second reporter codes from each arm of the branch chain, followed by the simultaneous synthesis of linker A and linker B, is enabled, terminating at the 5' ends of the first and second ends of the SSRT. The inline redundancy provided by two identical reporter codes separated by symmetric branched chains carrying the movement control elements has been found to offer several advantages during nanopore sequencing. For example, if the Xpandomer moves in either direction, it can potentially be read by the nanopore; i.e., the Xpandomer can be read either "forward" or "backward." This flexibility enables other methods of sequencing, such as the "ratchet" method and "flooring" based on the AC pattern of voltage application, which are discussed further herein.

[0081] Figure 12 shows one embodiment of a cleaved Xpandomer in the process of moving a nanopore. The nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two electrolyte chambers. A typical electrolyte has 1 M KCl buffered to pH 7.0. When a small voltage of typically 100 mV is applied across the bilayer, the nanopore provides the only channel for potential-induced ion flow and is the source primary resistance in the circuit. The Xpandomer reporter code is designed to give a specific ion flow cutoff level, and since pulses of potential result in an array of reporter code moving through the nanopore, array information can be read by measuring the array of ion flow levels. In the case of an α-hemolysin nanopore, the nanopore is typically embedded in the membrane so that movement occurs by entering on the cis-antibiotic side and exiting on the trans-basal side. As shown in Figure 12, the nanopore is oriented to first capture the Xpandomer from the basal side. As the Xpandomer moves the nanopore, the reporter enters the base until its movement control element stops at the base entrance. The reporter is held in the base until the TCE enters the base and can pass through it, after which the movement proceeds to the next reporter. In this embodiment, the passage of the TCE into the base is made possible by disengaging the movement control portion from the TCE. [Examples]

[0082] Various features and embodiments of this disclosure are shown in the following representative examples, which are illustrative and not limiting. Those skilled in the art will readily understand that certain embodiments are merely illustrative of the invention, which are described more fully in the subsequent claims. All embodiments and features described herein should be understood to be interchangeable and combinable with all embodiments contained herein.

[0083] Example 1: Effect of alternative buffer components and metallic materials on Prussian blue formation This example illustrates the study of the effects of buffer components and metal surface composition used in an electrochemical cell on the formation of Prussian blue.

[0084] material and method

[0085] The standard femtoliter-range chambers of the electrochemical cells used for SBX nanopore sequencing have internal stainless steel surfaces, including inlet and outlet ports, that come into contact with the solution contained in the cell, which includes ammonium chloride and potassium ferrocyanide and potassium ferricyanide, each at approximately 250 mM. To simulate the conditions occurring in such femtoliter nanopore sequencing chambers in larger volumes, scratch cannulas made from various types of metal alloys were immersed in buffers containing either ammonium chloride or ammonium acetate, varying amounts of potassium ferrocyanide and potassium ferricyanide, and various other buffer species such as sodium salicylate, sodium pyrophosphate, resorcinol, and sulfosalicylic acid. The cannulas were monitored for the formation of Prussian blue precipitate on the surface. Various conditions and results of the tests are summarized in Table 1 below. [Table 1]

[0086] As shown by the results summarized in Table 1, replacing stainless steel SS316 cannulas with non-ferrous nickel alloy C276 or C22 cannulas was highly effective in preventing Prussian blue formation over several days or even weeks.

[0087] Furthermore, changes in the buffer components had a significant impact on Prussian blue formation. Specifically, substituting ammonium chloride with ammonium acetate was highly effective in preventing Prussian blue formation. Similarly, reducing the levels of ferrocyanide and ferricyanide species by half significantly reduced Prussian blue formation.

[0088] Example 2: Effects of alternative buffer components and metallic materials on Prussian blue formation during nanopore sequencing This example describes the study of the effects of buffer components and metal surfaces on the formation of Prussian blue in an electrochemical cell during SBX nanopore sequencing.

[0089] As described elsewhere in this specification, SBX nanopore sequencing is performed using an array of electrochemical cells containing buffers containing ferricyanide and ferrocyanide ions. The solution enters and exits the cells through inlet and outlet ports during use.

[0090] Materials and Methods: Hundreds of SBX nanopore-based sequencing experiments were performed over several months using standard array nanopores containing electrochemical cells with inlet and outlet ports made of either stainless steel (316 stainless) or nickel alloy (alloy C276, C22, or 625), by flowing SBX sequencing buffer through the cells. Cells in the array were monitored for failures, and failed cells were analyzed for the presence of Prussian blue precipitate clogging in the ports.

[0091] Results: It was found that changing the port material from stainless steel to nickel alloy reduced cell failure due to clogging by Prussian blue precipitate from approximately 35% to less than 5%.

[0092] All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, or other document is individually indicated as being incorporated herein by reference for all purposes.

[0093] Although various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. It is an electrochemical cell, (a) A nanopore embedded in a membrane, the membrane separating the cell into cis and trans chambers connected by the nanopore, the cis and trans chambers each containing an electrode, a solution containing ferrocyanide ions and ferricyanide ions, and a buffer composition, (b) an inlet port and an outlet port operably connected to the cell, wherein the port includes metal, Includes, By applying alternating current (AC) in the voltage range of 450 mV to 1200 mV to the cell, no visible formation of Prussian blue occurs within the cell for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, or at least 10 hours. The aforementioned electrochemical cell.

2. The aforementioned metal is 1M NH ４ In a buffer solution of Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or in 1 M NH4O at pH 6.2–6.

8. ４ When polarized to a potential of approximately 0.3V relative to an Ag / AgCl reference electrode in a buffer solution of Cl, 800 mM urea, and 100 mM MES, the voltage is 10 －３ mA / cm ２ The cell according to claim 1, exhibiting the following current densities.

3. The cell according to claim 1 or 2, wherein the metal comprises less than 10% iron, less than 8% iron, less than 6% iron, or less than 5% iron.

4. The cell according to claim 1 or 2, wherein the metal is a nickel alloy.

5. The cell according to claim 4, wherein the metal is a nickel alloy selected from C276, C22, 625, and MP35N.

6. The cell according to claim 1 or 2, wherein the cell contains a solution volume between about 0.1 femtoliters and about 1,000 femtoliters.

7. The cell according to claim 1 or 2, wherein the total concentration of the ferrocyanide ions and ferricyanide ions is between approximately 25 mM and 250 mM.

8. The cell according to claim 1 or 2, wherein the buffer composition contains ammonium acetate at a concentration of about 0 mM to about 1500 mM.

9. The cell according to claim 1 or 2, wherein the buffer composition contains ammonium chloride at a concentration of less than 2000 mM or less than 1000 mM.

10. The cell according to claim 1 or 2, wherein the buffer composition comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM and ammonium chloride at a concentration of less than 2000 mM, and optionally comprises ammonium acetate at a concentration of 0 mM and ammonium chloride at a concentration of less than 1000 mM.

11. A method for sequencing a target nucleic acid, (a) an electrochemical cell comprising (i) a nanopore embedded in a membrane, the membrane separating the cell into cis and trans chambers operably connected by the nanopore, the cis and trans chambers each comprising an electrode, a solution containing ferrocyanide ions and ferricyanide ions, and a buffer composition, and (ii) an inlet port and an outlet port containing a metal connected to the cell, the electrochemical cell comprising (b) Adding a molecule derived from the target nucleic acid to the cis chamber, (c) Applying a voltage to the cell that moves at least a portion of the molecules through the nanopore, (d) Detecting a change in the voltage flow within the cell as the molecule moves through the nanopore, wherein the change in the voltage flow indicates the sequence of the target nucleic acid. Includes, By applying alternating current (AC) in the voltage range of 450 mV to 1200 mV to the cell, no visible formation of Prussian blue occurs within the cell for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, or at least 10 hours. The aforementioned method.

12. The aforementioned metal is 1M NH ４ In a buffer solution of Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or in 1 M NH4O at pH 6.2–6.

8. ４ When polarized to a potential of approximately 0.3V relative to an Ag / AgCl reference electrode in a buffer solution of Cl, 800 mM urea, and 100 mM MES, the voltage is 10 －３ mA / cm ２ The method according to claim 11, wherein the following current density is observed.

13. The method according to claim 11 or 12, wherein the metal comprises less than 10% iron, less than 8% iron, less than 6% iron, or less than 5% iron.

14. The method according to claim 11 or 12, wherein the metal is a nickel alloy, and optionally the metal is a nickel alloy selected from C276, C22, 625, and MP35N.

15. The method according to claim 11 or 12, wherein the cell contains a solution volume between approximately 0.1 femtoliters and 1,000 femtoliters.

16. The method according to claim 11 or 12, wherein the total concentration of the ferrocyanide ions and ferricyanide ions is between approximately 25 mM and 250 mM.

17. The method according to claim 11 or 12, wherein the buffer composition contains ammonium acetate at a concentration of about 0 mM to about 1500 mM.

18. The method according to claim 11 or 12, wherein the buffer composition contains ammonium chloride at a concentration of less than about 2000 mM or less than about 1000 mM.

19. The method according to claim 11 or 12, wherein the buffer composition comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM and ammonium chloride at a concentration of less than 2000 mM, and optionally comprises ammonium acetate at a concentration of 0 mM and ammonium chloride at a concentration of less than 1000 mM.

20. The method according to claim 11 or 12, wherein the applied voltage includes a baseline voltage, and optionally the baseline voltage is approximately 55 mV to approximately 95 mV.

21. The method according to claim 11 or 12, wherein the applied voltage includes a pulse voltage, and optionally the pulse voltage is approximately 320 mV to approximately 550 mV.

22. The method according to claim 21, wherein the pulse voltage has a duration of approximately 5 μs to approximately 15 μs.

23. The method according to claim 21, wherein the time between pulse voltages is approximately 0.5 ms to approximately 1.7 ms.

24. The method according to claim 11 or 12, wherein the applied voltage provides an alternating current, and optionally the alternating current has a periodicity of about 0.4 seconds to about 6 seconds.

25. The method according to claim 11 or 12, wherein the molecule derived from the target nucleic acid includes Xpandomer.

26. The method according to claim 25, wherein the Xpandomer comprises a plurality of XNTP subunits coupled with sequences corresponding to a sequence of all or part of the target nucleic acid, and each XNTP subunit of the chain comprises a reporter construct, nucleic acid base residues and selectively cleavable bonds, and cleavage of the selectively cleavable bonds results in an Xpandomer longer than the plurality of XNTP subunits of the chain.

27. The method according to claim 26, further comprising severing the selectively separable bond to obtain an Xpandomer.

28. The method according to claim 25, wherein the Xpandomer includes a reporter construct for analyzing genetic information in a sequence corresponding to all or part of a continuous nucleotide sequence of the target nucleic acid.

29. The method according to claim 28, wherein detecting the voltage change includes detecting the voltage change caused by the reporter construct of the Xpandomer that moves the nanopore.

30. The method according to claim 29, wherein the reporter construct for analyzing the genetic information comprises a reporter code and a movement control element, the movement control element providing sterically hindered movement control and pausing the movement of the Xpandomer when passing through a nanopore subjected to a baseline voltage, the movement control element engaging with the reporter code within the opening of the nanopore, and the reporter code being sensed by the nanopore.

31. The method according to claim 11 or 12, wherein the electrochemical cell is the electrochemical cell according to claim 1 or 2.

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