Compositions and methods for reducing prussian blue formation during nanopore sequencing - Patents.com

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 The present application relates to electrochemical cells and associated buffer compositions that reduce the deleterious formation of Prussian blue during nanopore-based assays, and methods of using the compositions in nanopore-based nucleic acid detection technologies, such as nanopore sequencing. [Background technology]

[0002] background Nanopore-based nucleic acid sequencing is a compelling approach that has been widely studied. In one approach, single-stranded polynucleotide sequences are detected by changes in ionic current as the polynucleotide translocates through a nanopore embedded in a lipid bilayer that separates the two sides of an electrochemical cell. During polynucleotide translocation, partial blockage of the nanopore opening alters the flow of ions over time, resulting in a change 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 transcribe the sequence of DNA into measurable polymer molecules called "Xpandomers". See, for example, U.S. Patent No. 7,939,259, entitled "High Throughput Nucleic Acid Sequencing by Expansion" and PCT Publication WO 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 the backbone Xpandomer sequence with reporter constructs spaced approximately 10 nm apart, designed to provide well-differentiated response signals with high signal-to-noise during nanopore translocation. The enhanced signal-to-noise provided by the distinct response signals provides a significant increase in sequence read efficiency and accuracy of Xpandomers compared to natural nucleic acid molecules.

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

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

[0006] Thus, there remains a need for electrochemical cell designs and materials, buffer and redox reagent compositions, and methods of using them to reduce or prevent the formation of deleterious by-products (e.g., Prussian blue precipitates) that interfere with accurate electrochemical signal detection in nanopore-based nucleic acid sequencing devices, systems, and methods. Summary of the Invention

[0007] overview The present disclosure generally relates to electrochemical cells and associated buffer compositions used in the cells that reduce the deleterious formation of Prussian blue or related precipitates during nanopore-based assays, and 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 the present disclosure, but does not exhaustively describe each and every embodiment, combination, or variation contemplated and described within the disclosure. Further embodiments are contemplated and described by the disclosure in the detailed description, drawings, and claims.

[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 comprising ferrocyanide ions, ferricyanide ions, and a buffer composition; and (b) inlet and outlet ports operably connected to the cell, the inlet and outlet ports comprising a metal.

[0009] In at least one embodiment of the electrochemical cell of the present disclosure, the metal has a potential of 10 when polarized to a potential of about 0.3 V versus an Ag / AgCl reference electrode in a buffer solution of 1 M NH4Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or a buffer solution of 1 M NH4Cl, 800 mM urea, 100 mM MES at pH 6.2-6.8. -3 mA / cm 2The following current densities are shown: In at least one embodiment, the metal comprises 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 of between about 0.1 and 1000 femtoliters.

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

[0012] In at least one embodiment of the electrochemical cell of the present disclosure, the buffer composition comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM. In at least one embodiment, the buffer composition comprises 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 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, application of an alternating current (AC) to the cell in the voltage range of 450 mV to 1200 mV results in no visible formation of Prussian blue in 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 comprising: (a) providing 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 comprising ferrocyanide ions and ferricyanide ions, and a buffer composition; and (ii) inlet and outlet ports comprising 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 that translocates at least a portion of the molecule through the nanopore; (d) detecting a change in voltage flow within the cell as the molecule translocates through the nanopore, the change in voltage flow being indicative of a sequence of the target nucleic acid; and Includes.

[0015] In at least one embodiment of the sequencing method of the present disclosure, the metal has a potential of 10 when polarized to a potential of about 0.3 V versus an Ag / AgCl reference electrode in a buffer solution of 1 M NH4Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or a buffer solution of 1 M NH4Cl, 800 mM urea, 100 mM MES at pH 6.2-6.8. -3 mA / cm 2 The following current densities are shown: In at least one embodiment, the metal comprises 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 method of the present disclosure, the cell contains a solution volume of between about 0.1 and 1000 femtoliters.

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

[0018] In at least one embodiment of the sequencing method of the present disclosure, the buffer composition comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM. In at least one embodiment, the buffer composition comprises 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 about 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.

[0019] In at least one embodiment of the sequencing method of the present disclosure, application of an alternating current (AC) in the voltage range of 450 mV to 1200 mV to the cell results in no visible formation of Prussian blue in 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 comprises a baseline voltage, and optionally, the baseline voltage is about 55 mV to about 95 mV. In at least one embodiment, the applied voltage comprises 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 the 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 comprises an Xpandomer.In at least one embodiment, the Xpandomer comprises a plurality of XNTP subunits coupled with a sequence corresponding to the continuous nucleotide sequence of all or part of the target nucleic acid, each XNTP subunit of the chain comprises a reporter construct, a nucleobase residue and a selectively cleavable bond, and the cleavage of the selectively cleavable bond produces an Xpandomer of a length 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 cleaving the selectively cleavable bond to obtain an Xpandomer.

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

[0024] In at least one embodiment of the sequencing method of the present disclosure, a reporter construct for analyzing genetic information comprises a reporter code and a translocation control element, where the translocation control element provides translocation control by steric hindrance and pauses translocation of the Xpandomer as it passes through a nanopore subjected to a baseline voltage, where the translocation control element engages with the reporter code within the opening of the nanopore, and where the reporter code is sensed by the nanopore. [Brief description of the drawings]

[0025] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG.") in which:

[0026] [Figure 1] 1 illustrates one embodiment of a cell 100 in a nanopore-based sequencing chip. [Diagram 2] 2 shows one embodiment of a cell 200 for performing nucleotide sequencing using Nano-SBS technology. [Diagram 3] 1 shows an embodiment of a cell in which nucleotide sequencing is to be performed with preloaded tags. [Figure 4] 4 shows one embodiment of a process 400 for nucleic acid sequencing using preloaded tags. [Diagram 5] 5 shows one embodiment of a circuit 500 within a cell of a nanopore-based sequencing chip. [Figure 6] One embodiment of circuitry 600 within a cell of a nanopore-based sequencing chip is shown, in which the voltage applied to the nanopore can be configured to vary over the period of time that the nanopore is in a particular detectable state. [Figure 7] 1 shows a schematic diagram of the generalized XNTP features and their function in Nano-SBX technology. [Figure 8] 1 shows a schematic diagram of the generalized XNTP features and their function in Nano-SBX technology. [Figure 9] 1 shows a schematic diagram of the generalized XNTP features and their function in Nano-SBX technology. [Figure 10] 1 shows a schematic diagram of the generalized XNTP features and their function in Nano-SBX technology. [Figure 11] FIG. 2 is a schematic diagram showing further details of one embodiment of XNTP. [Figure 12] FIG. 1 is a schematic diagram showing one embodiment of an Xpandomer passing through a biological nanopore. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description The present disclosure provides electrochemical cells and associated buffer compositions for use in the cells. The electrochemical cells are useful for performing nanopore-based methods of assaying nucleic acids, including nanopore-based sequencing. The features of the electrochemical cells and associated buffer compositions have the surprising and advantageous result of reducing the formation of harmful precipitates during the use of the cells in nanopore-based assays. Harmful precipitates include Prussian blue, which can precipitate as a solid in redox reactions involving ferricyanide and ferrocyanide ions and metallic iron found in components of the electrochemical cell, such as the inlet and outlet ports. The formation of such precipitates can rapidly clog the ports and / or nanopores, thereby significantly reducing the accuracy and efficiency of any nanopore-based assay. The present disclosure also provides methods for using the electrochemical cells 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 the description of this specification and the appended claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "protein" includes a plurality of proteins, and a reference to a "compound" refers to a plurality of compounds. The use of "comprise", "comprises", "comprising", "include", "includes", and "including" are interchangeable and are not intended to be limiting. It is further understood that where the description of various embodiments uses the term "comprising", one of ordinary skill in the art would understand that in some specific instances, the embodiments can alternatively be described using the term "consisting essentially of" or "consisting of".

[0029] Where a range of values ​​is provided, it is understood that each intervening integer in the value and each tenth of each intervening integer in the value, between the upper and lower limits of the range, and any other stated or intervening value within that stated range, is encompassed within the invention, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit within the stated range. Where a stated range includes one or both of the limits, ranges excluding either (i) or both (ii) of the included limits are also encompassed within 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 of skill in the art, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter "Sambrook"); and Current Protocols in Molecular Biology, edited by FMA Ausubel et al., first published in book form in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented periodically until 2011, and now available in online journal form as Current Protocols in Molecular Biology, Vols. 00-130 (1987-2020), and published in the Wiley Online Library by Wiley & Sons, Inc. (hereinafter "Ausubel").

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

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. It should be understood that the terms used herein are for describing specific embodiments only and are not intended to be limiting. For the purposes of interpreting this disclosure, the following definitions of terms are applied, and where appropriate, terms used in the singular form also include the plural form 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 methods of use are known in the art. See, for example, U.S. Pat. Nos. 9,494,554, 9,567,630, 9,557,294, and 9,605,309, each of which is incorporated herein by reference. These devices generally include an electrochemical cell having a chamber containing a nanopore embedded in a membrane. The membrane acts to separate the cell chamber into two subchambers, called the cis and trans sides of the cell, each of which contains an electrode. The membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane made of non-naturally occurring polymeric materials. The nanopore pore acts as a channel (or passage) in the membrane between the cis and trans sides of the cell. Depending on the nanopore used, the pore has a width or diameter that may range from about 1 angstrom to about 10,000 angstroms. The nanopore may be a naturally occurring pore-forming protein, such as the α-hemolysin from S. aureus, a non-naturally occurring mutant or variant of a wild-type pore-forming protein.A range of naturally occurring and non-naturally occurring nanopores with various pore sizes and characteristics are known in the art.See, for example, US Patent No. 10,351,908, US Patent No. 10,934,582, US Patent No. 10,227,645.

[0035] Within an electrochemical cell, a nanopore embedded in a membrane is placed in close proximity to electrodes coupled to a sensing circuit, for example, a complementary metal oxide semiconductor (CMOS) or field effect transistor (FET) circuit. When a potential is applied (via the electrodes) across the nanopore, which is immersed in a conducting fluid, a small current due to the flow of ions through the nanopore can be observed. This ion flow is sensitive to pore size, and thus molecules entering the pore affect the ion flow and voltage measured via this sensor circuit.

[0036] Electrochemical cells for nanopore-based sequencing of nucleic acids are typically used in a massively parallel manner, with thousands of such cells configured as an array in a single device, often referred to as a chip (or biochip). Typically, a nanopore-based sequencing chip device incorporates an array of more than one million electrochemical cells, and may include 1000 rows by 1000 columns of such cells (see, for example, the 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 Application Publication Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0368710, and 2018 / 0057870, and International Publication No. WO 2019 / 166457, each of which is incorporated herein by reference. Each well in the array is fabricated using standard CMOS processes with surface modifications that allow constant contact between the bioreagents and the conductive salts. Each well can support a phospholipid bilayer membrane with an embedded nanopore polymerase conjugate. The electrodes of each well are individually addressable by a computer interface. All reagents used are introduced into a simple flow cell above the array microchip using computer-controlled syringe pumps. The chip supports analog-to-digital conversion and reports electrical measurements independently from all electrodes at a rate of over 1000 points / second. Nanopore measurements can be made asynchronously at least once every 1 millisecond (msec) at each of the 8M addressable nanopore-containing membranes in the array and recorded to an interfaced computer. Further description of an exemplary electrochemical cell useful for nanopore-based nucleic acid assays such as sequencing is provided below, including chamber and electrode materials, buffers, sensing circuitry, array devices, and their use in various applications.

[0037] FIG. 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 a nanopore or "protein nanopore transmembrane molecular complex" (PNTMC) 104 is placed directly on this surface within the cell, and electroporation is used to insert a single PNTMC 104 into the membrane 102. The individual nanopore-embedded membranes in each cell of the array are not chemically or electrically connected to one another. Each cell is therefore an independent sequencing machine, generating data specific to a single polymer molecule associated with the PNTMC.

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

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

[0040] A thin film of electrolyte constitutes a femtoliter volume of solution in the transformer chamber in direct contact with the working electrode. This small volume of electrolyte, in contact with the electrode of the electrochemical cell, must undergo repeated voltage pulses in sensing ion flow through the nanopore. These repeated electrochemical measurements using electrodes in a small volume of electrolyte, and the need to maintain ion flow through the nanopore using repeated measurements corresponding to individual molecular moieties moving through the pore, require a highly sensitive sensor system. This electrochemical measurement system is highly sensitive to any precipitated or aggregated molecular entities that may clog the pore or otherwise disrupt ion flow between the working and counter electrodes. Thus, the electrolyte salt, electrochemical cell materials, and electrochemical measurement conditions should be carefully controlled to prevent or reduce deleterious precipitate formation or electrode wear in the system.

[0041] The electrolyte solution 108 may include 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). In some embodiments, the electrolyte solution film has a thickness of about 3 microns (μm). The thickness of the electrolyte solution film may range from 0 to 5 microns.

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

[0043] It has also been found that the salts used in the electrolyte solution of the electrochemical cell can affect the ability of the cell to be used for high sensitivity 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 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 discussed above. Thus, in some embodiments of the electrochemical cell of the present disclosure, the buffer composition 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 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 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] A dielectric material is typically used to form the oxide layer 106 that defines the transformer chamber of the cell. Useful dielectric materials can include glass, oxide, silicon mononitride (SiN), and the like. In some embodiments, the top surface of the dielectric oxide layer 106 in contact with the bulk electrolyte 114 can be silanized. The silanization forms a hydrophobic layer on the top surface 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 top of the dielectric layer.

[0045] As shown in Figure 1, the membrane is a lipid bilayer formed at least partially on a dielectric oxide layer 106, spanning 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 transitions to a lipid bilayer that spans across the opening of the well. The hydrophobic layer can facilitate the formation of a lipid monolayer on the dielectric layer and the transition from the lipid monolayer to a lipid bilayer.

[0046] The bulk electrolyte 114 may further include 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). Thus, in some embodiments of the electrochemical cell of the present disclosure, the buffer composition of the bulk electrolyte used in the cell includes 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 includes 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 of between about 25 mM and 250 mM.

[0047] As discussed above with respect to thin electrolyte films, the composition of the salt (e.g., ammonium chloride vs. ammonium acetate) and redox-active ions used in the bulk electrolyte can affect the accuracy and sensitivity of nanopore-based measurements made using the electrochemical cell. Depending on the type of electrochemical measurement being performed, particularly the potential applied to the electrodes in contact with the solution, it is believed that the composition of the solution can be adjusted to reduce or prevent the formation of deleterious precipitates.

[0048] To perform the necessary insertion and removal of solutions, the electrochemical cell of the present disclosure includes inlet and outlet ports operatively connected to the chambers or reservoirs of the cell. Although the schematic of the electrochemical cell in FIG. 1 does not show 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 making the membrane with the embedded nanopore, nucleotide synthesis reagents, and electrolytes and buffers. Typically, the inlet and outlet ports include metal tubes attached to the cell to provide a fluid connection from the outside to the chamber of the cell that can be used to insert or remove solutions. Thus, at least a portion of the inlet and outlet port tubes are in contact with the electrochemical solution within the cell chamber. Thus, the composition of the ports must be electrochemically inert at the voltages used to induce ionic flow through the nanopore and to make sensory measurements of changes in ionic flow as molecular moieties enter and / or move through the nanopore. Stainless steel tubes have been used to provide metal tubes for the inlet and outlet ports, but steel contains iron. One surprising advantage of the electrochemical cell of the present disclosure is that the inlet and outlet ports contain metals containing less than 10% iron, thereby reducing or preventing the formation of deleterious precipitates within the cell, such as Prussian blue. Without intending to be bound by theory or mechanism, it is believed that corrosion of stainless steel components (e.g., ports) of the electrochemical cell is caused by potassium ferricyanide (K3[Fe(CN6)3]), a strong oxidizing agent, and chloride ions, Cl, which induce pitting corrosion. - The loss of Fe from stainless steel (and other iron-containing metals) is accelerated by the presence of 3+ This is believed to cause the release of Fe ions. 3+ The ion is the ferrocyanide ion ([Fe(CN)6] 4-) to form the precipitate Prussian blue (Fe4[Fe(CN)6]3). Solutions used in electrochemical cells for nanopore sequencing typically require the presence of ferrocyanide or ferricyanide ions, and stainless steel components in contact with the solutions (e.g., inlet and outlet ports) are believed to result in the formation of the precipitate Prussian blue under electrochemical measurement conditions typically used in cells for nanopore-based sequencing. The formation of solid precipitates within an electrochemical cell results in clogging and typically leads to rapid failure of the cell over the course of a nanopore-based measurement.

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

[0050] Thus, in at least one embodiment of the electrochemical cell of the present disclosure, the inlet and outlet ports of the cell have a potential of 10 when polarized to a potential of about 0.3 V versus a Ag / AgCl reference electrode in a buffer solution of 1 M NH4Cl, 800 mM urea, 100 mM HEPES, pH 7.4, or in a buffer solution of 1 M NH4Cl, 800 mM urea, 100 mM MES at pH 6.2-6.8. -3 mA / cm 2The inlet and outlet ports are made of a metal that exhibits a current density of 0.1 to 1.0 μm. As mentioned above, in general, the metal should not contain iron, and ports with stainless steel that contact the solution in the cell during use are excluded. It has been found that ports made of nickel, such as nickel alloys that do not contain iron, 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 of 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 referred to as a common electrode. The common electrode can be configured to apply a common potential to the bulk liquid in contact with the nanopores in the multiple cells. The common potential and the common electrode are common to all measurement cells.

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

[0053] Thus, in at least one embodiment, the metallic working electrode 110 is a titanium nitride (TiN) metal electrode with enhanced 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., m 2 / kg) or volume units (e.g., m 2 / m 3 or m -1 ) or base area units (e.g., m 2 / m2 ) is the total surface area of ​​the electrode. With a larger surface area, the electrochemical capacitance of the metal working electrode is greater, allowing more ions to be transferred at the same applied potential before the capacitor becomes charged. The surface area of ​​the working electrode 110 can be increased by making the TiN electrode "spongy" or porous. The TiN sponge creates a large effective surface area that is saturated with and in contact with the electrolyte.

[0054] The capacitance associated with the membrane (C membrane ) and the capacitance associated with the working electrode (C electrochemical ) can be adjusted to achieve optimal overall system performance. The improvement in system performance is due to the electrochemical While maximizing C membrane This can be achieved by reducing C membrane is tuned to produce the required RC time constant without the need for additional on-chip capacitance, thereby allowing for a significant reduction in cell and chip size. The base surface area of ​​the metal working electrode 110 is equal to or greater than the surface area of ​​the opening of the trans-side well containing the electrolyte 108, whose walls are defined by the oxide layer 106. The two base surface areas are therefore membrane and C. electrochemical The ratio of C to C can be independently optimized to provide the desired ratio between C and C. 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 to the coated portion of the working electrode 1102 beneath the dielectric layer 1104. This results in a maximum available surface area of ​​TiN in contact with the electrolyte, resulting in a high C electrochemical is the maximum.

[0055] Further description of the design of non-Faradaic electrochemical cells, TiN working electrodes, and other nanopore-based cell designs useful in the devices, compositions, and methods of the present disclosure can be found, for example, in U.S. Pat. No. 10,174,371, which is incorporated herein by reference.

[0056] As described elsewhere herein, the electrochemical cell of the present disclosure can be used as a device for nanopore-based nucleic acid detection and measurement assays, including nucleic acid sequencing. In general, nanopore-based sequencing of target nucleic acids can be performed using an electrochemical cell of the above design and associated solution compositions that include electrolytes and buffers that reduce precipitate formation as described above. The method includes: (a) providing 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 comprising ferrocyanide ions, ferricyanide ions, and a buffer composition; and (ii) inlet and outlet ports comprising 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 that translocates at least a portion of the molecule through the nanopore; and (d) detecting a change in voltage flow in the cell as the molecule translocates through the nanopore, where the change in voltage flow is indicative of the sequence of the target nucleic acid.

[0057] As described elsewhere herein, the particular potentials applied, their timing and duration can significantly affect the highly sensitive changes in ion flow conductance through the nanopore that occur in the presence of nucleic acid molecule moieties to be detected in 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. Pat. Nos. 1,115,0216 and 1,102,9306, 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, resulting in the formation of deleterious precipitates such as Prussian blue. A surprising advantage of the electrochemical cells of the present disclosure and the associated solutions used therewith is that, in some embodiments, the method of nanopore sequencing using the electrochemical cells of the present disclosure can be performed using alternating current (AC) applied to the cell at a voltage range of 450 mV to 1200 mV for 10 hours without any visible formation of Prussian blue within the cell.

[0058] In some embodiments of the sequencing methods of the present disclosure, the applied voltage comprises applying a baseline potential to the cell. Typical baseline voltages applied may range from about 55 mV to about 95 mV.

[0059] In some embodiments of the sequencing method, the applied voltage can include a pulsed voltage. A pulsed voltage useful in the present method can be from about 320 mV to about 550 mV. The duration of the pulsed voltage is typically from about 5 μs to about 10 μs. In at least one embodiment, the time between the pulsed voltages is from about 0.5 ms to about 1.7 ms. In at least one embodiment, the applied voltage provides an alternating current to the electrochemical cell. For example, the resulting alternating current can have a periodicity of from about 0.4 seconds to about 6 seconds.

[0060] It is believed that in at least one embodiment, the array of electrochemical cells of the present disclosure can be used to perform a method of sequencing in parallel. Such parallel nanopore-based sequencing of nucleic acids using a parallel array of electrochemical cells has been used with nanopore-based sequencing by synthesis (Nano-SBS) technology. System compositions and methods for Nano-SBS are known in the art. See, for example, U.S. Patent Application Publication Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0368710, and 2018 / 0057870A1, and WO 2019 / 166457. It is believed that these known systems, compositions, and methods can be used or adapted for use with the electrochemical cells of the present disclosure. A description of the use of Nano-SBS in the context of the electrochemical cells and compositions of the present disclosure is provided below.

[0061] FIG. 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 to be sequenced and a primer are introduced into the electrochemical cell 200. To this template-primer complex, four differently tagged nucleotides 208 are added in the bulk aqueous phase. When a correctly tagged nucleotide is complexed with a polymerase 204, the tail of the tag is positioned within the barrel of the nanopore 206. The tag held within the barrel of the nanopore 206 generates a unique ion-blocking signal 210, thereby electronically distinguishing the added base due to the different chemical structures of the tags.

[0062] FIG. 3 illustrates one embodiment of a cell that is intended to perform nucleotide sequencing with preloaded tags. A nanopore 301 is formed in a membrane 302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) is associated with the nanopore. In some cases, the enzyme 303 is covalently attached to the nanopore 301. The polymerase 303 is associated with a nucleic acid molecule 304 to be sequenced. The associated nucleic acid molecule 304 may be linear or circular. In some embodiments, a nucleic acid primer 305 hybridizes to a portion of the nucleic acid molecule 304. The polymerase 303 catalyzes the incorporation of a nucleotide 306 into the primer 305, using the single-stranded nucleic acid molecule 304 as a template. As described above, the nucleotide 306 includes a tag species ("tag") 307 that allows it to be distinguished from the other three nucleotides.

[0063] FIG. 4 illustrates one embodiment of a process 400 for nucleic acid sequencing with preloaded tags. In step A, the tagged nucleotides (one of four different types: A, T, G, or C) are not associated with the polymerase. In step B, the tagged nucleotides are associated with the polymerase. In step C, the polymerase is in close proximity to the nanopore. The tags are drawn into the nanopore by an electric field generated by a voltage applied across the membrane and / or the nanopore. Some of the tagged nucleotides that are associated are not base-paired with the nucleic acid molecule. These unpaired nucleotides are typically rejected by the polymerase within a time scale that is shorter than the time scale that correctly paired nucleotides remain associated with the polymerase. Because the unpaired nucleotides are only transiently associated with the polymerase, the process 400 illustrated in FIG. 4 typically does not proceed beyond step B.

[0064] Before the polymerase docks into the nanopore, the conductance of the nanopore is about 300 picosiemens (300 pS). In stage C, the conductance of the nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS, corresponding to one of the four types of tagged nucleotides. The polymerase undergoes isomerization and transphosphorylation reactions to incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule. Notably, when the tag is retained within the nanopore, a unique conductance signal (see, e.g., signal 210 in FIG. 2) is generated due to the distinct chemical structure of the tag, thereby electronically identifying the added base. Repeating the cycle (i.e., stages A-E or stages A-F) allows sequencing of the nucleic acid molecule. In stage 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 incorporated into the growing nucleic acid molecule also pass through the nanopore. Although unincorporated nucleotides may in some cases be detected by the nanopore, the present method provides a means to distinguish between incorporated and unincorporated nucleotides based at least in part on the time the nucleotide is detected at the nanopore. Tags attached to unincorporated nucleotides pass through the nanopore quickly and are detected for a short period of time (e.g., less than 10 ms), whereas tags attached to incorporated nucleotides are loaded into the nanopore and detected over an extended period of time (e.g., at least 10 ms).

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

[0067] FIG. 6 illustrates 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 when the nanopore is in a particular detectable state. One possible state of the nanopore is an open channel state when a nanomolecular moiety (e.g., a tag) is present in the barrel of the nanopore. Other possible states of the nanopore correspond to states when four different types of molecular moieties are present in the barrel of the nanopore. Yet another possible state of the nanopore is when the membrane is ruptured. FIG. 6 illustrates a nanopore 602 inserted into a membrane 612, where the nanopore 602 and membrane 612 are positioned between a cell working electrode 614 and a counter electrode 616 such that a voltage is applied across the nanopore 602. The nanopore 602 is also in contact with a bulk liquid / electrolyte 618. Note that the nanopore 602 and membrane 612 are depicted upside down compared to the nanopore and membrane of FIG. 1. Hereinafter, an electrochemical cell is meant to include at least a membrane, a nanopore, a working electrode, and associated circuitry. In some embodiments, the counter electrode is shared between multiple cells (e.g., in an array) and is therefore also referred to as 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 the common electrode are common to all measurement cells. Within each measurement cell there is a metal working cell electrode, and in contrast to the common electrode, the metal working cell electrode 614 can be configured to apply a separate potential independent of the working cell electrodes of the other measurement cells.

[0068] In addition to the Nano-SBS sequencing described above, in some embodiments, the electrochemical cell of the present disclosure can be used to perform parallel sequencing of nucleic acids using nanopore-based sequencing-by-expansion (Nano-SBX) technology. See, for example, U.S. Patent No. 7,939,259. SBX technology is based on the polymerization of highly modified non-natural nucleotide analogs called "XNTPs." In general, SBX uses biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer." The transcribed sequence is encoded along the Xpandomer backbone in high signal-to-noise reporters spaced approximately 10 nm apart, designed for high signal-to-noise, highly differentiated response. These differences result in a significant performance improvement in sequence read efficiency and accuracy of the Xpandomer compared to natural DNA. A general description of the SBX process is shown in Figures 7, 8, 9, and 10.

[0069] XNTPs are extendable 5' triphosphate modified unnatural nucleotide analogs compatible with template-dependent enzymatic polymerization. A highly simplified version of XNTPs is shown in Figure 7, which highlights the unique features of these unnatural substrates. XNTPs 100 have two distinct functional regions: a selectively cleavable phosphoramidate bond 110 that connects the 5'α-phosphate 115 to the nucleobase 105, and a symmetrically synthesized reporter tether (SSRT) 120 that is attached within the nucleoside triphosphoramidate at a position that allows controlled extension by cleavage of the phosphoramidate bond. The SSRTs contain linkers 125A and 125B separated by selectively cleavable phosphoramidate bonds. Each linker is attached to one end of a reporter code 130. XNTPs 100 are shown in a "constrained configuration" characteristic of XNTP substrates and daughter strand products of template-dependent polymerization. The constrained conformation of polymerized XNTPs is the precursor to the extended conformation, as seen in the Xpandomer product. The transition from the constrained conformation to the extended conformation occurs upon cleavage of the phosphoramidate PN bond in the primary backbone of the daughter strand.

[0070] The synthesis of Xpandomer polymers is summarized in Figures 8 and 9. As shown in Figure 8, during assembly, monomeric XNTP substrates 145 (XATP, XCTP, XGTP and XTTP) are polymerized at the extendable end of a nascent daughter strand 150 by a process of template-directed polymerization using a single-stranded template 140 as a guide. Generally, the process starts from a primer and proceeds in the 5' to 3' direction. Generally, a DNA polymerase or other polymerase is used to form the daughter strand, and conditions are selected to result in a complementary copy of the template strand. After the daughter strand is synthesized, the coupled SSRT forms a constrained Xpandomer that further forms the daughter strand. The SSRT in the daughter strand has the "constrained configuration" of the XNTP substrate. The constrained configuration of the SSRT 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 results from the cleavage of a selectively cleavable phosphoramidate bond (indicated by an unshaded oval for simplicity) in the primary backbone of the daughter strand. In this embodiment, the SSRTs contain one or more reporters or reporter codes 130A, 130C, 130G or 130T specific to the nucleobase to which they are linked, thereby encoding the sequence information of the template. In this way, the SSRTs provide 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 Xpandomer 165 moving from cis chamber 175 to trans chamber 185 through nanopore 180. Upon passing through the nanopore, each reporter code of the linearized Xpandomer generates a distinct and reproducible electronic signal specific to the nucleobase to which it is linked. This signal is shown by overlay trace 190.

[0073] Figure 11 shows a generalized structure of one embodiment of XNTP in more detail. XNTP 200 comprises a nucleoside triphosphoramidate 210 with linker arm moieties 220A and 220B separated by a selectively cleavable phosphoramidate bond 230. SSRT 275 is linked to the nucleoside triphosphoramidate with linking groups 250A and 250B, with a first SSRT end linked to a heterocycle 260 (represented here by cytosine, but the heterocycle may be any one of the four standard nucleobases, A, C, G or T), and a second SSRT end linked to the alpha-phosphate 270 of the nucleobase backbone. Those skilled in the art will appreciate that many suitable coupling chemistries known in the art can be used to form the final XNTP substrate product, for example, SSRT conjugates can be achieved by the formation of triazole linking groups.

[0074] In this embodiment, 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, SSRT can include a single TCE. Each of these features performs a unique function during the translocation of Xpandomer through the nanopore, generating a set of unique and reproducible electronic signals. SSRT275 is designed to control the rate of translocation of Xpandomer by TCE through a combination of steric hindrance and / or electrical repulsion, as discussed elsewhere herein. The 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 typically used for 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 lengths of 1-12 or more ethylene glycol units, and aliphatic polymers having lengths of 1-12 or more carbon units. In certain embodiments, the SSRT contains a feature called a "polymerase enhancing region" at the end of the SSRT proximal to the nucleotide triphosphoramidate diester. The polymerase enhancing region can contain a positively charged polyamine spacer (e.g., a primary, secondary, tertiary or quaternary amine) or a triamine spacer (three secondary amines separated by three carbons each) that promotes incorporation of the XNTP structure by a nucleic acid polymerase. In certain embodiments, the polymerase enhancing region includes two repeating units of spermine, where the spermine portion is provided by a phosphoramidite monomer having the following structure (as one of skill in the art will recognize, the trifluoroacetamide protecting group is removed at the end of the SSRT synthesis to expose the amine group on the spermine): [ka]

[0076] As used throughout this disclosure, the term "reporter construct" refers to elements of the SSRT, including a reporter code, a symmetric chemical branching chain, and a migration control element. In certain embodiments, the reporter construct is a polymer that includes, consecutively from a first end to a second end, a first reporter code, a symmetric chemical branching chain carrying a migration control element, and a second reporter code. The term "carries" 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 two reporter codes attached to the arms of the Y and a migration control element attached to the stem of the Y. Thus, the two reporter codes are linked in-line by the branching chain, while the branching chain carries the migration control element perpendicular to the linear in-line SSRT.

[0077] As used throughout this disclosure, the terms "Linker A" and "Linker B" refer to regions of the SSRT that comprise a polymerase enhancing region and one or more translocation slowing features or regions, respectively, and in certain embodiments, a spacer region comprising a polymer, e.g., PEG6, that can be customized to modulate the length of the SSRT traversing the nanopore.

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

[0079] In one embodiment, R may be H, for example, when the compound is used to sequence a DNA template. In other embodiments, R may be OH, for example, when the compound is used to sequence an RNA template. In certain embodiments, the nucleobase is adenine, cytosine, guanine, thymine, uracil, or a nucleobase analog. As will be appreciated by those skilled in the art, adenine, cytosine, guanine, thymine, and uracil are naturally occurring nucleobases. As used herein, the term "nucleobase analog" refers to a non-naturally occurring nucleobase that can form a Watson-Crick base pair with a complementary nucleobase on an adjacent single-stranded nucleic acid template. Exemplary nucleobase analogs include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylketone, 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- These include, but are not limited to, mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxosine, pseudouracil, queosine, 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-deazainosine, and 8-aza-7-deazaadenine.

[0080] As discussed elsewhere herein, the reporter construct is a polymer with a first end and a second end, which includes, consecutively from the first end to the second end, a first reporter code, a symmetric chemical branch carrying a migration control element, and a second reporter code. This series 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 are linked in-line in reverse by the symmetric chemical branch. Briefly, synthesis proceeds in the 3' to 5' direction, starting at the 3' end of TCE. The addition of the symmetric branch to the 5' end of TCE allows for simultaneous polymerization of the first and second reporter codes from each arm of the branch, followed by simultaneous synthesis of linker A and linker B, terminating at the 5' end of the first and second ends of the SSRT. The in-line redundancy provided by two identical reporter codes separated by symmetrical branching strands carrying translocation control elements was found to offer several advantages during nanopore sequencing. For example, the Xpandomer could potentially be read by the nanopore when translocating in either direction, i.e., the Xpandomer could be read either "forward" or "backward". This flexibility allows for "ratcheting" methods of sequencing, discussed further herein, and other methods such as "flossing" based on AC patterns of voltage application.

[0081] FIG. 12 shows one embodiment of a cut Xpandomer in the process of translocating 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 a pH of 7.0. When a small voltage, typically 100 mV, is applied across the bilayer, the nanopore provides the only channel for the flow of ions induced by the potential and is the source primary resistance in the circuit. The Xpandomer reporter code is designed to give a specific ion flow blockage level, and a pulse of potential results in the sequence of the reporter code translocating through the nanopore, so that the sequence information can be read by measuring the sequence of ion flow levels. In the case of the α-hemolysin nanopore, the nanopore is typically embedded in the membrane such that translocation occurs by entering the cis vestibular side and exiting the trans basal side. As shown in FIG. 12, the nanopore is oriented to capture the Xpandomer from the basal side first. As the Xpandomer translocates the nanopore, the reporter enters the base until its translocation control element stops it at the base entrance. The reporter is held at the base until TCE is allowed to enter and pass through the base, after which translocation proceeds to the next reporter. In this embodiment, passage of TCE into the base is allowed by dissociating the translocation control moiety from the TCE. EXAMPLES

[0082] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily appreciate that the specific examples are merely illustrative of the invention as more fully described in the claims that follow. It should be understood that all embodiments and features described in this application are interchangeable and combinable with all embodiments contained therein.

[0083] Example 1: Effect of alternative buffer components and metal materials on Prussian blue formation This example illustrates a study of the effect 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 chamber of the electrochemical cell used for SBX nanopore sequencing has an interior stainless steel surface, including the inlet and outlet ports, that contacts the solution contained in the cell, which contains ammonium chloride and potassium ferrocyanide and potassium ferricyanide at approximately 250 mM, respectively. To simulate the conditions that occur within such femtoliter nanopore sequencing chambers in a larger volume, scratch cannulas made of various types of metal alloys were immersed in buffer solutions containing either ammonium chloride or ammonium acetate, various 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. The 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 the stainless steel SS316 cannula with the non-ferrous nickel alloy C276 or C22 cannula was highly effective in preventing the formation of Prussian blue over a period of days or even weeks.

[0087] Furthermore, changes in buffer composition had a significant effect on Prussian blue formation. Specifically, replacing 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: Effect of alternative buffer components and metal materials on Prussian blue formation during nanopore sequencing This example describes a 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 herein, SBX nanopore sequencing is carried out using an array of electrochemical cells containing buffers containing ferricyanide and ferrocyanide ions. During use, solutions enter and exit the cells via inlet and outlet ports.

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

[0091] Results: Changing the port material from stainless steel to a nickel alloy was found to reduce cell failures due to clogging by Prussian blue precipitate from approximately 35% to less than 5%.

[0092] All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference herein for all purposes.

[0093] While various particular 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 invention.

Claims

1. 1. An electrochemical cell comprising: (a) a nanopore embedded in a membrane, said membrane separating said cell into cis and trans chambers connected by said nanopore, said cis and trans chambers each containing an electrode, a solution containing ferrocyanide ions, ferricyanide ions, and a buffer composition; (b) inlet and outlet ports operably connected to the cell, the ports comprising a metal; and 1. An electrochemical cell comprising:

2. The metal is dissolved in 1M NH ４ Cl, 800 mM urea, 100 mM HEPES, in a buffer solution of pH 7.4, or 1 M NH ４ When polarized to a potential of about 0.3 V against a Ag / AgCl reference electrode in a buffer solution of 800 mM Cl, 800 mM urea, and 100 mM MES, －３ mA / cm ２ 2. The cell of claim 1 exhibiting a current density of:

3. 3. The cell of claim 1 or 2, wherein the metals comprise less than 10% iron, less than 8% iron, less than 6% iron, or less than 5% iron.

4. The cell of any one of claims 1 to 3, wherein the metal is a nickel alloy.

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

6. The cell of any one of claims 1 to 5, wherein the cell contains a solution volume of between about 0.1 femtoliters and about 1000 femtoliters.

7. The cell of any one of claims 1 to 6, wherein the ferrocyanide and ferricyanide ions are at a total concentration of between about 25 mM and 250 mM.

8. The cell of any one of claims 1 to 7, wherein the buffer composition comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM.

9. The cell of any one of claims 1 to 8, wherein the buffer composition comprises ammonium chloride at a concentration of less than 2000 mM or less than 1000 mM.

10. 10. The cell of any one of claims 1 to 9, 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, optionally comprising an ammonium acetate concentration of 0 mM and an ammonium chloride concentration of less than 1000 mM.

11. 11. The cell of any one of claims 1 to 10, wherein application of an alternating current (AC) to the cell in the voltage range of 450 mV to 1200 mV does not result in visible formation of Prussian blue in 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.

12. 1. A method for sequencing a target nucleic acid, comprising: (a) providing an electrochemical cell comprising: (i) a nanopore embedded in a membrane, said membrane separating said cell into cis and trans chambers operably connected by said nanopore, said cis and trans chambers each comprising an electrode, a solution comprising ferrocyanide and ferricyanide ions, and a buffer composition; and (ii) inlet and outlet ports comprising a metal connected to said cell; (b) adding a molecule derived from a target nucleic acid to the cis chamber; (c) applying a voltage to the cell that translocates at least a portion of the molecule through the nanopore; (d) detecting a change in voltage flow in the cell as the molecule translocates through the nanopore, the change in voltage flow being indicative of a sequence of the target nucleic acid; and A method comprising:

13. The metal is dissolved in 1M NH ４ Cl, 800 mM urea, 100 mM HEPES, in a buffer solution of pH 7.4, or 1 M NH ４ When polarized to a potential of about 0.3 V against a Ag / AgCl reference electrode in a buffer solution of 800 mM Cl, 800 mM urea, and 100 mM MES, －３ mA / cm ２ 13. The method of claim 12, which exhibits a current density of:

14. 14. The method of any one of claims 12 to 13, wherein the metal comprises less than 10% iron, less than 8% iron, less than 6% iron, or less than 5% iron.

15. 15. The method of any one of claims 12 to 14, wherein the metal is a nickel alloy, optionally the metal is a nickel alloy selected from C276, C22, 625, and MP35N.

16. The method of any one of claims 12 to 15, wherein the cell contains a solution volume of between about 0.1 and 1000 femtoliters.

17. 17. The method of any one of claims 12 to 16, wherein the ferrocyanide and ferricyanide ions are at a total concentration of between about 25 mM and 250 mM.

18. 18. The method of any one of claims 12 to 17, wherein the buffer composition comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM.

19. 19. The method of any one of claims 12 to 18, wherein the buffer composition comprises ammonium chloride at a concentration of less than about 2000 mM or less than about 1000 mM.

20. 20. The method of any one of claims 12 to 19, 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, optionally comprising an ammonium acetate concentration of 0 mM and an ammonium chloride concentration of less than 1000 mM.

21. 21. The method of any one of claims 12 to 20, wherein applying an alternating current (AC) to the cell in the voltage range of 450 mV to 1200 mV does not result in visible formation of Prussian blue in 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.

22. 22. The method of any one of claims 12 to 21, wherein the applied voltage comprises a baseline voltage, optionally wherein the baseline voltage is from about 55 mV to about 95 mV.

23. 22. The method of any one of claims 12 to 21, wherein the applied voltage comprises a pulsed voltage, and optionally, the pulsed voltage is from about 320 mV to about 550 mV.

24. The method of any one of claims 12 to 23, wherein the pulse voltage has a duration of about 5 μs to about 15 μs.

25. The method of any one of claims 12 to 24, wherein the time between the pulse voltages is from about 0.5 ms to about 1.7 ms.

26. 26. The method of any one of claims 12 to 25, wherein the applied voltage provides an alternating current, and optionally, the alternating current has a periodicity of from about 0.4 seconds to about 6 seconds.

27. The method of any one of claims 12 to 26, wherein the molecule derived from the target nucleic acid comprises an Xpandomer.

28. 28. The method of claim 27, wherein the Xpandomer comprises a plurality of XNTP subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, each XNTP subunit of the chain comprises a reporter construct, a nucleobase residue and a selectively cleavable bond, and cleavage of the selectively cleavable bond results in an Xpandomer of a length greater than the plurality of XNTP subunits of the chain.

29. 30. The method of claim 28, wherein the method further comprises cleaving the selectively cleavable bond to obtain an Xpandomer.

30. The method of any one of claims 27 to 29, wherein the Xpandomer comprises a reporter construct for analyzing genetic information in a sequence corresponding to the continuous nucleotide sequence of all or a portion of the target nucleic acid.

31. 31. The method of claim 30, wherein detecting the change in voltage comprises detecting the change in voltage due to the reporter construct of the Xpandomer translocating the nanopore.

32. The method of claim 31, wherein the reporter construct for analyzing the genetic information comprises a reporter code and a translocation control element, the translocation control element provides translocation control by steric hindrance and pauses translocation of the Xpandomer when passing through a nanopore subjected to a baseline voltage, the translocation control element engages with the reporter code within the opening of the nanopore, and the reporter code is sensed by the nanopore.

33. The method according to any one of claims 12 to 32, wherein the electrochemical cell is an electrochemical cell according to any one of claims 1 to 11.

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