Compositions and methods for reducing prussian blue formation during nanopore sequencing

Abstract

Disclosed herein are electrochemical cells, nanopore devices, and related buffer compositions useful for nanopore-based nucleic acid sequencing. Also disclosed are methods for using the electrochemical cells, devices, and compositions in nanopore-based nucleic acid sequencing methods, such as nanopore extension sequencing (Nano-SBX) and nanopore synthesis sequencing (Nano-SBS) methods.

Description

Technical Field

[0001] The present application relates to electrochemical cells and related buffer compositions that reduce 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 Art

[0002] Nanopore-based nucleic acid sequencing is an attractive approach that has been extensively studied. In one approach, the sequence of a single-stranded polynucleotide is 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. Partial blockage of the nanopore aperture during polynucleotide translocation alters the ion flow over time, resulting in current changes that can be measured by the cell.

[0003] Expanded sequencing ("SBX") is a nanopore-based nucleic acid sequencing method that uses a biochemical process to transcribe a DNA sequence onto a measurable polymer molecule called an "Xpandomer". See, for example, U.S. Patent No. 7,939,259, entitled "High-throughput nucleic acid sequencing by expansion"; and PCT Publication No. WO2020236526A1, entitled "Translocation control elements, reporter codes, and other means for translocation control for 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, so as to provide a high signal-to-noise ratio and well-differentiated response signal during nanopore translocation. The enhanced signal-to-noise ratio provided by the different response signals significantly improves the sequence reading efficiency and accuracy of Xpandomer relative to natural nucleic acid molecules.

[0004] Nanopore-based sequencing by synthesis ("SBS") uses a polymerase (or other chain extension enzyme) covalently linked to a nanopore to synthesize a DNA chain complementary to a target sequence template (i.e., a copy chain). A nanopore embedded in a membrane of an electrochemical cell is used to simultaneously detect the identity of each nucleotide monomer as it is added to the growing chain. See, for example, U.S. Patent Publications Nos. 2013 / 0244340A1, 2013 / 0264207A1, 2014 / 0134616A1, 2015 / 0368710A1, and 2018 / 0057870A1, and discloses International Application WO 2019 / 166457A1. When the tag portion attached to each added nucleotide monomer enters the nanopore and changes the ion flow, each added nucleotide monomer is detected by monitoring the signal due to the change in ion flow through the nanopore. For optimal performance, the tag moiety should reside in the nanopore long enough to provide a detectable, identifiable, and reproducible signal associated with altered ion flow through the nanopore (relative to a baseline "open current" flow) such that a specific nucleotide associated with the tag can be unambiguously distinguished from other tagged nucleotides in the SBS solution.

[0005] However, nanopore-based sequencing faces the burden of having to resolve small current signal differences immersed in significant background noise in a microvolume electrochemical cell. This measurement challenge is compounded by small variations 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 the same to reduce or prevent the formation of deleterious byproducts (e.g., Prussian blue precipitants) that interfere with accurate detection of electrochemical signals in nanopore-based nucleic acid sequencing devices, systems, and methods. Summary of the invention

[0007] The present disclosure generally relates to electrochemical cells and related buffer compositions used in cells to reduce the deleterious formation of Prussian blue during nanopore-based assays, and methods of 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 cover every embodiment, combination or variation contemplated and described in the present disclosure. Embodiments are further contemplated and described through the disclosure of 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 that divides the cell into a cis chamber and a trans chamber that are operably connected by the nanopore, wherein the cis chamber and the trans chamber each contain an electrode, a solution comprising ferrocyanide ions and ferrocyanide ions, and a buffer composition; and (b) an inlet port and an outlet port operably connected to the cell, wherein the ports comprise a metal.

[0009] In at least one embodiment of the electrochemical cell of the present disclosure, the metal is in 1M NH 4 Cl, 800 mM urea, 100 mM HEPES, pH 7.4 buffer or in 1 M NH 4 Cl, 800 mM urea, 100 mM MES, pH 6.2-6.8 buffer solution polarized to a potential of about 0.3 V relative to the Ag / AgCl reference electrode exhibited a 10 -3 mA / cm 2 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 encloses 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 concentration of ferrocyanide ions and ferrocyanide ions is 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, an alternating current (AC) in the voltage range of 450 mV to 1200 mV is applied to the cell such that no visible Prussian blue is formed 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, wherein the method comprises:

[0015] (a) providing an electrochemical cell comprising (i) a nanopore embedded in a membrane, the membrane separating the cell into a cis chamber and a trans chamber operably connected by the nanopore, wherein the cis chamber and the trans chamber each contain an electrode, a solution comprising ferrocyanide ions, ferrocyanide ions, and a buffer composition; and (ii) an inlet port and an outlet port operably connected to the cell comprising a metal;

[0016] (b) adding a molecule derived from the target nucleic acid to the cis chamber;

[0017] (c) applying a voltage to the cell such that at least a portion of the molecules are translocated through the nanopore; and

[0018] (d) detecting a change in voltage current in the cell as the molecule translocates through the nanopore, wherein the change in voltage current is indicative of the sequence of the target nucleic acid.

[0019] In at least one embodiment of the sequencing method disclosed herein, when 1M NH 4 Cl, 800 mM urea, 100 mM HEPES, pH 7.4 buffer or in 1 M NH 4 Cl, 800mM urea, 100mM MES, pH 6.2-6.8 buffer solution, relative to the Ag / AgCl reference electrode polarized to a potential of about 0.3V, exhibited a 10- 3 mA / cm 2 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.

[0020] In at least one embodiment of the sequencing method of the present disclosure, the cell encloses a solution volume between about 0.1 femtoliters and 1000 femtoliters.

[0021] In at least one embodiment of the sequencing method disclosed herein, the total concentration of ferrocyanide ions and ferrocyanide ions is between about 25 mM and 250 mM.

[0022] 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.

[0023] In at least one embodiment of the sequencing method of the present disclosure, an alternating current (AC) with a voltage ranging from 450 mV to 1200 mV is applied to the pool such that no visible Prussian blue is formed in the pool 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.

[0024] In at least one embodiment of the sequencing method of the present disclosure, the applied voltage comprises a baseline voltage; optionally, wherein the baseline voltage is about 55mV to about 95mV. In at least one embodiment, the applied voltage comprises a pulse voltage; optionally, wherein the pulse voltage is about 320mV to about 550mV. In at least one embodiment, the pulse voltage has a duration of about 5μs to about 10μs. In at least one embodiment, the time interval between the pulse voltages is about 0.5ms to about 1.7ms. In at least one embodiment, the applied voltage provides an alternating current; optionally, wherein the alternating current has a periodicity of about 0.4s to about 6s.

[0025] In at least one embodiment of the sequencing methods 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 in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual XNTP subunits of the chain comprise a reporter construct, a nucleobase residue, and a selectively cleavable bond, and wherein cleavage of the selectively cleavable bond produces an Xpandomer that is longer in length than the plurality of XNTP subunits of the chain.

[0026] In at least one embodiment of the sequencing method of the present disclosure, the method further comprises cleaving the selectively cleavable bond to generate an Xpandomer.

[0027] In at least one embodiment of the sequencing method of the present disclosure, the Xpandomer includes a reporter construct for resolving genetic information in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid. In at least one embodiment, detecting a change in voltage includes detecting a change in voltage caused by displacement of the nanopore by the reporter construct of the Xpandomer.

[0028] In at least one embodiment of the sequencing method of the present disclosure, a reporter construct for parsing genetic information includes a reporter code and a translocation control element, wherein the translocation control element provides translocation control by means of steric hindrance and pauses translocation when the Xpandomer passes through a nanopore subjected to a baseline voltage, wherein the translocation control element engages the reporter code within the orifice of the nanopore, wherein the reporter code is sensed by the nanopore. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The novel features and advantages of the present disclosure may be better understood by reference to the following detailed description, which sets forth illustrative embodiments utilizing the principles of the present disclosure, and the accompanying drawings (also referred to herein as "Figures" and "Diagrams"), in which:

[0030] Figure 1 An embodiment of a cell 100 in a nanopore-based sequencing chip is depicted.

[0031] Figure 2 An embodiment of a cell 200 for nucleotide sequencing using Nano-SBS technology is depicted.

[0032] Figure 3 An embodiment is depicted for a pool for nucleotide sequencing with pre-loaded tags.

[0033] Figure 4 An embodiment of a process 400 for nucleic acid sequencing with pre-loaded tags is depicted.

[0034] Figure 5 An embodiment of a circuit 500 in a cell of a nanopore-based sequencing chip is depicted.

[0035] Figure 6 An embodiment of a circuit 600 in a cell of a nanopore-based sequencing chip is depicted, wherein a voltage applied across a nanopore can be configured to vary over a period of time that the nanopore is in a particular detectable state.

[0036] Figure 7 Schematic diagram depicting the characteristics of generalized XNTPs and their function in Nano-SBX technology.

[0037] Figure 8 Schematic diagram depicting the characteristics of generalized XNTPs and their function in Nano-SBX technology.

[0038] Fig. 9 Schematic diagram depicting the characteristics of generalized XNTPs and their function in Nano-SBX technology.

[0039] Fig.10 Schematic diagram depicting the characteristics of generalized XNTPs and their function in Nano-SBX technology.

[0040] Fig.11 is a schematic diagram showing more details of an embodiment of XNTP.

[0041] Fig.12 is a schematic diagram showing an example of an Xpandomer passing through a biological nanopore. DETAILED DESCRIPTION

[0042] The present disclosure provides an electrochemical cell and a related buffer composition for the cell. The electrochemical cell can be used to perform a nucleic acid assay method based on a nanopore, including sequencing based on a nanopore. The characteristics of the electrochemical cell and the related buffer composition produce surprising favorable results, i.e., the formation of harmful precipitants is reduced during the use of the cell in the assay based on the nanopore. Harmful precipitants include Prussian blue, which can be precipitated as a solid in the redox reaction involving ferrocyanide ions and ferrocyanide ions and the metallic iron in the electrochemical cell components (such as inlet ports and outlet ports). The formation of this precipitant can quickly block ports and / or nanopores, thereby greatly reducing the accuracy and efficiency of any nanopore-based assay. The present disclosure also provides a method for using an electrochemical cell and a composition for a nucleic acid detection technology based on a nanopore, such as sequencing by synthesis (Nano-SBS) based on a nanopore and extended sequencing based on a nanopore.

[0043] For the description herein and in the appended claims, the singular forms "a", "an" and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound. "Comprising" and "including" are used interchangeably and are not intended to be limiting. It should be further understood that if the term "comprising" is used in the description of multiple embodiments, those skilled in the art will understand that in some specific cases, the language "consisting essentially of" or "consisting of" may be used to alternatively describe the embodiments.

[0044] If a range of values ​​is provided, then unless the context clearly specifies otherwise, it is understood that each intermediate integer of the value between the upper and lower limits of the range and each tenth of each intermediate integer of the value (unless the context clearly specifies otherwise) and any other specified value or intermediate value within the specified range are encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also encompassed by the present invention, subject to any explicitly excluded limitations within the specified range. If the specified range includes one or two limits, then ranges excluding (i) any one or (ii) two of those included limitations are also included in the present invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0045] In general, the nomenclature used herein and the techniques and procedures described herein include common techniques and methods that are well understood and commonly employed by ordinary technicians in the art, such as described in, 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 referred to as "Sambrook") and Current Protocols in Molecular Biology, edited by F.M. Ausubel et al., originally published as a book in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented in 2011, now available online in journal form as "Current Protocols in Molecular Biology", Volumes. 00-130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter referred to as "Ausubel").

[0046] All publications, patents, patent applications, and other documents referenced in this disclosure are incorporated herein 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 for all purposes.

[0047] Unless otherwise defined, the meaning of all scientific and technical terms used herein is the same as that generally understood by those of ordinary skill in the art to which the present disclosure belongs. It should be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. For the purpose of explaining the present disclosure, the following description of terms will be applied, and where appropriate, terms used in the singular will also include the plural, and vice versa.

[0048] Nanopore-based nucleic acid detection device

[0049] 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, US9494554B2, US9567630B2, US9557294B2, US9605309B2, each of which is incorporated herein by reference in its entirety. These devices generally include an electrochemical cell having a chamber containing a nanopore embedded in a membrane. The function of the membrane is to divide the cell chamber into two subchambers, referred to as the cis side and the cis side 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 a non-natural polymeric material. The pores of the nanopore serve as channels (or passages) in the membrane between the cis side and the cis side of the cell. Depending on the nanopore used, the width or diameter of the pore may be in the range of about 1 angstrom to about 10,000 angstroms. The nanopore may be a naturally occurring pore-forming protein, such as a non-natural mutant or variant of α-hemolysin, a wild-type pore protein from Staphylococcus aureus. A range of naturally and non-naturally occurring nanopores with different pore sizes and properties are known in the art. See, for example, US10351908B2, US10934582B2, US10227645B2.

[0050] In an electrochemical cell, a nanopore embedded in a membrane is placed close to an electrode coupled to a sensing circuit, such as, for example, a complementary metal oxide semiconductor (CMOS) or field effect transistor (FET) circuit. When a voltage potential is applied (via the electrode) across a nanopore immersed in a conducting fluid, a small current can be observed due to the flow of ions through the nanopore. The ion flow is sensitive to the pore size, so molecules entering the pore affect the ion flow and the voltage measured by the sensor circuit.

[0051] Electrochemical cells for nanopore-based nucleic acid sequencing are typically used in large-scale parallel fashion, with thousands of such cells configured as arrays in a single device, which is typically referred to as a chip (or biochip). A typical nanopore-based sequencing chip device contains an array of one million or more electrochemical cells, and may include 1000 rows × 1000 columns of such cells (see, for example, chips manufactured by Roche Sequencing Solutions, Santa Clara, CA, USA). Methods for making and using such nanopore array microchips can also be found in U.S. Patent Application Publications Nos. 2013 / 0244340A1, US2013 / 0264207A1, US2014 / 0134616A1, 2015 / 0368710A1, and 2018 / 0057870A1, and published International Application WO 2019 / 166457A1, each of which is incorporated herein by reference. Each well in the array is manufactured using a standard CMOS process with surface modification, which allows continuous contact with biological reagents and conductive salts. Each well can support a phospholipid bilayer membrane in which a nanopore-polymerase conjugate is embedded. The electrodes at each well can be addressed individually by a computer interface. A computer-controlled syringe pump is used to introduce all reagents used into a simple flow cell above the array microchip. The chip supports analog-to-digital conversion and reports electrical measurements from all electrodes at a rate of more than 1000 points per second. Nanopore measurements can be performed at each membrane in the 8M addressable membranes containing nanopores in the array, at least once asynchronously per millisecond (msec), and recorded on an interface computer. A further description of an exemplary electrochemical cell for nucleic acid determination (e.g., sequencing) based on nanopores is provided below, which includes chamber and electrode materials, buffer solutions, sensing circuits, array devices, and their uses in various applications.

[0052] Figure 1 An exemplary embodiment of an electrochemical cell 100 that can be used for nanopore-based sequencing is shown, and the electrochemical cell is present in an array of such cells. A membrane 102 is formed on a surface in the cell. In some embodiments, the membrane 102 is a lipid bilayer. A bulk electrolyte solution 114 or a "protein nanopore transmembrane molecular complex" (PNTMC) 104 containing nanopores is placed directly on the surface in the cell, and a single PNTMC 104 is inserted into the membrane 102 using electroporation. The membranes embedded with nanopores in each cell of the array are neither chemically nor electrically connected to each other. Therefore, each cell is an independent sequencer that generates data specific to a single polymer molecule associated with the PNTMC.

[0053] Continue to refer Figure 1, the thin film of electrolyte solution 108 is separated from the bulk electrolyte solution 114 by the ion-impermeable membrane 102. Thus, the membrane embedded with nanopores divides 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 a metal 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.

[0054] The pores of the PNTMC 104 provide a channel through the membrane 102 that allows the flow of analytes (e.g., nucleic acids) and ions, which results in the modulation of the ionic current across the impermeable bilayer. Thus, the pores of the PNTMC 104 provide a unique path for the ionic current to flow from the metal 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, which further improves its sensitivity as an electrochemical potential sensor.

[0055] The thin film of electrolyte constitutes a femtoliter volume of solution in the trans chamber, which is in direct contact with the working electrode. The electrodes in contact with the electrochemical cell must undergo repeated voltage pulses when sensing the ion flow through the nanopore. These repeated electrochemical measurements using electrodes in a tiny volume of electrolyte, and the need to maintain the ion flow through the nanopore in repeated measurements corresponding to the displacement of a single molecule part through the nanopore require a sensor system with high sensitivity. The electrochemical measurement system is highly sensitive to any precipitated or aggregated molecular entities that may block the pores or otherwise interfere with the ion flow between the working electrode and the counter electrode. Therefore, the electrolyte salt, electrochemical cell materials, and electrochemical measurement conditions should be carefully controlled to prevent or reduce the formation of harmful precipitates or electrode wear in the system.

[0056] The electrolyte solution 108 may include 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 (CaCl 2 ), Strontium chloride (SrCl 2 ), manganese chloride (MnCl 2 ) and magnesium chloride (MgCl 2 ). In some embodiments, the film of the electrolyte solution has a thickness of about three micrometers (μm). The thickness of the film of the electrolyte solution may be in the range of 0 μm to 5 μm.

[0057] In addition to the above-mentioned salts, the electrolyte membrane may also contain redox active ions, such as ferrocyanide ions and ferrocyanide ions. These redox ions are used in certain nanopore-based sequencing methods performed in electrochemical cells. For example, the Nano-SBX measurement performed in the cell of the present disclosure may contain ferrocyanide ions and ferrocyanide ions. Such redox active ions may undergo irreversible reactions, depending on their concentration and the specific electrochemical conditions at the working electrode and the counter electrode. In fact, as described elsewhere herein, the irreversible formation of the precipitant Prussian blue in the electrochemical cell is highly harmful to any nanopore-based measurement in the cell. Therefore, the concentration of redox active ions such as ferrocyanide and ferrocyanide should be carefully controlled. In at least one embodiment of the present disclosure, any ferrocyanide ions and ferrocyanide ions in the solution in the electrochemical cell should be maintained at a total concentration between about 25mM and 250mM.

[0058] It is also found that the salt used in the electrolyte solution of the electrochemical cell can affect the ability of the cell to be used for sensitive nanopore-based nucleic acid measurements. For example, many sequencing schemes based on nanopores use ammonium chloride as a buffer salt in the electrolyte solution provided in the cell chamber. It has been found that in a nucleic acid sequencing method based on nanopores (such as Nano-SBX), by replacing the salt in the electrolyte solution with ammonium acetate to control the concentration of ammonium chloride, it can help reduce or prevent the formation of redox precipitates (such as Prussian blue), as described above, redox precipitates are very harmful to the accuracy and sensitivity of the electrochemical cell. Therefore, in some embodiments of the electrochemical cell of the present disclosure, the buffer composition used in the cell includes an ammonium acetate concentration of about 0mM to about 1500mM, and / or an ammonium chloride concentration of less than 2000mM. In at least one embodiment, the buffer composition includes an ammonium acetate concentration of about 0mM to about 1500mM, and / or an ammonium chloride concentration of less than 2000mM. In at least one embodiment, the concentration of ammonium acetate is 0mM and the concentration of ammonium chloride is about 600mM.

[0059] Dielectric materials are typically used to form an oxide layer 106 that defines the trans chamber of the cell. Available dielectric materials may include glass, oxide, silicon nitride (SiN), etc. In some embodiments, the top surface of the dielectric oxide layer 106 in contact with the main electrolyte 114 may be silanized. Silanization forms a hydrophobic layer over the top surface of the dielectric layer. In some embodiments, the hydrophobic layer has a thickness of about 1.5 nanometers (nm). Alternatively, a hydrophobic dielectric material (such as hafnium oxide) may be used to form the top of the dielectric layer.

[0060] like Figure 1As shown, the membrane is a lipid bilayer at least partially formed on the dielectric oxide layer 106 and spans the well of the thin film containing the electrolyte 108. For example, the membrane can be formed on top of the hydrophobic layer on the dielectric oxide membrane, and when the membrane reaches the opening of the well, the lipid monolayer is transformed into a lipid bilayer spanning the opening of the well. The hydrophobic layer facilitates the formation of the lipid monolayer above the dielectric layer and the transition from the lipid monolayer to the lipid bilayer.

[0061] The main electrolyte 114 may further include 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 (CaCl 2 ), Strontium chloride (SrCl 2 ), manganese chloride (MnCl 2 ) and magnesium chloride (MgCl 2 ). Therefore, in some embodiments of the electrochemical cell of the present disclosure, the buffer composition of the main electrolyte used in the cell comprises ammonium acetate at a concentration of about 0mM to about 1500mM, and / or ammonium chloride at a concentration of less than 2000mM. In at least one embodiment, the buffer composition comprises ammonium acetate at a concentration of about 0mM to about 1500mM, and / or ammonium chloride at a concentration of less than 2000mM. In at least one embodiment, the concentration of ammonium acetate is 0mM and the concentration of ammonium chloride is about 600mM. Similarly, in some embodiments, any ferricyanide ions and ferrocyanide ions in the main electrolyte solution of the electrochemical cell should be maintained at a total concentration of between about 25mM and 250mM.

[0062] As discussed above with respect to thin electrolyte membranes, 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 made using an electrochemical cell. It is expected that depending on the type of electrochemical measurement being performed, and in particular the potential applied to the electrodes in contact with the solution, the composition of the solution can be adjusted to reduce or prevent the formation of deleterious precipitates.

[0063] To allow for the necessary introduction and removal of solutions, the electrochemical cells of the present disclosure include inlet and outlet ports operably connected to a chamber or reservoir of the cell. Figure 1The schematic diagram of the electrochemical cell does not depict the inlet port and the outlet port, but it is well known in the art to add these ports to the electrochemical cell. The inlet port and the outlet port allow the reagents required for the nucleic acid detection based on the nanopore to be inserted, including materials for creating the membrane embedded in the nanopore, nucleotide synthesis reagents, and electrolytes and buffer solutions. Typically, the inlet port and the outlet port include a metal tube installed to the cell so as to provide a fluid connection from the outside to the chamber of the cell, which can be used to introduce or remove the solution. In this way, at least a portion of the inlet port and the outlet port are in contact with the electrochemical solution in the cell chamber. Therefore, under the voltage for inducing ion flow through the nanopore, the composition of the port should be electrochemically inert, and the change in ion flow is measured by sensory measurement when the molecular part enters and / or shifts through the nanopore. Stainless steel pipes have been used to provide metal pipes for the inlet port and the outlet port, however, steel contains iron. A surprising advantage of the electrochemical cell of the present disclosure is that the inlet port and the outlet port contain a metal with an iron content of less than 10%, and this in turn reduces or prevents the harmful formation of precipitates (such as Prussian blue) in the cell. Without being 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 ferrocyanide (K 3 [Fe(CN 6 ) 3 ]) is accelerated by the presence of potassium ferrocyanide, which is a strong oxidant. Chloride ions Cl- will induce pitting corrosion, causing stainless steel (and other ferrous metals) to release Fe 3+ Ion. Fe 3+ Ions can react with ferrocyanide ions ([Fe(CN) 6 ] 4- ) reaction, resulting in the formation of Prussian blue precipitate (Fe 4 [Fe(CN) 6 ] 3 ). The solutions used in electrochemical cells for nanopore sequencing typically require the presence of ferrocyanide ions or ferrocyanide ions, and it is believed that stainless steel parts in contact with the solution (e.g., inlet and outlet ports) lead to the formation of the precipitant Prussian blue under the electrochemical measurement conditions typically used in cells for nanopore-based sequencing. The formation of solid precipitants in electrochemical cells can lead to blockages and often cause rapid failure of the cell during nanopore-based measurements.

[0064] Although it has been determined that ferrous metals (such as stainless steel) are problematic for Prussian blue formation, more specifically, it has been found that the formation of Prussian blue or other metal precipitates can be reduced or prevented by using metal inlet and outlet ports, wherein the precipitates are formed in 1 M NH 4 Cl, 800 mM urea, 100 mM HEPES, pH 7.4 buffer or in 1 M NH 4Cl, 800 mM urea, 100 mM MES, pH 6.2-6.8 buffer solution compared to Ag / AgCl reference electrode polarized to a potential of about 0.3 V showed less than or equal to 10 -3 mA / cm 2 of current density.

[0065] Thus, in at least one embodiment of the electrochemical cell of the present disclosure, the inlet port and the outlet port of the cell are made of metal, wherein when in 1M NH 4 Cl, 800 mM urea, 100 mM HEPES, pH 7.4 buffer or in 1 M NH 4 C1, 800 mM urea, 100 mM MES, pH 6.2-6.8 buffer solution compared to Ag / AgCl reference electrode polarized to a potential of about 0.3 V showed less than or equal to 10 -3 mA / cm 2 As described above, generally, the metal should not contain iron, which excludes ports having stainless steel that are in contact with the solution in the cell during use. It has been found that when the cell is used with a solution containing ferrocyanide ions and ferrocyanide ions, ports made of iron-free nickel (such as a nickel alloy) play an excellent role in reducing and / or preventing the formation of Prussian blue. In particular, inlet ports and outlet ports made of a nickel alloy selected from C276, C22, 625 and MP35N provide excellent nanopore-based sequencing measurements and lifespan.

[0066] The electrochemical cell 100 includes a counter electrode (CE) 116 and a reference electrode 117, which are used as electrochemical potential sensors. In some embodiments, the counter electrode 116 is shared between 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 units.

[0067] In at least one embodiment, a non-Faraday electrochemical cell with a titanium nitride (TiN) working electrode has been found to be advantageous for nanopore-based nucleic acid sequencing. Figure 1 The cell of is similar to that of , but includes a TiN working electrode that exhibits increased electrochemical capacitance. The electrochemical cell also includes a conductive or metal layer that connects the working electrode of the cell to the circuitry of the array to which the cell belongs.

[0068] Thus, in at least one embodiment, the metal working electrode 110 is a titanium nitride (TiN) metal electrode having increased electrochemical capacitance. The electrochemical capacitance associated with the metal working electrode 110 can be increased by maximizing the specific surface area of ​​the electrode. The specific surface area of ​​the working electrode 110 is the total surface area per unit mass (e.g., m2 / kg) or total surface area per unit volume (e.g. m 2 / m 3 or m -1 ) or total surface area per unit base area (e.g. m 2 / m 2 ). As the surface area increases, the electrochemical capacitance of the working electrode increases, and a greater number of ions can be displaced at the same applied potential before the capacitor charges. The surface area of ​​the working electrode 110 can be increased by making the TiN electrode "spongy" or porous. The TiN sponge absorbs the electrolyte and creates a large effective surface area in contact with the electrolyte.

[0069] The capacitance associated with the membrane (C 膜 ) and the capacitance associated with the working electrode (C 电化学 ) ratio to achieve the best overall system performance. This can be achieved by reducing C 膜 At the same time, C 电化学 Maximize to increase system performance. Adjust C 膜 to form the desired RC time constant without the need for additional on-chip capacitance, thereby allowing for significant reductions in cell size and chip size. The base surface area of ​​the metal working electrode 110 is greater than or equal to the surface area of ​​the opening of the trans side well containing the electrolyte 108 having a well defined by the oxide layer 106. Therefore, the two base surface areas can be optimized independently to provide the desired C 膜 and C 电化学 By using a sponge-like porous TiN working electrode, the electrolyte can diffuse through the spaces between the columnar TiN structures and diffuse vertically downward to the uncovered portion of the working electrode, and then diffuse horizontally to the covered portion of the working electrode 1102 located below the dielectric layer 1104. As a result, the effective surface area of ​​the TiN in contact with the electrolyte is maximized, and C 电化学 is maximized.

[0070] Further description of non-Faraday electrochemical cells, TiN working electrodes, and other nanopore-based cell designs that can be used with the devices, compositions, and methods of the present disclosure can be found, for example, in US 10174371 B2, which is incorporated herein by reference.

[0071] As described elsewhere herein, the electrochemical cell of the present disclosure can be used as a device for nucleic acid detection and measurement determination (including nucleic acid sequencing) based on nanopores. In general, the sequencing of target nucleic acids based on nanopores can be performed using the electrochemical cell of the above-mentioned design and the related solution composition, and the solution composition includes an electrolyte and a buffer solution that reduce the formation of precipitants as described above. The method includes: (a) providing an electrochemical cell, the electrochemical cell including (i) a nanopore embedded in a membrane, the membrane separating the cell into a cis chamber and a trans chamber operably connected by the nanopore, wherein the cis chamber and the trans chamber each include an electrode, a solution including ferrocyanide ions, ferrocyanide ions, and a buffer composition; and (ii) including an inlet port and an outlet port operably connected to the cell, the inlet port and the outlet port including a metal; (b) adding a molecule derived from the target nucleic acid to the cis chamber; (c) applying a voltage to the cell so that at least a portion of the molecule is displaced through the nanopore; and (d) detecting a change in the voltage flow in the cell when the molecule is displaced through the nanopore, wherein the change in the voltage flow indicates the sequence of the target nucleic acid.

[0072] As described elsewhere herein, the timing and duration of the specific potential applied can have a significant effect on the highly sensitive changes in the ion flow conductance through the nanopore, which occurs in the presence of the nucleic acid molecule portion detected in the sequencing method. Exemplary methods and parameters for applying a voltage potential on an electrochemical cell array across a nanopore for sequencing are known in the art and are described in, for example, US11150216 and US11029306, each of which is incorporated herein by reference. The applied potential, their pulse timing and duration can also affect the redox chemistry in the pool, resulting in the formation of harmful precipitates, such as Prussian blue. The surprising advantage of the electrochemical cell of the present disclosure and the related solutions therein is that, in some embodiments, the nanopore sequencing method using the electrochemical cell of the present disclosure can apply alternating current (AC) to the pool for 10 hours in a voltage range of 450mV to 1200mV, so that no visible Prussian blue is formed in the pool.

[0073] In some embodiments of the sequencing methods of the present disclosure, the applied voltage comprises applying a baseline voltage potential to the cell. A typical baseline voltage applied can be in the range of about 55 mV to about 95 mV.

[0074] In some embodiments of the method for sequencing, the applied voltage may include a pulse voltage. The pulse voltage useful in the method may be about 320mV to about 550mV. The duration of the pulse voltage is generally about 5μs to about 10μs. In at least one embodiment, the time interval between the pulse voltages is about 0.5ms to 1.7ms. In at least one embodiment, the applied voltage provides an alternating current to the electrochemical cell. For example, the generated alternating current may have a periodicity of about 0.4s to about 6s.

[0075] In at least one embodiment, it is expected that the electrochemical cell array of the present disclosure can be used to perform sequencing methods in a parallel manner. This nanopore-based parallel nucleic acid sequencing using a parallel array of electrochemical cells has been used together 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 Publications Nos. 2013 / 0244340A1, 2013 / 0264207A1, 2014 / 0134616A1, 2015 / 0368710A1, and 2018 / 0057870A1, and International Application WO 2019 / 166457A1 is disclosed. It is expected that these known systems, compositions, and methods can be used with the electrochemical cell of the present disclosure or modified to be used with the electrochemical cell of the present disclosure. A description of using Nano-SBS in the context of the electrochemical cell and composition of the present disclosure is provided below.

[0076] Figure 2 An embodiment of an electrochemical cell 200 for nanopore-based nucleic acid sequencing using the Nano-SBS technique is shown. In the Nano-SBS technique, a template 202 to be sequenced and a primer are introduced into the electrochemical cell 200. In the template-primer complex, four nucleotides 208 with different tags are added to the bulk aqueous phase. When the correctly tagged nucleotides are complexed with the polymerase 204, the tail of the tag is positioned in the barrel of the nanopore 206. The tags held in the barrel of the nanopore 206 generate a unique ion blockade signal 210, thereby electronically identifying the added base due to the different chemical structures of the tags.

[0077] Figure 3 An embodiment of a pool in which nucleotide sequencing is to be performed using preloaded tags is shown. 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 polymerase 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 can be linear or circular. In some embodiments, a nucleic acid primer 305 hybridizes with 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 a nucleotide 306 into the primer 305. As described above, the nucleotide 306 contains a tag species ("tag") 307, thereby allowing it to be distinguished from the other three nucleotides.

[0078] Figure 4An embodiment of a process 400 for nucleic acid sequencing with pre-loaded tags is shown. In stage A, a tagged nucleotide (one of four different types: A, T, G, or C) is not associated with the polymerase. In stage B, the tagged nucleotide is associated with the polymerase. In stage C, the polymerase is in close proximity to the nanopore. The electric field generated by the voltage applied across the membrane and / or the nanopore pulls the tag into the nanopore. Some of the associated tagged nucleotides do not base pair with the nucleic acid molecule. These unpaired nucleotides are typically rejected by the polymerase within a shorter time frame than the time frame in which the correctly paired nucleotides remain associated with the polymerase. Since unpaired nucleotides are only transiently associated with the polymerase, Figure 4 The process 400 shown in FIG. 4 is typically not performed beyond stage B.

[0079] Before the polymerase docks with the nanopore, the conductance of the nanopore is about 300 pico-Siemens (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 labeled nucleotides. The polymerase performs isomerization and transphosphorylation reactions to incorporate the nucleotide into the growing nucleic acid molecule and release the label molecule. In particular, when the label is held in the nanopore, a unique conductivity signal is generated due to the different chemical structures of the label (e.g., see Figure 2 The signal 210 in the molecule is detected by the PCR reaction, thereby electronically identifying the added base. Repeating this cycle (i.e., stage A to stage E or stage A to stage F) allows the nucleic acid molecule to be sequenced. In stage D, the released tag passes through the nanopore.

[0080] In some cases, labeled nucleotides that are not incorporated into the growing nucleic acid molecule will also pass through the nanopore, e.g. Figure 4 In some cases, unincorporated nucleotides may be detected by the nanopore, but the method provides a means for distinguishing incorporated nucleotides from unincorporated nucleotides based at least in part on the time at which the nucleotides are detected in the nanopore. Tags bound to unincorporated nucleotides pass through the nanopore quickly and are detected in a short time (e.g., less than 10 ms), while tags bound to incorporated nucleotides are loaded into the nanopore and detected in a long time (e.g., at least 10 ms).

[0081] Figure 5 An embodiment of a circuit 500 in an electrochemical cell of a nanopore-based sequencing chip is shown. As described above, when a molecular moiety (e.g., a tag) enters the nanopore 502, a unique conductance signal is generated due to the unique chemical structure and how it fits into the nanopore (e.g., see Figure 2 signal 210 in the DNA), thereby electronically identifying the added base. Figure 5The circuit in Figure 5 maintains a constant voltage across the nanopore 502 while measuring the ion flow conductance. Specifically, the circuit includes an operational amplifier 504 and a pass device 506, which maintains a voltage across the nanopore 502 equal to V a or V b The ion flow through the nanopore 502 is integrated on the capacitor n cap 508 and measured by analog-to-digital (ADC) converter 510. However, circuit 500 only measures unidirectional current. In addition, temperature drift in operational amplifier 504 may cause the actual voltage applied across nanopore 502 to vary across different electrochemical cells in the array. The actual voltage applied across nanopore 502 may drift tens of millivolts from the expected value, resulting in significant measurement inaccuracies. Shrinking the size of the operational amplifier in a large array may cause other performance issues.

[0082] Figure 6 An embodiment of a circuit 600 for use in an electrochemical cell of a nanopore-based sequencing chip is shown, wherein a voltage applied across a nanopore can be configured to vary during a time period when the nanopore is in a particular detectable state. One of the possible states of the nanopore is an open channel state when a nanomolecular moiety (such as a tag) is present in the barrel of the nanopore. Other possible states of the nanopore correspond to states in which four different types of molecular fragments are present in the barrel of the nanopore. Yet another possible state of the nanopore is a state when the diaphragm is ruptured. Figure 6 A nanopore 602 is shown inserted into a membrane 612, and the nanopore 602 and membrane 612 are located 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 Figure 1 Nanopore 602 and membrane 612 are drawn upside down compared to the nanopore and membrane in . In the following, an electrochemical cell means at least a membrane, a nanopore, a working electrode and associated circuitry. In some embodiments, the counter electrode is shared between 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 nanopore in the measuring cell. The common potential and the common electrode are common to all measuring cells. There is a metal working cell electrode in each measuring cell; in contrast to the common electrode, the metal working cell electrode 614 can be configured to apply a different potential independent of the working cell electrodes in other measuring cells.

[0083] In addition to the Nano-SBS sequencing described above, in some embodiments, the electrochemical cells of the present disclosure can be used to perform parallel sequencing of nucleic acids using nanopore-based extended sequencing (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 in high signal-to-noise ratio reporters along the Xpandomer backbone, which are approximately 10 nm apart and are designed for high signal-to-noise ratio, well-differentiated reactions. These differences provide significant performance enhancements in the efficiency and accuracy of sequence reads of Xpandomers relative to natural DNA. An overview of the SBX process is provided in Figure 7 , Figure 8 , Fig. 9 and Fig.10 Described in.

[0084] XNTPs are expandable, 5' triphosphate-modified, non-natural nucleotide analogs that are compatible with template-dependent enzymatic polymerization. Figure 7 , which emphasizes the unique features of these non-natural substrates: XNTP 100 has two different functional regions; namely: a selectively cleavable phosphoramidite bond 110, connecting 5'α-phosphate 115 to a nucleobase 105; and a symmetrically synthesized reporter tether (SSRT) 120 connected to certain positions within the nucleoside triphosphoramidite, which allows controlled unfolding by cutting the phosphoramidite bond. SSRT includes linkers 125A and 125B, which are separated by a selectively cleavable phosphoramidite bond. Each linker is connected to one end of the reporter code 130. XNTP100 is illustrated in a "constrained configuration", which is a feature of the daughter chain product of the XNTP substrate and template-dependent polymerization. The constrained configuration of the polymerized XNTP is a precursor to the extended configuration, as seen in the Xpandomer product. The transition from the constrained configuration to the extended configuration occurs when the PN bond of the phosphoramidite within the main backbone of the daughter chain is broken.

[0085] The synthesis of Xpandomer polymers is summarized in Figure 8 and Fig. 9 In. Figure 8As shown, during assembly, monomeric XNTP substrates 145 (XATP, XCTP, XGTP and XTTP) are polymerized at the extendable end of the nascent daughter chain 150 by a template-directed polymerization process using a single-stranded template 140 as a guide. Typically, the process starts with a primer and proceeds in the 5' to 3' direction. Typically, a DNA polymerase or other polymerase is used to form the daughter chain, and conditions are selected so as to obtain a complementary copy of the template chain. After the daughter chain is synthesized, the coupled SSRT forms a constrained Xpandomer that further forms the daughter chain. The SSRT in the daughter chain has a "constrained configuration" of the XNTP substrate. The constrained configuration of the SSRT is a precursor to the unfolded configuration, as seen in the Xpandomer product.

[0086] like Fig. 9 As shown, the transition from the constrained configuration 160 to the expanded configuration 165 is caused by the cleavage of a selectively cleavable phosphoramidate bond (represented by an unshaded ellipse for simplicity) within the primary backbone of the daughter strand. In this embodiment, the SSRT comprises one or more reporters or reporter codes, 130A, 130C, 130G, or 130T, that are specific to the nucleobases to which they are attached, thereby encoding the sequence information of the template. In this way, the SSRT provides a means to expand the length of the Xpandomer and reduce the linear density of the parent strand sequence information.

[0087] Fig.10 Xpandomer 165 is shown translocating from cis chamber 175 to trans chamber 185 through nanopore 180. After passing through the nanopore, each reporter code of the linearized Xpandomer generates a unique and reproducible electronic signal specific for the nucleobase to which it is attached. The signal is shown by superimposed trace 190.

[0088] Fig.11 The general structure of one embodiment of an XNTP is depicted in more detail. XNTP 200 comprises a nucleoside triphosphoramidate 210 having a linker arm portion 220A and 220B, which are separated by a selectively cleavable phosphoramidate bond 230. SSRT 275 is attached to the nucleoside triphosphate at the bonding groups 250A and 250B, wherein the first SSRT end is attached to a heterocycle 260 (represented here by cytosine, although the heterocycle can be any of the four standard nucleobases A, C, G, or T), and the second SSRT end is attached 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 conjugation can be accomplished by the formation of a triazole bonding group.

[0089] In this embodiment, SSRT 275 includes several functional elements or "features", such as polymerase enhancement 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 the Xpandomer through the nanopore to produce a series of unique and repeatable electronic signals. SSRT 275 is designed to control the rate of Xpandomer translocation caused by the TCE through a combination of steric hindrance and / or electrical repulsion, as discussed elsewhere herein. The size of different reporter codes can prevent ions from flowing through the nanopore at different measurable levels.

[0090] Specific SSRT polymeric sequences can be efficiently synthesized using phosphoramidite chemistry commonly used for oligonucleotide synthesis. Report subcodes and other features can be designed by selecting sequences of specific phosphoramidites from commercially available and / or proprietary libraries. Such libraries include, but are not limited to, polyethylene glycols with a length of 1 to 12 or more ethylene glycol units and aliphatic polymers with a length of 1 to 12 or more carbon units. In certain embodiments, SSRT includes a feature called a "polymerase enhancement region" at the end of the SSRT near the nucleotide triphosphoramidite diester. The polymerase enhancement region may include a positively charged polyamine spacer (e.g., a primary amine, a secondary amine, a tertiary amine, or a quaternary amine) or a triamine spacer (three secondary amines, each separated by three carbons), which promotes incorporation of the XNTP structure by a nucleic acid polymerase. In certain embodiments, the polymerase enhancement region includes two spermine repeating units, wherein the spermine portion is provided by a phosphoramidite monomer having the following structure (a person skilled in the art will recognize that the trifluoroacetamide protecting group is removed at the end of SSRT synthesis to expose the amine group on the spermine):

[0091]

[0092] As used throughout this disclosure, the term "reporter construct" refers to an element of an SSRT including a reporter code, a symmetrical chemical branch, and a shift control element. In certain embodiments, the reporter construct is a polymer comprising a first reporter code, a symmetrical chemical branch with a shift control element, and a second reporter code connected in series from the first end to the second end. The term "with" refers to a covalent connection between a symmetrical branch and a shift control element, which produces a favorable orientation of the shift control element relative to the two reporter codes. The symmetrical chemical branch can be represented by the letter "Y", wherein the two reporter codes are joined to the arms of the Y, and the shift control element is joined to the stem of the Y. Therefore, the two reporter codes are joined online by a branch, and the branch carries a shift control element in a vertical orientation relative to a linear, online, SSRT.

[0093] As used throughout this disclosure, the terms "Linker A" and "Linker B" refer to SSRT regions that each include a polymerase enhancement region and one or more translocation deceleration features or regions, and in certain embodiments, include a spacer region containing, for example, a PEG6 polymer, which can be customized to adjust the length of the SSRT that passes through the nanopore.

[0094] In certain embodiments, the XNTP may be a compound having the following general structure:

[0095]

[0096] In one embodiment, R can be H, for example, when the compound is used to sequence a DNA template. In another embodiment, R can 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. It will be appreciated by those skilled in the art that 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 and Crick base pair with a complementary nucleobase on an adjacent single-stranded nucleic acid template. Exemplary nucleobase analogs include, but are not limited to, 5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactoside, 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, β-D -mannosyl quercetin, 5′-methoxycarboxymethyl uracil, 5-methoxy uracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), whibutoxin, pseudouracil, quercetin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyl uracil, 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.

[0097] As discussed elsewhere herein, the reporter construct is a polymer having a first end and a second end, and comprises a first reporter code, a symmetrical chemical branch with a shift control element, and a second reporter code connected in series from the first end to the second end. This series of features reflects the symmetrical structure of the reporter construct (and the entire SSRT, including a symmetrical linker, a linker A, and a linker B), wherein the sequences of the two reporter codes are identical and connected in series in opposite orientations through symmetrical chemical branches. In short, the synthesis is carried out in the 3′ to 5′ direction, starting from the 3′ end of the TCE. Adding a symmetrical branch to the 5′ end of the TCE causes the first reporter code and the second reporter code to polymerize simultaneously from each arm of the branch, and then simultaneously synthesizing a linker A and a linker B, terminating at the 5′ end of the first end and the second end of the SSRT. It has been found that the online redundancy provided by two identical reporter codes separated by a symmetrical branch with a shift control element provides several advantages in the nanopore sequencing process. For example, Xpandomer may be read by the nanopore when shifted in either direction, i.e., Xpandomer can be read “forward” or “backward”. This flexibility enables the "ratchet" sequencing method discussed further herein and other methods such as "flossing" based on an alternating pattern of voltage application.

[0098] Fig.12 An embodiment of an Xpandomer cut during translocation of a nanopore is shown. The nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two electrolyte compartments. A typical electrolyte is 1M KCl buffered to a pH of 7.0. When a small voltage (typically 100mV) is applied across the bilayer, the nanopore provides the only channel for ion flow induced by the potential and is the source primary resistance in the circuit. The Xpandomer reporter code is designed to provide a specific level of blockage of ion flow, and the sequence information can be read by measuring the translocation of ions through the nanopore as the voltage potential pulse causes the sequence of the reporter code. In the case of the α-hemolysin nanopore, the nanopore is typically embedded in the membrane so that translocation occurs by entering the cis-vestibular side and leaving the trans-vestibular side. As Fig.12 As shown, the nanopore is oriented to capture the Xpandomer from the stem side first. As the Xpandomer displaces the nanopore, the reporter enters the stem until its translocation control element stops at the entrance of the stem. The reporter is held in the stem until the TCE is able to enter and pass through the stem, and then the translocation proceeds to the next reporter. In this embodiment, the entry of the TCE into the stem is achieved by the dissociation of the translocation control portion from the TCE.

[0099] Examples

[0100] Various features and embodiments of the present disclosure are shown in the following representative examples, which are intended to be illustrative rather than limiting. Those skilled in the art will readily appreciate that the specific examples are only intended to illustrate the present invention, as more fully described in the claims that follow. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each embodiment contained therein.

[0101] Example 1: Effects of alternative buffer components and metal materials on the formation of Prussian blue

[0102] This example illustrates a study of the effects of the buffer composition used in the electrochemical cell and the metal surface composition on the formation of Prussian blue.

[0103] Materials and methods

[0104] The standard femtoliter range chamber of the electrochemical cell for SBX nanopore sequencing has an internal stainless steel surface, including an inlet port and an outlet port, in contact with a solution contained in the cell, which includes ammonium chloride and about 250 mM each of potassium ferrocyanide and potassium ferrocyanide. In order to simulate the conditions that occur in a higher volume femtoliter nanopore sequencing chamber, scratched cannulas made of different types of metal alloys were immersed in a buffer containing ammonium chloride or ammonium acetate, different amounts of potassium ferrocyanide and potassium ferrocyanide, and various other buffer substances (such as sodium salicylate, sodium pyrophosphate, resorcinol, and sulfosalicylic acid). The formation of Prussian blue precipitates on the cannula surface was monitored. Table 1 below summarizes the changes in conditions and the results of the study.

[0105] Table 1

[0106]

[0107]

[0108] As shown by the results summarized in Table 1, replacing the stainless steel SS316 cannula with the iron-free nickel alloy C276 or C22 cannula is very effective in preventing the formation of Prussian blue over days and even weeks.

[0109] Additionally, changing the buffer composition had a large effect on Prussian blue formation. Specifically, replacing ammonium chloride with ammonium acetate was very effective in preventing Prussian blue formation. Similarly, reducing the levels of ferrocyanide and ferrocyanide species by half also significantly reduced Prussian blue formation.

[0110] Example 2: Effects of alternative buffer components and metal materials on the formation of Prussian blue during nanopore sequencing

[0111] The impact of

[0112] This example illustrates the study of the effects of buffer composition and metal surface on Prussian blue formation in an electrochemical cell during SBX nanopore sequencing.

[0113] As described elsewhere herein, SBX nanopore sequencing is performed using an array of electrochemical cells containing a buffer solution containing ferrocyanide and ferrocyanide ions. During use, the solution flows into and out of the cell via an inlet port and an outlet port.

[0114] Materials and methods: Over a period of several months, a series of hundreds of SBX nanopore-based sequencing experiments were performed using a standard array nanopore containing an electrochemical cell with inlet and outlet ports made of stainless steel (316 stainless steel) or nickel alloy (alloy C276, C22, or 625) that flowed SBX sequencing buffer through the cell. Cells in the array were monitored for failure and the failed cell ports were analyzed for the presence of Prussian blue precipitate blockages.

[0115] result: The study found that pool failures due to Prussian blue precipitant plugging were reduced from about 35% to less than 5% as the port material was changed from stainless steel to nickel alloy.

[0116] All publications, patents, patent applications, and other records cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other record was individually indicated to be incorporated by reference for all purposes.

[0117] While various specific embodiments have been shown and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. An electrochemical cell comprising: (a) a nanopore embedded in a membrane, the membrane dividing the cell into a cis chamber and a trans chamber connected by the nanopore, wherein the cis chamber and the trans chamber each contain an electrode, a solution comprising ferrocyanide ions, ferrocyanide ions, and a buffer composition; and (b) operably connected to an inlet port and an outlet port of the cell, wherein the ports comprise metal.

2. The cell of claim 1 , wherein the metal exhibits a relative electrochemical activity of less than or equal to 10 when polarized to a potential of about 0.3 V relative to 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, pH 6.2-6.

8. -3 mA / cm 2 of current density.

3. The pool of any one of claims 1 to 2, wherein the metal contains less than 10% iron, less than 8% iron, less than 6% iron or less than 5% iron.

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

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

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

7. The cell of any one of claims 1 to 6, wherein the total concentration of the ferrocyanide ions and the ferrocyanide ions is 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. 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, ammonium acetate at a concentration of 0 mM and ammonium chloride at a concentration of less than 1000 mM.

11. A pond according to any one of claims 1 to 10, wherein an alternating current (AC) in the range of 450 mV to 1200 mV is applied to the pond such that no visible Prussian blue forms in the pond 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. 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 dividing the cell into a cis chamber and a trans chamber operably connected by the nanopore, wherein the cis chamber and the trans chamber each contain an electrode, a solution comprising ferrocyanide ions and ferrocyanide ions, and a buffer composition; and (ii) an inlet port and an outlet port connected to the cell comprising a metal; (b) adding a molecule derived from a target nucleic acid to the cis chamber; (c) applying a voltage to the cell such that at least a portion of the molecules are translocated through the nanopore; as well as (d) detecting a change in voltage flow in the cell when the molecule translocates through the nanopore, The change in the voltage current indicates the sequence of the target nucleic acid.

13. The method of claim 12, wherein the metal exhibits a relative electrochemical conductivity of less than or equal to 10 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, pH 6.2-6.8 when polarized to a potential of about 0.3V relative to a Ag / AgCl reference electrode. -3 mA / cm 2 of current density.

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

15. The method according to 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 encloses a solution volume of between about 0.1 and about 1000 femtoliters.

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

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. 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. 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, ammonium acetate at a concentration of 0 mM and ammonium chloride at a concentration of less than 1000 mM.

21. The method of any one of claims 12 to 20, wherein an alternating current (AC) in the voltage range of 450 mV to 1200 mV is applied to the cell such that no visible Prussian blue forms 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. 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. The method of any one of claims 12 to 21, wherein the applied voltage comprises a pulse voltage; optionally, wherein the pulse 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 pulse voltages is from about 0.5 ms to about 1.7 ms.

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

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

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, wherein individual XNTP subunits of a chain comprise a reporter construct, a nucleobase residue, and a selectively cleavable bond, and wherein cleavage of the selectively cleavable bond produces an Xpandomer that is longer in length than the plurality of XNTP subunits of the chain.

29. The method of claim 28, wherein the method further comprises: The selectively cleavable bond is cleaved to produce an Xpandomer.

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

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

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