Hybrid triblock copolymer film composition and method for determining nanopore arrangement

Abstract

This application discloses hybrid lipid bilayer compositions comprising phospholipids, triblock copolymers, and molecules having pores connecting both sides of the lipid bilayer, as well as the use of these lipid bilayer compositions in electrochemical cells in nanopore-based nucleic acid detection technologies such as nanopore expansion (NANO-SBX) and nanopore synthesis (NANO-SBS).

Description

[Technical Field]

[0001] [Cross-reference of related applications] This disclosure asserts the interests as of the filing date of U.S. Provisional Patent Application No. 63 / 464,051 dated May 4, 2023, which is incorporated herein by reference in its entirety.

[0002] [Technical field] This application relates to hybrid lipid bilayer compositions comprising phospholipids, triblock copolymers, and molecules having pores that connect both sides of the lipid bilayer, as well as the use of these lipid bilayer compositions in electrochemical cells for nanopore-based nucleic acid detection techniques such as nanopore sequencing. [Background technology]

[0003] Nanopore-based nucleic acid sequencing is a widely studied and compelling approach. In one approach, single-stranded polynucleotide sequences are detected by changes in ionic current as the polynucleotides move through nanopores embedded in a lipid bilayer membrane that separates the two sides of an electrochemical cell. During the movement of the polynucleotides, partial occlusion of the nanopore opening alters the flow of ions over time, resulting in changes in current that can be measured by the cell.

[0004] Sequencing by Expansion ("SBX") is a nanopore-based nucleic acid sequencing method that uses a biochemical process to transfer the DNA sequence to a measurable polymer molecule called "Xpandomer." See, for example, U.S. Patent No. 7,939,259 entitled "High Throughput Nucleic Acid Sequencing by Expansion" and PCT Publication WO2020236526A1 entitled "Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing." In the SBX process, the target nucleic acid sequence is encoded along a mainchain Xpandomer sequence having a reporter construct approximately 10 nm apart, designed to provide a well-distinguished signal-to-noise response signal with a high signal-to-noise ratio during nanopore transfer. The enhanced signal-to-noise ratio provided by the distinct response signals significantly improves the readability and accuracy of Xpandomers sequencing compared to native nucleic acid molecules.

[0005] Nanopore-based sequencing-by-synthesis ("SBS") uses a polymerase (or other chain elongation enzyme) covalently bound to a nanopore to synthesize a DNA strand (i.e., a copy strand) complementary to a target sequence template. Nanopores embedded in a membrane within an electrochemical cell are used to simultaneously detect the identity of each nucleotide monomer as each monomer is added to its growth strand. See, for example, U.S. Patent Publications 2013 / 0244340A1, 2013 / 0264207A1, 2014 / 0134616A1, 2015 / 0368710A1, and 2018 / 0057870A1, and International Publication 2019 / 166457A1. Each attached nucleotide monomer is detected by monitoring the signal resulting from the change in ion flow through the nanopore as the tag portion attached to each attached nucleotide monomer enters the nanopore and alters the ion flow. For optimal performance, the tag portion must be present in the nanopore for a sufficient time to provide a detectable, identifiable, and reproducible signal related to the change in ion flow through the nanopore, allowing the specific nucleotide associated with the tag to be clearly distinguished from other tagged nucleotides in the SBS solution (relative to the baseline "open current" flow).

[0006] However, nanopore-based sequencing is burdensome because it must resolve small current-signal differences immersed in significant background noise within microvolume electrochemical cells. This measurement challenge is complicated by subtle changes in materials and parameters affecting the electrochemical cell, including, but not limited to, the performance of lipid bilayers with embedded nanopores.

[0007] There remains a need for improved lipid bilayer materials that enable more efficient and accurate electrochemical signal detection in nanopore-based nucleic acid sequencing devices, systems, and methods. [Overview of the Initiative]

[0008] The present disclosure relates, in a broad sense, to a lipid bilayer composition comprising a phospholipid, a triblock copolymer, and a molecule having pores that connect both sides of the lipid bilayer, and to the use of these lipid bilayer compositions in an electrochemical cell 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 all embodiments, combinations, or variations contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0009] In at least one embodiment, the present disclosure provides a lipid bilayer composition comprising: (a) a mixture of a phospholipid and a triblock copolymer (or TBC), wherein the triblock copolymer occupies from about 2% to about 40% of the solids mass in the mixture, and (b) a pore-forming molecule having pores that connect both sides of the bilayer. In at least one embodiment, the triblock copolymer has a molecular weight of from about 3500 Daltons to about 6500 Daltons and / or the triblock copolymer has a polydispersity of from about 1.1 to about 1.8.

[0010] In at least one embodiment of the lipid bilayer composition, the triblock copolymer is a poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) triblock copolymer, which is herein referred to as (PMOXA) m -(PDMS) n -(PMOXA) m and is also referred to as a triblock copolymer.

[0011] In at least one embodiment of the lipid bilayer composition, the triblock copolymer has the following formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (wherein R 1R is the terminal head group, PMOXA is the poly(2-methyl-2-oxazoline) subunit, PDMS is the poly(dimethylsiloxane) subunit, 2 (where is an -OH or terminal head group, m is 4-20, and n is 20-60). In at least one embodiment, the triblock copolymer is as follows: m=12 and n=21, m=8 and n=34, m=7 and n=21, m=9 and n=30, m=11 and n=40, m=9 and n=37, m=5 and n=18, m=5 and n=17, m=4 and n=18, m=11 and n=53, m=9 and n=37, m=12 and n=53, m=9 and n=37, m=13 and n=59, m=13 and n=52, m=11 and n=49, m=12 and n=50, m=9 and n=36, m=9 and n=42, m=12 and n= The set has a mean m-value and n-value pair selected from 55, m=9 and n=39, m=11 and n=49, m=10 and n=38, m=10 and n=40, m=12 and n=44, m=12 and n=41, m=12 and n=37, m=12 and n=45, m=10 and n=31, m=11 and n=37, m=12 and n=24, m=13 and n=31, m=12 and n=40, m=9 and n=33, m=11 and n=34, m=9 and n=32, m=11 and n=33, m=9 and n=39, m=11 and n=36, and mixtures thereof.

[0012] In at least one embodiment, formula R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 In a lipid bilayer composition comprising a triblock copolymer having R 1 This is the terminal head group, R 2 is -OH (i.e., terminal hydroxyl group). Formula R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2In at least one embodiment of a lipid bilayer composition containing a triblock copolymer, R 1 and R 2 Both are terminal head bases.

[0013] In at least one embodiment, formula R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 In a lipid bilayer composition containing a triblock copolymer, R 2 and / or R 1 The terminal head group contains a triazole group. In at least one embodiment, the terminal head group R 2 and / or R 1 The group comprises a group selected from the following groups: hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine. In at least one embodiment, the terminal head group comprises a fluorescent moiety such as a coumarin moiety or a coumarin derivative moiety.

[0014] In at least one embodiment, the terminal head group is selected from the following: TIFF2026522806000001.tif221156 TIFF2026522806000002.tif233156 TIFF2026522806000003.tif239156

[0015] In at least one embodiment of the lipid bilayer composition of this disclosure, the triblock copolymer is amphiphilic.

[0016] In at least one embodiment of the lipid bilayer composition of the present disclosure, the phospholipid is selected from DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DSPE, or a mixture thereof.

[0017] In at least one embodiment of the lipid bilayer composition of the present disclosure, the pore-forming molecules connecting both sides of the bilayer are protein nanopores, which optionally include naturally occurring α-hemolysin and / or variants of α-hemolysin.

[0018] In another embodiment, the lipid bilayer compositions of the present disclosure can be incorporated into an electrochemical cell, which can be used in a nanopore-based detection method. For example, in at least one embodiment, the present disclosure provides an electrochemical cell comprising: (a) a nanopore embedded in a lipid bilayer, wherein the lipid bilayer comprises a mixture of phospholipids and triblock copolymers; and (b) a cis-side chamber and a trans-side chamber separated by the lipid bilayer and operably connected by the nanopore, the cis-side chamber and the trans-side chamber comprising an electrode and an electrolyte solution, respectively.

[0019] In another embodiment, the Disclosure also provides a nanopore-based sequencing method for a target nucleic acid, the method incorporating the use of an electrochemical cell comprising the lipid bilayer composition of the Disclosure. For example, in at least one embodiment, the Disclosure provides a method for sequencing a target nucleic acid, the method comprising: (a) an electrochemical cell, the following: (i) Nanopores embedded in a lipid bilayer, wherein the lipid bilayer comprises a mixture of phospholipids and triblock copolymers; (ii) a cis-side chamber and a trans-side chamber separated by a lipid bilayer and operably connected by a nanopore, each containing an electrode and an electrolyte solution; and (iii) Inlet and outlet ports operably connected to the cell; To provide an electrochemical cell containing, (b) Adding a molecule derived from the target nucleic acid to the cis chamber, (c) Applying a voltage to the cell that moves at least a portion of the molecule through the nanopore, (d) Detecting changes in the voltage flow within the cell that indicate the sequence of the target nucleic acid as the molecule moves through the nanopore.

[0020] In at least one embodiment, the sequencing method of the present disclosure is a nanopore-based SBX sequencing method, wherein 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 sequences corresponding to all or part of a continuous nucleotide sequence of the target nucleic acid, wherein each XNTP subunit of the chain comprises a reporter construct, nucleic acid base residues and selectively cleavable bonds, and cleavage of the selectively cleavable bonds yields an Xpandomer longer than the plurality of XNTP subunits of the chain.

[0021] In at least one embodiment, the sequencing method further comprises: cleaving selectively cleavable bonds to obtain an Xpandomer, which optionally includes a reporter construct for analyzing genetic information in sequences corresponding to a contiguous nucleotide sequence of all or part of a target nucleic acid.

[0022] In at least one embodiment, detection of voltage changes includes detection of voltage changes by a reporter construct of Xpandomer that penetrates a nanopore, and optionally, the reporter construct for analyzing genetic information includes a reporter code and a dislocation control element, the dislocation control element providing sterically hindrance-based dislocation control and pausing Xpandomer dislocations as it passes through a nanopore to which a baseline voltage has been applied, and the dislocation control element is coupled with the reporter code within the opening of the nanopore, the reporter code being detected by the nanopore. [Brief explanation of the drawing]

[0023] A better understanding of the novel features and advantages of this disclosure will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of this disclosure are utilized, and to the accompanying drawings (also referred to herein as "Figure" and "FIG.").

[0024] [Figure 1] This shows one embodiment of a cell 100 in a nanopore-based sequencing chip. [Figure 2] This document describes one embodiment of a cell 200 that performs nucleotide sequencing using Nano-SBS technology. [Figure 3] This shows one embodiment of a cell in which nucleotide sequencing is to be performed using a preloaded tag. [Figure 4] This document illustrates one embodiment of process 400 for nucleic acid sequencing using preloaded tags. [Figure 5] This shows one embodiment of the circuit 500 within a nanopore-based sequencing chip cell. [Figure 6] One embodiment of the circuit 600 within the cell of a nanopore-based sequencing chip is shown, in which the voltage applied to the nanopore can be configured to vary over a period of time during which the nanopore is in a specific detectable state. [Figure 7] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 8] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 9] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 10] This diagram shows the generalized characteristics and functions of XNTP in Nano-SBX technology. [Figure 11] This is a schematic diagram showing further details of one embodiment of XNTP. [Figure 12] This is a schematic diagram showing one embodiment of Xpandomer passing through biological nanopores. [Figure 13A] These are heatmap images and plots showing the effect of differences in TBC content in DPhPE films on the sequenceable cell population within nanopores. Figure 13A shows results using films containing only DPhPE. A bimodal distribution on the vertical axis in the left inner of each image indicates the presence of cell populations that cannot be sequenced. A single distribution indicates the presence of a single cell population that can be sequenced. [Figure 13B] These are heatmap images and plots showing the effect of different TBC content in DPhPE films on the sequenceable cell population within nanopores. Figure 13B shows results using a hybrid film composed of the following ratios of TBC Ch111 to DPhPE: 20% Ch111 (Figure 13B). A bimodal distribution on the left inner vertical axis of each image indicates the presence of cell populations that cannot be sequenced. A single distribution indicates the presence of a single cell population that can be sequenced. [Figure 13C] These are heatmap images and plots showing the effect of different TBC content in DPhPE films on the sequenceable cell population within nanopores. Figure 13C shows results using a hybrid film composed of the following ratios of TBC Ch111 to DPhPE: 30% Ch111 (Figure 13C). A bimodal distribution on the left inner vertical axis of each image indicates the presence of cell populations that cannot be sequenced. A single distribution indicates the presence of a single cell population that can be sequenced. [Figure 13D] These are heatmap images and plots showing the effect of different TBC content in DPhPE films on the sequenceable cell population within nanopores. Figure 13D shows results using a hybrid film composed of the following ratios of TBC Ch111 to DPhPE: 40% Ch111 (Figure 13D). A bimodal distribution on the left inner vertical axis of each image indicates the presence of cell populations that cannot be sequenced. A single distribution indicates the presence of a single cell population that can be sequenced. [Figure 14A]An example of the ¹H-NMR spectrum in CD2Cl2 of the "living" polymer TBC compound α,ω-(hydroxy-2-methyl-2-oxazolinium triflate)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) in Scheme 1 after evaporation of the reaction solvent is shown. This compound has the TBC formula PMOXA8-PDMS39-PMOXA8 and contains 68 wt% PDMS, Mn=4.2kDa, f(2-methyl-2-oxazolinium triflate)-=70%, fhydroxy-=30%, and normalized with 4H(m,0.55-0.45ppm,-CH2- adjacent to-Si(-CH3)2-O-). [Figure 14B] The representative 1H-NMR spectra of the "Ch111 type" TBC compound in CDCl3 after dialysis and fractionation are shown. This compound has the TBC formula PMOXA12-PDMS50-PMOXA12, 64 wt% PDMS, MnCH111=5.7kDa, facetoxy-=7%, fhydroxy-=93%, normalized with 4H(m, 0.55-0.45 ppm, -CH2- adjacent to -Si(-CH3)2-O-). [Figure 14C] An example of the 1H-NMR spectrum in CD2Cl2 of the purified α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) of Scheme 3, as described, is shown. This compound has the TBC formula PMOXA11-PDMS56-PMOXA11, 69 wt% PDMS, molecular weight Mn=6kDa, f-azido-approximately 90%, f-HO-approximately 10%, normalized with 4H(m,0.58-0.46 ppm, -CH2- adjacent to -Si(-CH3)2-). [Figure 14D] This image shows the superimposed FT-IR spectra of α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline)TBC after solvent evaporation (gray), ultrafiltration (blue), and cosolvent fractionation (red) in Scheme 3. [Figure 14E]An example of the 1H-NMR spectrum in CD2Cl2 after dialysis and fractionation of the "Ch127 / 128 type" TBC polymer α,ω-(hydroxy-,5-(1-polymer-1H-1,2,3-triazole-4-yl)isophthalic acid)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) (Scheme 4) is shown. This compound has the TBC formula PMOXA9-PDMS30-PMOXA9, 58 wt% PDMS, MnCH127=3.8kDa, fH5-(1-polymer-1H-1,2,3-triazole-4-yl)isophthalic acid-=76%, fhydroxy-=24%, and normalized with 4H(m,0.59-0.46 ppm, -CH2- adjacent to -Si(-CH3)2-O-). [Modes for carrying out the invention]

[0025] This disclosure provides hybrid lipid bilayer compositions comprising phospholipids, triblock copolymers, and molecules having pores connecting both sides of a lipid bilayer, as well as the use of these lipid bilayer compositions in electrochemical cells. Electrochemical cells are useful for carrying out nanopore-based methods for assaying nucleic acids, including nanopore-based sequencing. The features of the hybrid lipid bilayer compositions and the electrochemical cells incorporating them result in a remarkablely beneficial outcome: a reduction in the adverse error-inducing effects when using the cells in nanopore-based assays. Adverse effects (also known as protopore formation) can lead to undesirable orientations in sequencing and cause rapid blockage of ports and / or nanopores, thereby significantly reducing the accuracy and efficiency of any nanopore-based assay. This disclosure also provides methods for using 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.

[0026] In this specification and the accompanying claims, the singular forms "a" and "an" include multiple references unless the context explicitly indicates otherwise. Thus, for example, a reference to "protein" includes multiple proteins, and a reference to "compound" refers to multiple compounds. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" is interchangeable and not intended to be limiting. Where the description of various embodiments uses the term "comprising," it should be further understood that in some particular examples the embodiment could be described alternatively using the terms "essentially consisting of" or "consisting of."

[0027] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intervening integer of the value and each tenth of each intervening integer of the value are understood to be included in the invention, unless the context explicitly indicates otherwise, between the upper and lower limits of that range, and any other listed or intervening values ​​within that stated range. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in the invention, subject to any specifically excluded limitations within the stated range. If a stated range includes one or both limits, the range excluding (i) one or (ii) both of the included limits is also included in the invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0028] In a broad sense, the nomenclature and techniques and procedures described herein include those that are understood and commonly used by those skilled in the art, such as Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter "Sambrook"), and Current Protocols in Molecular Biology, FMAusubel et al., eds. (first published as a book in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., with regular supplements published until 2011, and now published by Wiley & Sons, Inc., and available as an online journal in the Wiley Online Library (hereinafter "Ausubel") as Current Protocols in Molecular Biology, Vols. 00-130, (1987-2020)).

[0029] All publications, patents, patent applications, and other documents referenced in this disclosure are incorporated herein by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, or other document is individually indicated as being incorporated herein by reference for all purposes.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. It should be understood that the terms used herein are intended to describe only specific embodiments and are not intended to limit them. For the purposes of interpreting this disclosure, the following definitions of terms apply, and where appropriate, singular terms are also included in their plural forms and vice versa.

[0031] membranes containing triblock copolymers Membranes, as materials and components, are essential to the functionality of a wide variety of processes and devices. In at least one embodiment, this disclosure provides compositions, devices, applications, and methods based on hybrid membranes, which are lipid bilayers comprising a mixture of phospholipids and triblock copolymers (TBCs). The remarkable properties of these TBC-containing hybrid lipid bilayer compositions are that they can improve accuracy and efficiency when used in nanopore-based devices, particularly in nucleic acid detection and / or sequencing applications. As described in the examples and elsewhere in this specification, when incorporated into nanopore-based devices for nucleic acid sequencing, the hybrid lipid bilayer compositions of this disclosure can increase the number of molecules sequenced per experiment by 1.1 to 5 times compared to the same device using a standard non-hybrid lipid bilayer without TBCs.

[0032] The hybrid membrane compositions of this disclosure exhibiting these enhanced properties are based on a lipid bilayer comprising a mixture of phospholipids and triblock copolymers (TBCs). Typically, the amount of triblock copolymer is about 2% to about 40% of the solids mass in the mixture with the phospholipids forming the bilayer. Methods for preparing hybrid membranes containing TBCs are well known to those skilled in the art and are described in the examples. Generally, the desired phospholipid and TBC composition is prepared as a mixture in a solution of silicone oil and hexadecane.

[0033] The molecular weight of the triblock copolymer used in the mixture can range from approximately 3500 daltons to approximately 6500 daltons. Triblock copolymers typically have a polydispersity of approximately 1.1 to approximately 1.8. However, a wider range of polydispersity is expected to be available in various hybrid membrane applications.

[0034] As described in the examples and elsewhere in this specification, the broad exemplary hybrid films of this disclosure are poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) triblock copolymers, as expressed herein (PMOXA). m -(PDMS) n -(PMOXA) m Also known as triblock copolymer. Structural formula (PMOXA) m -(PDMS) n -(PMOXA) m While triblock copolymers are known in the art, compositions incorporating TBCs on nanopore-embedded lipid bilayers, and the use of these hybrid membrane structures in nanopore-based sequencing methods, are not known.

[0035] Furthermore, this disclosure describes a novel class of (PMOXA) with modified terminal head groups. m -(PDMS) n -(PMOXA) m The invention provides triblock copolymers, for example, those in which the terminal head group contains a triazole group (PMOXA). m -(PDMS) n -(PMOXA) m This is a class of triblock copolymers. As described in the examples and elsewhere in this specification, TBCs having these novel terminal head group modifications provide improved properties for nanopore-based devices used for sequencing when incorporated into device films.

[0036] A useful TBC for the lipid bilayer composition disclosed herein is given by the following formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (In the formula, R 1 This is the terminal head group, R 2The formula includes TBCs (where is a terminal head group or hydroxyl group). In this formula, "(PMOXA)" is a poly(2-methyl-2-oxazoline) subunit of the polymer structure. Two (PMOXA) polymers, each having m subunits, are covalently bonded to a central poly(dimethylsiloxane) (or "PDMS") polymer having n subunits. 1 Position, and optionally R 2 The terminal head groups (or "HG") at each position are chemical groups covalently linked to the terminal (PMOXA) subunit. In at least one embodiment, TBC is symmetric, and R 1 and R 2 Both are identical terminal HGs. In at least one embodiment, the TBC is asymmetric, and R 1 and R 2 Both are different terminal HGs. In at least one embodiment, the TBC is asymmetric, and R 2 The group is a terminal hydroxyl group (or "-OH"), R 1 The base is terminal HG.

[0037] formula R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 The number of PMOXA and PDMS subunits in the polymer structure is defined by integer values ​​"m" and "n". In the exemplary TBCs described herein, m is between 4 and 20, and n is between 20 and 60. As shown in the table illustrating the exemplary TBCs, the structure of a TBC can be represented by two m values ​​and one n value. For example, "12-50-12" is a TBC of the formula R 1 -(PMOXA) 12 -(PDMS) 50 -(PMOXA) 12 -R 2The following illustrates the TBC. It is understood that the values ​​of m and n shown in the exemplary TBC formulas herein are the average values ​​of the range of m and n values ​​that occur in the polymer molecule population resulting from the TBC synthesis process herein. That is, the TBC shown as "12-50-12" is actually a distribution of TBC molecules where the average values ​​of m and n are 12 and 50, respectively. As disclosed herein, including in the examples described later, the specific m and n values ​​that determine the ratio of PMOX and PDMS polymer units in the TBC can be varied to provide a TBC with optimized functional properties when used in hybrid membranes.

[0038] Furthermore, a remarkable advantage of the TBCs described herein is that the functional properties of the TBCs when used in hybrid membranes can be improved by modifying and optimizing the chemical structure of the head groups of the TBCs. As described elsewhere in this specification, including in the following examples, formula R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 Various different head groups can be envisioned and used for the TBC represented herein. In at least one embodiment, the terminal head group R 2 and / or R 1 The group comprises a group selected from the following groups: hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine. In at least one embodiment, the terminal head group R 2 and / or R 1 It is assumed that the triazole moiety may be included, and a wide range of TBCs modified with various head groups can be prepared using standard CuAAC click chemistry. In at least one embodiment, the terminal head group R 2 and / or R 1This can include fluorescent groups such as coumarin groups or coumarin derivative groups. Various fluorescent groups are known in the art and can be incorporated as HG into the TBC of this disclosure using standard CuAAC click chemistry as described in the examples and elsewhere in this specification.

[0039] Specific exemplary terminal head structures useful for the TBC of this disclosure are shown in Table 1 below.

[0040] [Table 1] TIFF2026522806000005.tif100158

[0041] The preparation and use of TBCs containing the specific exemplary head groups shown in Table 1 are described in the Examples. Furthermore, it is envisioned that additional head groups based on chemical derivatives of these exemplary head group structures may be prepared, tested, and used for various TBC applications, such as hybrid membranes for nanopore sequencing. Such chemical derivatives can be prepared from alkyne versions of the desired compounds using the standard CuAAC click chemistry described in the Examples. Chemical derivatives may include any of the above specific structures further modified with desired chemical groups such as halogens, hydroxyls, amines, or charged groups (e.g., sulfates, carboxylates) on an alkyl chain or aryl ring.

[0042] Nanopore-based devices for detecting nucleic acids Nanopore-based devices for detecting nucleic acids have been developed for rapid sequencing, and various designs and uses are known in the art. See, for example, U.S. Patents 9,494,554,B2, 9,567,630,B2, 9,557,294,B2, and 9,605,309,B2, respectively, which are incorporated herein by reference. These devices generally include an electrochemical cell having a chamber containing nanopores embedded in a membrane. The membrane acts to separate the cell chamber into two subchambers, called the cis and trans sides of the cell, each containing electrodes.

[0043] Generally, the membrane of such a device can be an organic membrane, such as a lipid bilayer, or a synthetic membrane made of a non-natural polymer material. This disclosure envisions a nanopore-based device comprising a hybrid membrane comprising a bilayer mixture of phospholipids and triblock copolymers. As the examples herein demonstrate, such a hybrid membrane comprising TBCs can be used to embed nanopores and improve nucleic acid detection and sequencing performance when used in a nanopore-based device. Therefore, the following general disclosures relating to nanopore-based devices, their manufacture, and use should be interpreted as including the hybrid membranes described herein.

[0044] The pores of this device are provided by nanopores embedded in the membrane and function as passages (or channels) connecting the cis and trans sides of the cell. Depending on the nanopores used, the pores may have a width or diameter ranging from about 1 angstrom to about 10,000 angstroms. The nanopores may be naturally occurring pore-forming proteins such as α-hemolysin from Staphylococcus aureus (S. aureus), or non-naturally occurring variants or variants of wild-type pore-forming proteins. A range of naturally occurring and non-naturally occurring nanopores with various pore sizes and properties is known in the art. See, for example, U.S. Patent Nos. 10351908B2, 10934582B2, and 10227645B2.

[0045] Within an electrochemical cell, nanopores embedded in a membrane are positioned in close proximity to electrodes coupled to a sensing circuit, such as a complementary metal-oxide-semiconductor (CMOS) or field-effect transistor (FET) circuit. When a potential is applied across the nanopores (through the electrodes) immersed in a conductive fluid, a small current can be observed due to the flow of ions through the nanopores. This ion flow is sensitive to the pore size, and therefore, molecules entering the pores affect the ion flow and voltage measured through this sensor circuit.

[0046] Electrochemical cells for nanopore-based sequencing of nucleic acids are typically used in a large-scale parallel configuration, where thousands of such cells are configured as an array within a single device, often referred to as a chip (or biochip). Typically, a nanopore-based sequencing chip device may incorporate an array of more than one million electrochemical cells, containing 1,000 rows x 1,000 columns of such cells (see, for example, a chip manufactured by Roche Sequencing Solutions, Santa Clara, California, USA). Methods for manufacturing and using such nanopore array microchips can also be found in U.S. Patent Applications Publications 2013 / 0244340A1, 2013 / 0264207A1, 2014 / 0134616A1, 2015 / 0368710A1, and 2018 / 0057870A1, as well as International Publication 2019 / 166457A1, which are incorporated herein by reference, respectively. Each well in the array is fabricated using a standard CMOS process with surface modifications that allow constant contact between the bioreagent and the conductive salt. Each well can support a phospholipid bilayer membrane embedded with a nanopore polymerase conjugate. The electrodes in each well are individually addressable via a computer interface. All reagents used are introduced into a simple flow cell above the array microchip using a computer-controlled syringe pump. The chip supports analog-to-digital conversion and reports electrical measurements independently from all electrodes at a rate exceeding 1000 points / second. Nanopore measurements are performed asynchronously at least once every 1 millisecond (msec) on each of the 8M addressable nanopore-containing membranes in the array and can be recorded on the interfaced computer. A further description of exemplary electrochemical cells useful for nanopore-based nucleic acid assays such as sequencing, including the chamber and electrode materials, buffers, sensing circuits, array devices, and their use in various applications, is provided below.

[0047] Figure 1 shows an exemplary embodiment of an electrochemical cell 100 found in an array of such cells useful for nanopore-based sequencing. A membrane 102 is formed on the surface of the cell. In some embodiments, the membrane 102 is a lipid bilayer. A bulk electrolyte solution 114 containing nanopores or "protein nanopore transmembrane complexes" (PNTMCs) 104 is placed directly on this surface within the cell, and a single PNTMC 104 is inserted into the membrane 102 using electroporation. The individual nanopore-embedded membranes within each cell of the array are not chemically or electrically linked to one another. Thus, each cell is an independent sequencing machine, generating data specific to a single polymer molecule associated with the PNTMC.

[0048] Continuing to refer to Figure 1, the thin film of electrolyte solution 108 is isolated from the bulk electrolyte solution 114 by the ion-impermeable membrane 102. Thus, the nanopore-embedded membrane separates the electrochemical cell 100 into two chambers: a cis chamber containing the bulk electrolyte solution 114 and a transformer chamber containing the thin film of electrolyte solution 108. The thin film of electrolyte solution 108 in the transformer chamber is in contact with the metal working electrode 110 connected to the analog measurement circuit 112. The bulk electrolyte solution in the cis chamber is in contact with the counter electrode 116 and the reference electrode 117.

[0049] The pores in PNTMC 104 provide channels through membrane 102 that allow analytes (e.g., nucleic acids) and ions to flow, resulting in modulation of the ionic current across the impermeable bilayer. Thus, the pores in PNTMC 104 provide the sole pathway for the ionic current to flow from the 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 to further enhance its sensitivity as an electrochemical potential sensor.

[0050] A thin film of electrolyte constitutes a femtoliter volume of solution in a transformer chamber that is in direct contact with the working electrode. This small amount of electrolyte in contact with the electrode of the electrochemical cell must receive repeated voltage pulses to sense the ion flow through the nanopore. The need to maintain the ion flow through the nanopore using these repeated electrochemical measurements with the electrode in a small amount of electrolyte, and repeated measurements corresponding to individual molecular parts moving through the pore, requires a highly sensitive sensor system. This electrochemical measurement system is highly sensitive to any precipitate or aggregated molecular entities that could clog the pore or otherwise interfere with the ion flow between the working and counter electrodes. Therefore, the electrolyte salt, electrochemical cell material, and electrochemical measurement conditions should be carefully controlled to prevent or reduce harmful precipitate formation or electrode wear in the system.

[0051] The electrolyte solution 108 may contain any of the following: lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, ammonium chloride, ammonium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), or magnesium chloride (MgCl2). In some embodiments, the film of the electrolyte solution has a thickness of about 3 microns (μm). The thickness of the film of the electrolyte solution may be in the range of 0 to 5 microns.

[0052] In addition to the salts described above, the electrolyte membrane may contain redox-active ions such as ferrocyanide ions and ferricyanide ions. These redox ions are used in certain nanopore-based sequencing methods performed in electrochemical cells. For example, Nano-SBX measurements performed in the cells of this disclosure may contain ferricyanide and ferrocyanide ions. Such redox-active ions may undergo irreversible reactions depending on their concentration and the specific electrochemical conditions at the working and counter electrodes. Indeed, as described elsewhere in this specification, the irreversible formation of Prussian blue, a precipitate in electrochemical cells, is highly detrimental to any nanopore-based measurements in the cell. Therefore, the concentrations of redox-active ions such as ferricyanide and ferrocyanide should be carefully controlled. In at least one embodiment of this disclosure, the ferricyanide and ferrocyanide ions in the solution within the electrochemical cell should be maintained at a total concentration between approximately 25 mM and 250 mM.

[0053] It has also been found that the salt used in the electrolyte solution of an electrochemical cell can affect the cell's ability to be used for highly sensitive nanopore-based nucleic acid measurements. For example, many nanopore-based sequencing protocols use ammonium chloride as a buffer salt in the electrolyte solution provided within the cell chamber. It has been found that controlling the concentration of ammonium chloride by substituting it with ammonium acetate as the salt in the electrolyte solution during nanopore-based nucleic acid sequencing methods such as Nano-SBX can help reduce or prevent the formation of redox precipitates such as Prussian blue, which are highly detrimental to the accuracy and sensitivity of the electrochemical cell, as described above. Therefore, in some embodiments of the electrochemical cell of this disclosure, the buffer composition used in the cell contains ammonium acetate at a concentration of about 0 mM to about 1500 mM and / or ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the buffer composition contains ammonium acetate at a concentration of about 0 mM to 1500 mM and ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the concentration of ammonium acetate is 0 mM and the concentration of ammonium chloride is about 600 mM.

[0054] Dielectric materials are typically used to form an oxide layer 106 that defines the transformer chamber of the cell. Useful dielectric materials include glass, oxides, and silicon mononitride (SiN). In some embodiments, the upper surface of the dielectric oxide layer 106 in contact with the bulk electrolyte 114 can be silanized. Silanization forms a hydrophobic layer on top of the dielectric layer. In some embodiments, this hydrophobic layer has a thickness of about 1.5 nanometers (nm). Alternatively, a hydrophobic dielectric material such as hafnium oxide can be used to form the upper part of the dielectric layer.

[0055] As shown in Figure 1, the membrane is a lipid bilayer at least partially formed on the dielectric oxide layer 106 and spans a well containing a thin film of electrolyte 108. For example, the membrane can be formed on a hydrophobic layer on the dielectric oxide layer, and when the membrane reaches the opening of the well, the lipid monolayer migrates into a lipid bilayer that extends across the opening of the well. The hydrophobic layer can facilitate the formation of a lipid monolayer on the dielectric layer and the migration from the lipid monolayer to a lipid bilayer.

[0056] The bulk electrolyte 114 may further comprise one of lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2). Therefore, in some embodiments of the electrochemical cells of this disclosure, the buffer composition of the bulk electrolyte used in the cell comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM and / or ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the buffer composition comprises ammonium acetate at a concentration of about 0 mM to 1500 mM and ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the concentration of ammonium acetate is 0 mM and the concentration of ammonium chloride is about 600 mM. Similarly, in some embodiments, the ferricyanide and ferrocyanide ions in the bulk electrolyte solution of the electrochemical cell should be maintained at a total concentration between approximately 25 mM and 250 mM.

[0057] As mentioned above regarding electrolyte thin films, the composition of the salts used in the bulk electrolyte (e.g., ammonium chloride versus ammonium acetate) and the redox-active ions can affect the accuracy and sensitivity of nanopore-based measurements performed using electrochemical cells. Depending on the type of electrochemical measurement being performed, particularly the potential applied to the electrode in contact with the solution, it is possible to adjust the composition of the solution to reduce or prevent the formation of harmful precipitates.

[0058] To perform the necessary insertion and removal of solutions, the electrochemical cells of this disclosure include inlet and outlet ports operably connected to the cell's chamber or reservoir. Although the schematic diagram of the electrochemical cell in Figure 1 does not show the inlet and outlet ports, the incorporation of such ports into electrochemical cells is known in the art. The inlet and outlet ports allow for the insertion of reagents necessary for nanopore-based nucleic acid detection, including materials for fabricating membranes with embedded nanopores, nucleotide synthesis reagents, and electrolytes and buffers. Typically, the inlet and outlet ports include metal (e.g., stainless steel) tubes attached to the cell to provide a fluid connection from the outside to the cell's chamber, which can be used to insert or remove solutions. Thus, at least portions of the inlet and outlet port tubes are in contact with the electrochemical solution in the cell chamber. Optimally, the composition of the ports should be electrochemically inert at a voltage used to induce ion flow through the nanopores and to perform perceptual measurements of the change in ion flow as molecular portions enter and / or move through the nanopores.

[0059] The electrochemical cell 100 includes a counter electrode (CE) 116 and a reference electrode 117 that functions as an electrochemical potential sensor. In some embodiments, the counter electrode 116 is shared among multiple cells and is therefore also called a common electrode. The common electrode can be configured to apply a common potential to the bulk liquid in contact with nanopores in the multiple cells. The common potential and common electrode are common to all measurement cells.

[0060] In at least one embodiment, a non-Faraday electrochemical cell having a titanium nitride (TiN) working electrode has been found to be advantageous for nanopore-based sequencing of nucleic acids. The cell has a general structure similar to the cell in Figure 1, but includes a TiN working electrode that exhibits increased electrochemical capacity. The electrochemical cell further includes a conductive or metallic layer that connects the working electrode of the cell to a circuit of an array in which the cell is part.

[0061] Thus, in at least one embodiment, the metal working electrode 110 is a titanium nitride (TiN) metal electrode with an 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 metal working electrode 110 is the total surface area of the electrode per unit mass (e.g., m 2 / kg) or per unit volume (e.g., m 2 / m 3 or m -1 ) or per unit base area (e.g., m 2 / m 2 ). As the surface area increases, the electrochemical capacitance of the metal working electrode increases, and more ions can be moved at the same applied potential before the capacitor is charged. The surface area of the working electrode 110 can be increased by making the TiN electrode "sponge-like" or porous. The TiN sponge impregnates the electrolyte and creates a large effective surface area for contact with the electrolyte.

[0062] The ratio of the capacitance (C membrane ) associated with the membrane to the capacitance (C electrochemical ) associated with the working electrode can be adjusted to achieve optimal overall system performance. Improvement in system performance can be achieved by reducing C electrochemical while maximizing C membrane . C membrane is adjusted to generate the required RC time constant without the need for additional on-chip capacitance, thereby allowing significant reduction 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 walls defined by the oxide layer 106. Thus, the two base surface areas are related to C membrane and C electrochemicalIt can be independently optimized to provide a desired ratio between and . By using a spongy, porous TiN working electrode, the electrolyte can diffuse vertically down the uncoated portion of the working electrode through the spaces between the columnar TiN structures, and then horizontally into the coated portion of the working electrode 1102 beneath the dielectric layer 1104. As a result, the effective surface area of ​​the TiN in contact with the electrolyte is maximized, and C electrochemical This is the maximum value.

[0063] Further descriptions of non-Faraday electrochemical cells, TiN working electrodes, and other nanopore-based cell designs useful for the devices, compositions, and methods of this disclosure can be found, for example, in U.S. Patent No. 1,0174,371,B2, which is incorporated herein by reference.

[0064] As described elsewhere in this specification, the electrochemical cell of this disclosure can be used as a device for nanopore-based nucleic acid detection and measurement assays, including nucleic acid sequencing. Generally, nanopore-based sequencing of a target nucleic acid can be performed using the electrochemical cell of the above design and associated solution compositions comprising electrolytes and buffers that reduce the above precipitate formation. The method provides an electrochemical cell comprising (a) an electrochemical cell, (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 nanopore comprising a solution comprising ferrocyanide ions, ferricyanide ions, and a buffer composition, and (ii) an inlet port and an outlet port comprising 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 moves at least a portion of the molecule through the nanopore; and (d) detecting a change in the voltage flow within the cell indicating the sequence of the target nucleic acid as the molecule moves through the nanopore.

[0065] As described elsewhere in this specification, the specific potentials applied, their timing, and duration can significantly affect highly sensitive changes in ion flow conductance through the nanopore in the presence of nucleic acid molecular portions to be detected when performing the sequencing method. Exemplary methods and parameters for applying voltage potentials to nanopore-based electrochemical cell arrays for sequencing are known in the Art and are described, for example, in U.S. Patents 1,1150216 and 1,1029306, which are incorporated herein by reference, respectively. The applied potentials, their pulse timing, and duration also affect the redox chemistry within the cell, which can lead to the formation of harmful precipitates such as Prussian blue. A remarkable advantage of the electrochemical cells of this disclosure and the associated solutions used therein is that, in some embodiments, the nanopore sequencing method using the electrochemical cells of this disclosure can be performed using alternating current (AC) applied to the cell for 10 hours in a voltage range of 450 mV to 1200 mV, without the visible formation of Prussian blue within the cell.

[0066] In some embodiments of the sequencing method of this disclosure, the applied voltage includes applying a baseline potential to the cells. A typical baseline voltage applied may be in the range of about 55 mV to about 95 mV.

[0067] In some embodiments of the array determination method, the applied voltage may include a pulsed voltage. A pulsed voltage useful for this method may be approximately 320 mV to approximately 550 mV. The duration of the pulsed voltage is typically approximately 5 μs to approximately 15 μs. In at least one embodiment, the time between pulses is approximately 0.2 ms to 5 ms. In at least one embodiment, the applied voltage supplies alternating current to an electrochemical cell. For example, the resulting alternating current may have a periodicity of approximately 0.4 seconds to approximately 6 seconds.

[0068] In at least one embodiment, it is conceivable that a parallel sequencing method can be performed using an array of electrochemical cells of this disclosure. Such parallel nanopore-based sequencing of nucleic acids using a parallel array of electrochemical cells has been used in conjunction with nanopore-based sequencing by synthesis (Nano-SBS) techniques. System compositions and methods for Nano-SBS are known in the art. See, for example, U.S. Patent Application Publications 2013 / 0244340A1, 2013 / 0264207A1, 2014 / 0134616A1, 2015 / 0368710A1, and 2018 / 0057870A1, and International Publication 2019 / 166457A1. These known systems, compositions and methods are conceivable to be used or adapted for use with the electrochemical cells of this disclosure. A description of the use of Nano-SBS in the context of the electrochemical cells and compositions of this disclosure is provided below.

[0069] Figure 2 shows an embodiment of an electrochemical cell 200 used to perform nanopore-based nucleic acid sequencing using Nano-SBS technology. In Nano-SBS technology, a template 202 and primers to be sequenced are introduced into the electrochemical cell 200. Four different tagged nucleotides 208 are added to the bulk aqueous phase to this template-primer complex. When the correctly tagged nucleotides form a complex with polymerase 204, the tails of the tags are positioned within the barrel of the nanopore 206. The tags held within the barrel of the nanopore 206 generate a unique ion-barrier signal 210, thereby electronically identifying the added bases due to the different chemical structures of the tags.

[0070] Figure 3 shows one embodiment of a cell in which nucleotide sequencing is to be performed using a preloaded tag. A nanopore 301 is formed within a membrane 302. An enzyme 303 (e.g., a polymerase such as DNA polymerase) associates with the nanopore. In some cases, the enzyme 303 is covalently bound to the nanopore 301. The polymerase 303 associates with the nucleic acid molecule 304 to be sequenced. The associated nucleic acid molecule 304 may be linear or cyclic. In some embodiments, a nucleic acid primer 305 hybridizes to a portion of the nucleic acid molecule 304. The polymerase 303 uses the single-stranded nucleic acid molecule 304 as a template to catalyze the incorporation of nucleotide 306 into the primer 305. As described above, nucleotide 306 contains a tag species ("tag") 307 that allows it to be distinguished from the other three nucleotides.

[0071] Figure 4 shows one embodiment of process 400 for nucleic acid sequencing using a preloaded tag. In step A, the tagged nucleotide (one of four types: A, T, G, or C) is not associated with the polymerase. In step B, the tagged nucleotide associates with the polymerase. In step C, the polymerase is close to the nanopore. The tag is drawn into the nanopore by an electric field generated by a voltage applied across the membrane and / or the nanopore. Some of the associated tagged nucleotides do not form base pairs with the nucleic acid molecule. These unpaired nucleotides are typically rejected by the polymerase within a timescale shorter than the timescale in which properly paired nucleotides remain associated with the polymerase. Since the unpaired nucleotides only transiently associate with the polymerase, process 400 shown in Figure 4 typically does not proceed beyond step B.

[0072] Before polymerase docks with the nanopore, the nanopore conductance is approximately 300 picosiemens (300 pS). In step C, the nanopore conductance is approximately 60 pS, 80 pS, 100 pS, or 120 pS, corresponding to one of four types of tagged nucleotides. Polymerase undergoes isomerization and transphosphorylation reactions to incorporate the nucleotide into the growing nucleic acid molecule and release the tag molecule. In particular, once the tag is retained within the nanopore, a unique conductance signal (see, e.g., signal 210 in Figure 2) is generated due to the different chemical structures of the tag, thereby electronically identifying the added base. By repeating the cycle (i.e., steps A-E or A-F), sequencing of the nucleic acid molecule becomes possible. In step D, the released tag passes through the nanopore.

[0073] In some cases, as seen in step F of Figure 4, tagged nucleotides that are not integrated into the growing nucleic acid molecule also pass through the nanopore. While unintegrated nucleotides may, in some cases, be detected by the nanopore, this method provides a means for distinguishing between integrated and unintegrated nucleotides based at least partially on the time it takes for the nucleotides to be detected in the nanopore. Tags bound to unintegrated nucleotides pass through the nanopore rapidly and are detected for a short period (e.g., less than 10 ms), while tags bound to integrated nucleotides are loaded into the nanopore and are detected for a longer period (e.g., at least 10 ms).

[0074] FIG. 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 (e.g., see signal 210 in FIG. 2) is generated due to the different chemical structures and how it fits into the nanopore, whereby the added base is electronically discriminated. The circuit of FIG. 5 maintains a constant voltage across the nanopore 502 while the ion current conductance is being measured. In particular, the circuit includes an operational amplifier 504 and a pass device 506 that maintain a constant voltage equal to V a or V b across the nanopore 502. The ion current passing through the nanopore 502 is integrated by capacitor n cap 508 and measured by an analog-to-digital (ADC) converter 510. However, the circuit 500 measures only the flow of unidirectional current. Further, temperature drift within the operational amplifier 504 can vary the actual voltage applied across the nanopore 502 across different electrochemical cells in the array. The actual voltage applied across the nanopore 502 can drift by tens of millivolts above or below the desired value, thereby causing significant measurement inaccuracies. Shrinking the size of the operational amplifier in a large array can cause other performance issues.

[0075] Figure 6 shows one embodiment of a circuit 600 for use in an electrochemical cell of a nanopore-based sequencing chip, where the voltage applied to the nanopore can be configured to vary over a period of time in which the nanopore is in a particular detectable state. One possible state of the nanopore is the open channel state, when no molecular parts (e.g., tags) are present within the barrel of the nanopore. Other possible states of the nanopore correspond to the states when four different types of molecular parts are present within the barrel of the nanopore. Yet another possible state of the nanopore is when the membrane has ruptured. Figure 6 shows a nanopore 602 inserted into a membrane 612, where the nanopore 602 and membrane 612 are positioned between the cell working electrode 614 and the counter electrode 616, such that a voltage is applied across both ends of the nanopore 602. The nanopore 602 is also in contact with the bulk liquid / electrolyte 618. Note that the nanopore 602 and membrane 612 are drawn upside down compared to the nanopore and membrane in Figure 1. Hereinafter, an electrochemical cell means including at least a membrane, a nanopore, a working electrode, and associated circuitry. In some embodiments, the counter electrode is shared among multiple cells (e.g., in an array) and is therefore also called a common electrode. The common electrode may be configured to apply a common potential to the bulk liquid in contact with the nanopore within the measurement cell. The common potential and common electrode are common to all measurement cells. Within each measurement cell there is a metallic working cell electrode, and in contrast to the common electrode, the metallic working cell electrode 614 may be configured to apply a separate potential independent of the working cell electrodes of other measurement cells.

[0076] In addition to the Nano-SBS sequencing described above, in some embodiments, parallel sequencing of nucleic acids can be performed using the electrochemical cell of this disclosure with nanopore-based Sequencing-by-Expansion (Nano-SBX) technology. See, for example, U.S. Patent No. 7,939,259. The SBX technology is based on the polymerization of highly modified non-natural nucleotide analogs called "XNTPs". Typically, SBX uses biochemical polymerization to transfer the DNA template sequence onto a measurable polymer called "Xpandomer". The transferred sequence is encoded along the Xpandomer backbone in a high-signal-versus-noise reporter about 10 nm away, designed for high-signal-versus-noise, high-differentiation response. These differences result in a significant performance improvement in sequence read efficiency and accuracy of Xpandomer compared to natural DNA. A general description of the SBX process is shown in Figures 7, 8, 9, and 10.

[0077] XNTPs are expandable 5'-triphosphate modified unnatural nucleotide analogs that are compatible with template-dependent enzymatic polymerization. A highly simplified XNTP is shown in Figure 7, which highlights the intrinsic characteristics of these unnatural substrates. XNTP100 has two distinct functional regions: a selectively cleavable phosphoramide bond 110 that links the 5'-α-phosphate 115 to the nucleic acid base 105, and a symmetrically synthesized reporter tether (SSRT) 120 bound within the nucleoside triphosphoramide at a position that allows for controlled expansion by cleavage of the phosphoramide bond. The SSRT contains linkers 125A and 125B separated by the selectively cleavable phosphoramide bond. Each linker is bound to one end of the reporter code 130. XNTP100 is shown in a "constrained configuration" characteristic of XNTP substrates and daughter chain products of template-dependent polymerization. The constrained structure of polymerized XNTPs is a precursor to the extended structure, as seen in the Xpandomer product. The transition from the constrained to the extended structure occurs when the PN bond of the phosphoramide in the primary skeleton of the daughter chain is cleaved.

[0078] The synthesis of the Xpandomer polymer is summarized in Figures 8 and 9. As shown in Figure 8, during assembly, the monomer XNTP substrates 145 (XATP, XCTP, XGTP, and XTTP) are polymerized at the elongable ends of the nascent daughter strands 150 by a template-directed polymerization process using a single-strand template 140 as a guide. Generally, this process is initiated from the primer and proceeds in the 5' to 3' direction. Generally, DNA polymerase or other polymerases are used to form the daughter strands, and conditions are selected to obtain complementary copies of the template strand. After the daughter strands are synthesized, the coupled SSRTs form constrained Xpandomer, which further forms the daughter strands. The SSRTs in the daughter strands have a "constrained configuration" of the XNTP substrates. The constrained configuration of the SSRTs is a precursor to the extended configuration, as seen in the Xpandomer product.

[0079] As shown in Figure 9, the transition from the constrained configuration 160 to the extended configuration 165 is due to the cleavage of selectively cleavable phosphoramide bonds (indicated by unshaded ellipses for simplification) within the primary backbone of the daughter strand. In this embodiment, the SSRT includes one or more reporters or reporter codes 130A, 130C, 130G, or 130T that are specific to the nucleic acid bases to which they are linked, thereby encoding the sequence information of the template. In this way, the SSRT provides a means to extend the length of the Xpandomer and reduce the linear density of the sequence information of the parent strand.

[0080] Figure 10 shows the Xpandomer 165 moving from the cis chamber 175 to the trans chamber 185 through the nanopore 180. Upon passing through the nanopore, each reporter code of the linearized Xpandomer generates a distinct and reproducible electronic signal specific to the nucleic acid base to which it is linked. This signal is indicated by the superposition trace 190.

[0081] Figure 11 shows in more detail a generalized structure of one embodiment of XNTP. XNTP 200 comprises a nucleoside triphosphoramide 210 having linker arm portions 220A and 220B separated by selectively cleavable phosphoramide bond 230. SSRT 275 is linked to the nucleoside triphosphoramide by linking groups 250A and 250B, with the first SSRT terminus linked to a heterocycle 260 (represented here by cytosine, although the heterocycle may be any one of the four standard nucleic acid bases, A, C, G, or T), and the second SSRT terminus linked to an α-phosphate 270 of the nucleic acid base backbone. Those skilled in the art will understand that the final XNTP substrate product can be formed using many suitable coupling chemicals known in the art, for example, that the SSRT conjugate can be achieved by the formation of a triazole linking group.

[0082] In this embodiment, the SSRT275 includes several functional elements, or “features,” such as polymerase-enhancing regions 280A and 280B, reporter codes 285A and 285B, and translation control elements (TCEs) 290A and 290B. In other embodiments, the SSRT may include a single TCE. Each of these features plays a unique function during the migration of the Xpandomer through the nanopore, producing a series of distinctive and reproducible electronic signals. The SSRT275 is designed to control the rate of Xpandomer migration by the TCE via a combination of steric hindrance and / or electrorepulsion, as discussed elsewhere in this specification. Different reporter codes are sized to block ion flow through the nanopore at different measurable levels.

[0083] Specific SSRT polymer sequences can be efficiently synthesized using phosphoramidite chemistry, which is typically used in oligonucleotide synthesis. Reporter codes and other features can be designed by selecting specific phosphoramidite sequences from commercially available and / or proprietary libraries. Such libraries include, but are not limited to, polyethylene glycols having a length of 1 to 12 or more ethylene glycol units, and aliphatic polymers having a length of 1 to 12 or more carbon units. In certain embodiments, the SSRT includes a feature called a “polymerase-enhancing region” at the terminal of the SSRT proximal to the nucleotide triphosphoramidate diester. The polymerase-enhancing region may include a positively charged polyamine spacer (e.g., primary, secondary, tertiary, or quaternary amine) or a triamine spacer (three secondary amines separated by three carbons each) that facilitates the uptake of the XNTP structure by nucleic acid polymerase. In certain embodiments, the polymerase-enhancing region comprises two repeating units of spermine, the spermine portion of which is provided by a phosphoramidite monomer having the following structure (as those skilled in the art will recognize, the trifluoroacetamide protecting group is removed at the end of the SSRT synthesis to expose the amine group on spermine). [ka]

[0084] Where used throughout this disclosure, the term “reporter construct” refers to the elements of an SSRT comprising reporter codes, symmetric chemical branching chains, and translocation control elements. In certain embodiments, the reporter construct is a polymer comprising a first reporter code, symmetric chemical branching chains supporting translocation control elements, and a second reporter code, continuous from a first end to a second end. The term “supports” refers to a covalent bond between the symmetric branching chains and the translocation control elements, which results in a favorable orientation of the translocation control elements relative to the two reporter codes. The symmetric chemical branching chains may be represented by the letter “Y”, with the two reporter codes linked to the arms of the Y and the translocation control elements linked to the stem of the Y. Thus, the two reporter codes are linked inline by the branching chains, which carry the translocation control elements in a linear, inline, and perpendicular orientation to the SSRT.

[0085] As used throughout this disclosure, the terms “linker A” and “linker B” refer to regions of the SSRT that include a polymerase-enhancing region and one or more migration-decelerating features or regions, as well as, in certain embodiments, a spacer region containing a polymer such as PEG6, which can be customized to adjust the length of the SSRT traversing within the nanopore.

[0086] In certain embodiments, XNTP may be a compound having the following generalized structure. [ka]

[0087] In one embodiment, for example, when a compound is used to sequence a DNA template, R may be H. In another embodiment, for example, when a compound is used to sequence an RNA template, R may be OH. In certain embodiments, the nucleic acid base is adenine, cytosine, guanine, thymine, uracil, or a nucleic acid base analog. As those skilled in the art will understand, adenine, cytosine, guanine, thymine, and uracil are naturally occurring nucleic acid bases. As used herein, the term “nucleic acid base analog” refers to a non-naturally occurring nucleic acid base that can form a Watson-Crick base pair with a complementary nucleic acid base on an adjacent single-stranded nucleic acid template. Exemplary nucleic acid base analogs include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- This list includes, but is not limited to, mannosylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wibutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, 3-nitropyrrole, 8-aza-7-deazaguanine, 8-aza-7-deazynosine, and 8-aza-7-deazaadenine.

[0088] As discussed elsewhere in this specification, the reporter construct is a polymer having a first end and a second end, comprising, continuously from the first end to the second end, a first reporter code, a symmetric chemical branch chain carrying a movement control element, and a second reporter code. This set of features reflects the symmetric structure of the reporter construct (as well as the entire SSRT including the symmetric linkers, linker A and linker B), where the sequences of the two reporter codes are identical and inline linked in opposite directions by the symmetric chemical branch chain. Briefly, synthesis proceeds in the 3' to 5' direction, starting at the 3' end of the TCE. By adding the symmetric branch chain to the 5' end of the TCE, simultaneous polymerization of the first and second reporter codes from each arm of the branch chain, followed by the simultaneous synthesis of linker A and linker B, is enabled, terminating at the 5' ends of the first and second ends of the SSRT. The inline redundancy provided by two identical reporter codes separated by symmetric branched chains carrying the movement control elements has been found to offer several advantages during nanopore sequencing. For example, if the Xpandomer moves in either direction, it can potentially be read by the nanopore; i.e., the Xpandomer can be read either "forward" or "backward." This flexibility enables other methods of sequencing, such as the "ratchet" method and "flooring" based on the AC pattern of voltage application, which are discussed further herein.

[0089] Figure 12 shows one embodiment of a cleaved Xpandomer in the process of moving a nanopore. The nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two electrolyte chambers. A typical electrolyte is 1M ammonium chloride buffered to pH 6.0–8.0. When a small voltage of typically 85mV is applied across the bilayer, the nanopore provides the only channel for potential-induced ion flow and is the source primary resistance in the circuit. The Xpandomer reporter code is designed to give a specific ion flow cutoff level, and since pulses of potential result in an array of reporter code moving through the nanopore, array information can be read by measuring the array of ion flow levels. In the case of an α-hemolysin nanopore, the nanopore is typically embedded in the membrane so that movement occurs by entering on the cis-antibiotic side and exiting on the trans-basal side. As shown in Figure 12, the nanopore is oriented to first capture the Xpandomer from the basal side. As the Xpandomer moves the nanopore, the reporter enters the base until its movement control element stops at the base entrance. The reporter is held in the base until the TCE enters the base and can pass through it, after which the movement proceeds to the next reporter. In this embodiment, the passage of the TCE into the base is made possible by disengaging the movement control portion from the TCE. [Examples]

[0090] Various features and embodiments of this disclosure are shown in the following representative examples, which are illustrative and not limiting. Those skilled in the art will readily understand that certain embodiments are merely illustrative of the invention, which are described more fully in the subsequent claims. All embodiments and features described herein should be understood to be interchangeable and combinable with all embodiments contained herein.

[0091] Example 1: Synthesis of a triblock copolymer having a modified head group This example demonstrates the synthesis of various triblock copolymer compounds having modified head groups, which are used in the nanopore-embedded lipid bilayers described herein.

[0092] A. Synthesis of α,ω-(hydroxy-,2-methyl-2-oxazolinium triflate)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline).

[0093] This "living" triblock copolymer (TBC) compound, belonging to the PMOXA-PDMS-PMOXA family, has the structural formula shown in Scheme 1 and is a synthetic precursor for other α,ω-(hydroxyl- / methylester-)- and α,ω-(hydroxyl- / azido-)-terminated TBC compounds described later. [ka]

[0094] The synthesis of the TBC compound in Scheme 1 was carried out largely according to the method described in Lorcher, S.; Meier, W. “Cosolvent fractionation of PMOXA-b-PDMS-b-PMOXA: Bulk separation of triblocks from multiblocks”. European Polymer Journal 2017, 88, 575-585, as follows: 6.42 g of dried α,ω-bis(carbinol)-terminated polydimethylsiloxane (2.29 mmol, Mn 1H-NMRDissolve 0.67 mL (4.84 mmol) of dried triethylamine (2800 g / mol) in 100 mL of dry hexane and cool to -25°C. A solution of 0.83 mL (4.91 mmol) of trifluoromethanesulfonic acid anhydride dissolved in 15 mL of dry hexane was added dropwise to a cooled polydimethylsiloxane solution. The reaction mixture was stirred at -25°C for 3 hours under an inert atmosphere. The resulting triethylammonium trifluoromethanesulfonate precipitate was filtered at -20°C under an inert atmosphere, and the hexane was removed by evaporation at room temperature and 0.3 mbar. The obtained α,ω-bis(triflate)-terminated polydimethylsiloxane was further dried at 30°C and 0.3 mbar for 1 hour, and then 45 mL of anhydrous chloroform, 19 mL of anhydrous acetonitrile, and 3.3 mL (38.9 mmol) of anhydrous 2-methyl-2-oxazoline were added. The reaction mixture was held at 42°C for 65 hours under an inert atmosphere, and then slowly cooled to room temperature. Polymerization was stopped before complete monomer conversion using various termination agents (nucleophiles) under established reaction conditions.

[0095] Parameters of the "living" polymer obtained by polymerization of Scheme 1 1 The 1H-NMR (400MHz, CD2Cl2, δ) is as follows: 4.97(t, 2.51H, -CH2-2-methyl-2-oxazolinium triflate salt head group -N + (adjacent to =), 4.40 (adjacent to -O- in the head group of -CH2-2-methyl-2-oxazolinium triflate), 3.96 (-N in the head group of br,2.73H,-CH2-2-methyl-2-oxazolinium triflate) += adjacent to), 3.85-3.70 (br,4H,-CH2-(adjacent to -OH) overlaps with -CH2-(adjacent to -CH2-OH)), 3.7-3.2 (br,72.55H,-CH2-CH2-(adjacent to -NC(O)CH3 of the PMOXA main chain) overlaps with -CH2-CH2-O-CH2-CH2-CH2-linker-derived -CH2-), 2.48 (br,4.11H,2-methyl-2-oxazolinium triflate head group - CH3), 2.26-1.98 (br, 49.57H, -CH3 in the side chain of 2-methyl-2-oxazoline), 1.63-1.5 (br, 3.98H, -CH2- adjacent to -CH2-Si(-CH3)2-O-), 0.59-0.47 (m, 4H(ref.), -CH2- adjacent to -Si(-CH3)2-O-), and 0.08 (br, 235.46H, CH3- in the PDMS skeleton) are all in ppm units and are shown in Figure 14A.

[0096] B. Synthesis of α,ω-(acetyloxy-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline).

[0097] This member of the PMOXA-PDMS-PMOXA system of TBC compounds has the structural formula shown in Scheme 2 and is referred to here as the "Ch111 type" TBC. [ka]

[0098] Polymerization of 2-methyl-2-oxazoline was terminated by adding 4.3 molar equivalents of triethylamine and water (1:4 v / v) as a polydimethylsiloxane macroinitiator and mixing at room temperature for 18 hours. The volatile components were evaporated, and the polymer was dried at 50°C and 0.3 mbar for 3 hours. The polymer was dissolved in water:ethanol (1:1 v / v, 7 mg / mL), and low molecular weight ethanol / water-soluble impurities were removed by ultrafiltration (e.g., dialysis with ethanol:water (1:1 v / v) using a centrifugal filter with a regenerated cellulose membrane with a molecular weight cutoff of 2 kDa). After ultrafiltration, the water and ethanol were evaporated, and the polymer was dried at 50°C and 0.3 mbar for 3 hours. The dialyzed polymer was dissolved in methanol:hexane (1:1, 6 mg / mL), and cosolvent fractionation was performed by mixing at room temperature for 18 hours and at -60°C for 3 hours. The cooled solution was warmed to room temperature in a separation funnel and allowed to stand until clear phase separation was obtained. The lower fraction was collected, the solvent was evaporated, and the polymer was dried at 50°C and 0.3 mbar for 3 hours.

[0099] The Ch111 TBC compound prepared above 1 The H-NMR (400MHz, CDCl3, δ) parameters are as follows: 4.38 (br, 0.71H, -CH2- adjacent to -COOCH3), 3.77 (br, 3.92H, -CH2- adjacent to -OH), 3.68-3.25 (br, 98.24H, -CH2-CH2- (adjacent to -NC(O)CH3 of the PMOXA main chain) are -CH2- (adjacent to -CH2-OH), -CH2- (adjacent to -CH2-COOCH3), and -CH2- (-CH2- The values ​​are as follows: adjacent to CH2-Si(-CH3)2-O-, overlapping with CH2-Si(-CH3)2-O-, 2.2-2.04 (br, 67.93H, -NC(O)CH3), 1.65-1.5 (br, 4.51H, -CH2- adjacent to -CH2-Si(-CH3)2-O-), 0.55-0.45 (m, 4H(ref.), -CH2- adjacent to -Si(-CH3)2-O-), and 0.07 (br, 309.71H, CH3- of the PDMS skeleton), all in ppm units, as shown in Figure 14B.

[0100] Synthesis of C.α,ω-(azide group-,hydroxy group)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline)

[0101] The members of this PMOXA-PDMS-PMOXA-based TBC compound have the structural formula shown in Scheme 3 and are precursors for preparing TBC compounds having the modified end groups described later by CuAAC click chemistry. [ka]

[0102] The reaction solvent (chloroform, acetonitrile, etc.) was evaporated and removed under Schlenk conditions, and the resulting α,ω-bis(2-methyl-2-oxazolinium triflate)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) "living" polymer was dried at 30°C at 0.03 mbar for approximately 30 minutes. The dried solid "living" polymer was redissolved in acetonitrile (approximately 90 mg / mL) and then mixed with sodium azide (70 equivalents of sodium azide per 1 equivalent of PDMS) under an inert atmosphere at 80°C for 45 hours. After the reaction mixture cooled to room temperature, the unreacted sodium azide was filtered off, and the solvent was evaporated. The resulting polymer was redissolved in ethanol, and the precipitated sodium azide was removed by centrifugation. Finally, the ethanol was evaporated, and the resulting polymer was dried at 30°C at 1 mbar for 1 hour, and then stored at -20°C before use.

[0103] The generated α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) 1The measured parameters of the H-NMR (400 MHz, CD2Cl2, δ) were as follows: 3.7 (br, the -CH2- adjacent to -OH overlaps with the -CH2-CH2- derived from the PMOXA skeleton), 3.76-3.15 (br, 101.14H, -CH2-CH2- (adjacent to -NC(O)CH3 of the PMOXA skeleton) overlaps with -CH2- (adjacent to -CH2-OH), -CH2- (adjacent to -OH), -CH2- (adjacent to -N3), (overlapping with -CH2-(-CH2-CH2-Si(-CH3)2-O-)), 2.22-1.96 (br, 66.61H, -NC(O)-CH3), 1.64-1.50 (br, 5.12H, -CH2- adjacent to -CH2-Si(-CH3)2-O-), 0.58-0.46 (m, 4H(ref.), -CH2- adjacent to -Si(-CH3)2-O-) and 0.08 (br, 334.64H, CH3- of the PDMS skeleton). An exemplary NMR spectrum is shown in Figure 14C. FT-IR spectra were obtained for α,ω-(azido-,hydroxy-)-terminated-poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) after solvent evaporation (gray), ultrafiltration (blue), and cosolvent fractionation (red) (shown in Figure 14D). In all samples, the azido-terminated group was approximately 2100 cm⁻¹. -1 It can be detected.

[0104] Synthesis of α,ω-(hydroxy-,5-(1-polymer-1H-1,2,3-triazole-4-yl)isophthalic acid)-terminated-poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) using D.CuAAC.

[0105] This member of the PMOXA-PDMS-PMOXA system of TBC compounds has the structural formula shown in Scheme 4 and is referred to here as the "Ch127 / 128 type" TBC. [ka]

[0106] The reaction conditions for CuAAC in PMOXA-PDMS-PMOXA synthesis (using α,ω-bis(azide)-terminated PDMS and alkyne-terminated PMeOxa) (using ethanol and copper nanoparticles, reaction time, and temperature) have already been reported (e.g., Isaacman, MJ; Barron, KA; Theogarajan, LS. Clickable amphiphilic triblock copolymers. Journal of Polymer Science Part A: Polymer Chemistry 2012, 50(12), 2319-2329), but the application of CuAAC to modify the head group of TBC has not been reported until now.

[0107] The synthesis of Ch127 / 128 type polymers using CuAAC was carried out as follows: 0.45 g (0.1 mmol) of α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) and 0.54 g (2.85 mmol) of alkyne (5-ethynyl-1,3-benzenedicarboxylic acid in this example) were dissolved in 4 mL of ethanol, and the resulting solution was purged with an inert gas for 30 minutes. After degassing, 60-100 mg of copper nanoparticles (particle size 25-60 nm) were added to the polymer solution under an inert atmosphere, and the reaction mixture was stirred at 60°C under an inert atmosphere for 2-3 hours. After cooling to room temperature, the reaction mixture was diluted with ethanol, and the copper nanoparticles were removed by centrifugation. Trace amounts of copper were further removed by passing the polymer solution through a copper removal column several times. Unreacted alkynes and ethanol / water-soluble impurities were removed by ultrafiltration using a centrifugal filter with a 2 kDa MWCO regenerated cellulose membrane (e.g., dialysis with ethanol:water (0.8:0.2~1:1 v / v)). After ultrafiltration, water and ethanol were evaporated, and the polymer was dried at 50°C and 0.3 mbar for 3 hours. The dialyzed polymer was dissolved in methanol:hexane (1:1, 6 mg / mL) and cosolvent fractionation was performed by mixing at room temperature for 18 hours and at -60°C for 3-5 hours. The cooled solution was warmed at room temperature in a separation funnel and allowed to stand until clear phase separation was obtained. The lower fraction was collected, the solvent was evaporated, and the polymer was dried at 50°C and 0.3 mbar for 3 hours.

[0108] The above CH127 / 128 type polymer 1¹H-NMR (400MHz, CD2Cl3, δ): 8.7-8.2 (br, 6.1H, aromatic proton derived from triazole and aromatic ring), 4.7 (br, 2.96H, -CH2- adjacent to triazole), 3.93 (br, 3.31H, -CH2- adjacent to triazole), 3.79-2.95 (br, 82.44H, -CH2-CH2- (adjacent to -NC(O)CH3 of PMOXA main chain) is -CH2--CH2-O- CH2-CH2-CH2- (overlapping with -CH2- from the linker), 2.32-1.86 (br, 55.31H, -NC(O)-CH3), 1.68-1.48 (br, 4.46H, -CH2- adjacent to -CH2-Si(-CH3)2-O-), 0.59-0.46 (m, 4H(ref.), -CH2- adjacent to -Si(-CH3)2-O-), 0.08 (br, 177.35H, CH3- of the PDMS main chain), all in ppm units. An example NMR spectrum is shown in Figure 14E.

[0109] Table 2 (below) provides alkynes that can be used for the synthesis of additional PMOXA-PDMS-PMOXA TBCs having different molecular weights (MW), PDMS content, and different head groups.

[0110] [Table 2] TIFF2026522806000013.tif113157

[0111] The various alkynes shown in Table 1 can be used in CuAAC-based synthesis using α,ω-(azido-,hydroxy-)-terminated PMOXA-PDMS-PMOXA, producing various TBCs with different terminal groups including a triazole group, as shown in scheme 5-17 in the figure below. The reaction conditions are the same as those for CuAAC using 5-ethynyl-1,3-benzenedicarboxylic acid.

[0112] Scheme 5. Chemical structure of Ch118 / 122 [ka]

[0113] Scheme 6. Chemical structure of Ch112 / 113 / 114 [ka]

[0114] Scheme 7. Chemical structure of Ch119 / 123s [ka]

[0115] Scheme 8. Chemical structure of Ch115 / 116 [ka]

[0116] Scheme 9. Chemical structure of Ch120 / 124 [ka]

[0117] Scheme 10. Chemical structure of Ch125 / 126 [ka]

[0118] Scheme 11. Chemical structure of TBC with phosphate head group - Ch129 / Ch130 [ka]

[0119] Scheme 12. Chemical structure of TBC having a propanediic acid head group - Ch131 / Ch132 [ka]

[0120] Scheme 13. Chemical structure of TBC with butanediic acid head group - Ch133 / Ch134 [ka]

[0121] Scheme 14. Chemical structure of TBC having an aminoheptino acid head group - Ch135 / Ch136 [ka]

[0122] Scheme 15. Chemical structure of TBC with octinic acid head group - Next synthesis [ka]

[0123] Scheme 16. Chemical structure of Ch108 type TBC [ka]

[0124] Scheme 17. Chemical Structures of Ch137 and Ch138 Type TBCs [ka]

[0125] Example 2: Nanopore-based nucleic acid detection using a hybrid triblock copolymer (TBC) lipid bilayer This example demonstrates the use of hybrid triblock copolymer (TBC) lipid bilayers in electrochemical cells for SBX nanopore sequencing. A series of experiments were conducted to show the effects of triblock copolymer size and concentration on sequencing results.

[0126] material and method: A. Preparation of DPhPE / TBC solution: Dissolve 30 mg of DPhPE in 10 mL of a 9:1 mixture of PDM20 silicone oil and hexadecane in a vial. Stir the solution and sonicate at room temperature until completely dissolved to obtain a solution containing 3 mg / mL of DPhPE in a 9:1 silicone oil:hexadecane mixture. Add 9 mg of PMOXA-PDMS-PMOXA TBC to another vial. Add 2.7 mL of PDM20 silicone oil to the solid, followed by 0.3 mL of hexadecane. Sonicate the resulting suspension at room temperature for 1 hour, or until completely dissolved, to obtain a copolymer solution containing 3 mg / mL of DPhPE in a 9:1 silicone oil:hexadecane mixture. To prepare a 15% TBC and 85% DPhPE solution, add 150 μL of the 3 mg / mL polymer solution to 850 μL of the 3 mg / mL lipid solution.

[0127] B. Preparation of electrochemical cells having nanopore-embedded hybrid lipid bilayers: Preparation of nanopore-embedded lipid bilayers placed on nanopore-based electrochemical cell arrays for sequencing is described, for example, in U.S. Patent Publication Nos. 2013 / 0244340A1 and 2014 / 0134616A1. Standard nanopore-embedded bilayer preparation methods utilize DPhPE lipids as the sole component for membrane formation and subsequent sequencing. In this example, a hybrid bilayer-forming solution is used, which includes the aforementioned standard DPhPE lipids and synthetic triblock copolymer compounds in varying amounts. The resulting hybrid lipid / TBC solution is mixed with α-hemolysine nanopores, and the hybrid lipid bilayer is formed on the electrochemical cell array according to a standard method (see, for example, U.S. Patent Publication No. 2013 / 0244340A1).

[0128] C. Nanopore-based SBX sequencing using hybrid lipid bilayers: Nanopore-based SBX sequencing experiments are performed using an electrochemical cell array with a buffer solution containing ferricyanide and ferrocyanide ions, as described, for example, in U.S. Patent No. 7,939259, International Publication No. 2020236526A1, and U.S.SN 63 / 337,413 filed May 2, 2022. The main modification of the protocol is the composition of the membrane solution used when inserting biological nanopores and performing sequencing experiments. Variations in the properties of the triblock copolymer and the amount added to DPhPE can result in different sequencing characteristics, as detailed below.

[0129] We conducted and compared a series of nanopore-based SBX sequencing experiments over several months using electrochemical cell arrays in which nanopores were embedded in standard phospholipid bilayers and arrays in which nanopores were embedded in hybrid triblock copolymer lipid bilayers (Table 3, Experiments 1-8).

[0130] [Table 3] TIFF2026522806000028.tif26157

[0131] Results: Embedding nanopores in a hybrid triblock copolymer lipid bilayer reduced cell damage caused by the formation of non-sequencing "protopores" by at least twofold. In some cases, the use of specific polymer additives allowed for the complete elimination of non-sequencing "protopore" populations, which could account for up to 50% of potential pores when using lipid-only membranes.

[0132] In the initial test in Experiment 1 (Table 3), it was shown that adding polymer Ch111 in various amounts to DPhPE lipids improved sequencing throughput, measured as the number of sequencing molecules per cell per minute. With a standard DPhPE membrane alone, a rate of approximately 10 molecules / min (per cell) was achieved. With a 10% composition (or 0.3 mg / mL Ch111 + 2.7 mg / mL DPhPE), an improvement in sequencing speed of more than 19 molecules per cell per minute was observed. With DPhPE alone, although the number of cells to be sequenced was high, the total number of sequencing molecules increased. Tests with alternative polymers (e.g., Experiments 2 and 3) showed that this trend was consistent across other polymer sizes, and particularly with 10% polymer compositions on DPhPE lipids, it was clear that improved sequencing throughput (measured as total sequencing molecules or sequencing molecules per cell per minute) was achievable. In high-concentration tests (Experiment 4), it was shown that the 15% polymer composition was the optimal condition for throughput improvement when using polymer Ch111, compared to other conditions. Experiments 5-8 demonstrated the properties of other polymer species with varying monomer amounts, particularly their effects on throughput and the number of cells used for sequencing. A notable result was observed with (PMOXA)7-(PDMS). 21 -When using the (PMOXA)7 scaffold, Ch15 provided a higher level of sequencing run cell count and improved throughput compared to other polymers. The use of smaller scaffolds (such as Ch72 and Ch74 in Experiment 8) resulted in a decrease in the number of cells to perform sequencing and the number of molecules sequenced per cell per minute compared to Ch15. Experiment 6 shows that polymers of comparable size also yield similar results in throughput.

[0133] In the following experiment, we investigated the modification of polymer head groups. Using a novel synthesis method for PMOXA-PDMS-PMOXA polymers, we generated azide-containing polymers that allow head group modification through reaction with alkyne species. A series of sequencing experiments were conducted using these polymers as hybrid compositions, and the sequencing characteristics were evaluated as shown in Table 4.

[0134] [Table 4] TIFF2026522806000030.tif220157 TIFF2026522806000031.tif213157 TIFF2026522806000032.tif123157

[0135] The experimental results in Table 4 confirm that modifications of various head groups can also regulate the yield of sequencing cells and the number of sequencing molecules per cell per minute. Compared to unmodified lipids, modified head groups as a sequencing hybrid membrane can improve the number of sequencing molecules per cell per minute (Table 4, Experiment 1), or it is possible to increase the number of cells being sequenced while maintaining the number of sequencing molecules per cell per minute at a comparable level (Table 4, Experiment 2). Head group-modified polymers allow for a wider range of compositional tolerance than unmodified polymers, and the characteristics of the head group significantly affect yield and throughput. A comparison of Experiment 1 with Experiments 2-4 showed that cell yield decreased significantly with phospholipid choline head groups, while phospholipid ethanolamine head group modification was necessary to obtain good cell yield. Carboxylic acid or phosphate-containing head groups (Experiments 5, 6, 15, 17, 18, 19) can achieve higher throughput and comparable cell yields than unmodified or standard polymers. Triazole-alcohol and triazole-octylamine head groups are beneficial for cell yield, but reduce throughput compared to carboxylate head groups. Sulfonate and sulfobetaine head groups may result in lower cell yield compared to other polymers.

[0136] The third experimental set was conducted to understand the properties of membranes to which one or more synthetic triblock copolymers were added, and the results are shown in Table 5 below.

[0137] [Table 5] TIFF2026522806000034.tif60157

[0138] Table 5, Experiment 1, again demonstrates the ability of these mixtures, containing DPhPE lipids and multiple additional triblock copolymers, to improve the number of sequenced molecules per minute per cell compared to standard DPhPE without the addition of triblock copolymers. In this case, more complex mixtures also result in a significant increase in the number of cells to be sequenced. The polymer composition in the membrane, as a result of mixing unmodified polymers with head-modified polymers, can modulate the cell yield and throughput in molecular sequencing within each cell.

[0139] A further advantage obtained in all experiments using synthetic triblock copolymers is the elimination of undesirable nanopore populations that cannot provide sequencing data. When sequencing experiments are performed with DPhPE lipids alone, a considerable amount of non-sequencing pores are generated. As is clear from the results shown in the image in Figure 13A, when using a lipid bilayer of DPhPE alone, a bimodal distribution is obtained on the vertical axis of the graph tangent to the left of each, indicating that there are many nanopores embedded in the array cell that prevent the sequencing experiment from being performed. As the results shown in Figures 13B, 13C, and 13D show, adding 20%, 30%, or 40% TBC Ch111 relative to DPhPE in the lipid bilayer eliminates non-functional populations and improves the probability of successful sequencing data.

[0140] All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, or other document is individually indicated as being incorporated herein by reference for all purposes.

[0141] Although various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. A lipid bilayer composition, (a) A mixture of phospholipids and triblock copolymers, wherein the triblock copolymer accounts for about 2% to about 40% of the solid content mass in the membrane solution mixture, (b) A lipid bilayer composition comprising a pore-forming molecule having pores connecting both sides of the bilayer.

2. The composition according to claim 1, wherein the molecular weight of the triblock copolymer is about 3,500 daltons to about 6,500 daltons.

3. The composition according to any one of claims 1 to 2, wherein the polydispersity of the triblock copolymer is about 1.1 to about 1.

8.

4. The composition according to any one of claims 1 to 3, wherein the triblock copolymer is poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline)triblock copolymer.

5. The aforementioned triblock copolymer is given by the following formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (In the formula, R 1 This is the terminal head base, PMOXA is a poly(2-methyl-2-oxazoline) subunit, PDMS is a poly(dimethylsiloxane) subunit, R 2 is -OH or terminal head group, and (m is between 4 and 20, and n is between 20 and 60) A composition according to any one of claims 1 to 4, having the following characteristics.

6. The composition according to claim 5, wherein the triblock copolymer has average m and n values ​​selected from the following: m=12, n=21; (Ch03) m=8, n=34; (Ch05) m=7, n=21; (Ch15) m=9, n=30; (Ch20 / Ch127) m=11, n=40; ​​(Ch42) m=9, n=37; (Ch67) m=5, n=18; (Ch71) m=5, n=17; (Ch72) m=4, n=18; (Ch74) m=11, n=53; (Ch93) m=9, n=37; (Ch103) m=12, n=53; (Ch104) m=9, n=37; (Ch106 / Ch118) m=13, n=59; (Ch108) m=13, n=52; (Ch109) m=11, n=49; (Ch110) m=12, n=50; (Ch111) m=9, n=36; (Ch112) m=9, n=42; (Ch113) m=12, n=55; (Ch114) m=9, n=39; (Ch115 / Ch117) m=11, n=49; (Ch116) m=10, n=38; (Ch119) m=10, n=40; ​​(Ch120) m=12, n=44; (Ch121) m=12, n=41; (Ch122) m=12, n=37; (Ch123) m=12, n=45; (Ch124) m=10, n=31; (Ch125) m=11, n=37; (Ch126) m=12, n=24; (Ch128) m=13, n=31; (Ch129) m=12, n=40; ​​(Ch130) m=9, n=33; (Ch131) m=11, n=34; (Ch132) m=9, n=32; (Ch133) m=11, n=33; (Ch134) m=9, n=39; (Ch135) m=11, n=36; (Ch136) and mixtures thereof.

7. The composition according to claims 5 to 6, wherein the terminal head group includes a triazole moiety.

8. The composition according to claims 5 to 7, wherein the terminal head group comprises a group selected from hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine.

9. The composition according to claims 5 to 8, wherein the terminal head group is selected from the following.

10. The composition according to any one of claims 1 to 9, wherein the triblock copolymer is amphiphilic.

11. The composition according to any one of claims 1 to 10, wherein the phospholipid is selected from DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DSPE, or a mixture thereof.

12. The composition according to any one of claims 1 to 11, wherein the pore-forming molecule connecting both sides of the bilayer is a protein nanopore, and optionally the protein nanopore comprises naturally occurring α-hemolysin and / or a variant of α-hemolysin.

13. A composition containing approximately 0.1 mg / mL to approximately 1 mg / mL of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) triblock copolymer and approximately 1 mg / mL to approximately 3 mg / mL of phospholipid in a solution in which the ratio of silicone oil to hexadecane is approximately 9:1, approximately 14:1, or approximately 19:

1.

14. The composition according to claim 13, wherein the molecular weight of the triblock copolymer is about 3,500 daltons to about 6,500 daltons.

15. The composition according to any one of claims 13 to 14, wherein the polydispersity of the triblock copolymer is about 1.1 to about 1.

8.

16. The composition according to any one of claims 13 to 15, wherein the triblock copolymer is poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline)triblock copolymer.

17. The aforementioned triblock copolymer is given by the following formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (In the formula, R 1 This is the terminal head base, PMOXA is a poly(2-methyl-2-oxazoline) subunit, PDMS is a poly(dimethylsiloxane) subunit, R 2 is -OH or terminal head group, and (m is between 4 and 20, and n is between 20 and 60) A composition according to any one of claims 13 to 16, having the following characteristics.

18. The composition according to claim 17, wherein the triblock copolymer has average m and n values ​​selected from the following: m=12, n=21; (Ch03) m=8, n=34; (Ch05) m=7, n=21; (Ch15) m=9, n=30; (Ch20 / Ch127) m=11, n=40; ​​(Ch42) m=9, n=37; (Ch67) m=5, n=18; (Ch71) m=5, n=17; (Ch72) m=4, n=18; (Ch74) m=11, n=53; (Ch93) m=9, n=37; (Ch103) m=12, n=53; (Ch104) m=9, n=37; (Ch106 / Ch118) m=13, n=59; (Ch108) m=13, n=52; (Ch109) m=11, n=49; (Ch110) m=12, n=50; (Ch111) m=9, n=36; (Ch112) m=9, n=42; (Ch113) m=12, n=55; (Ch114) m=9, n=39; (Ch115 / Ch117) m=11, n=49; (Ch116) m=10, n=38; (Ch119) m=10, n=40; ​​(Ch120) m=12, n=44; (Ch121) m=12, n=41; (Ch122) m=12, n=37; (Ch123) m=12, n=45; (Ch124) m=10, n=31; (Ch125) m=11, n=37; (Ch126) m=12, n=24; (Ch128) m=13, n=31; (Ch129) m=12, n=40; ​​(Ch130) m=9, n=33; (Ch131) m=11, n=34; (Ch132) m=9, n=32; (Ch133) m=11, n=33; (Ch134) m=9, n=39; (Ch135) m=11, n=36; (Ch136) and mixtures thereof.

19. The composition according to claims 17 to 18, wherein the terminal head group includes a triazole moiety.

20. The composition according to claims 17 to 18, wherein the terminal head group comprises a group selected from hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine.

21. The composition according to claims 17 to 20, wherein the terminal head group is selected from the following.

22. The composition according to any one of claims 13 to 21, wherein the triblock copolymer is amphiphilic.

23. The composition according to any one of claims 13 to 22, wherein the phospholipid is selected from DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DSPE, or a mixture thereof.

24. The composition according to any one of claims 13 to 23, further comprising a composition in which the pore-forming molecules connecting both sides of the bilayer are protein nanopores, and optionally the protein nanopores include naturally occurring α-hemolysin and / or variants of α-hemolysin.

25. It is an electrochemical cell, (a) Nanopores embedded in a lipid bilayer, wherein the lipid bilayer contains a mixture of phospholipids and triblock copolymers, and (b) A cis-side chamber and a trans-side chamber separated by the lipid bilayer and operably connected by the nanopore, each containing an electrode and an electrolyte solution. An electrochemical cell, including one.

26. The cell according to claim 25, wherein the triblock copolymer accounts for about 2% to about 40% of the solid content mass in the membrane solution mixture.

27. The cell according to any one of claims 25 to 26, wherein the molecular weight of the triblock copolymer is about 3,500 daltons to about 6,500 daltons, and / or the polydispersity of the triblock copolymer is about 1.1 to about 1.

8.

28. The cell according to any one of claims 25 to 27, wherein the triblock copolymer is poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline)triblock copolymer.

29. The aforementioned triblock copolymer is given by the following formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (In the formula, R 1 This is the terminal head base, PMOXA is a poly(2-methyl-2-oxazoline) subunit, PDMS is a poly(dimethylsiloxane) subunit, R 2 is -OH or terminal head group, and (m is between 4 and 20, and n is between 20 and 60) A cell according to any one of claims 25 to 28, having the following characteristics.

30. The cell according to claim 29, wherein the triblock copolymer has average m and n values ​​selected from the following: m=12, n=21; (Ch03) m=8, n=34; (Ch05) m=7, n=21; (Ch15) m=9, n=30; (Ch20 / Ch127) m=11, n=40; ​​(Ch42) m=9, n=37; (Ch67) m=5, n=18; (Ch71) m=5, n=17; (Ch72) m=4, n=18; (Ch74) m=11, n=53; (Ch93) m=9, n=37; (Ch103) m=12, n=53; (Ch104) m=9, n=37; (Ch106 / Ch118) m=13, n=59; (Ch108) m=13, n=52; (Ch109) m=11, n=49; (Ch110) m=12, n=50; (Ch111) m=9, n=36; (Ch112) m=9, n=42; (Ch113) m=12, n=55; (Ch114) m=9, n=39; (Ch115 / Ch117) m=11, n=49; (Ch116) m=10, n=38; (Ch119) m=10, n=40; ​​(Ch120) m=12, n=44; (Ch121) m=12, n=41; (Ch122) m=12, n=37; (Ch123) m=12, n=45; (Ch124) m=10, n=31; (Ch125) m=11, n=37; (Ch126) m=12, n=24; (Ch128) m=13, n=31; (Ch129) m=12, n=40; ​​(Ch130) m=9, n=33; (Ch131) m=11, n=34; (Ch132) m=9, n=32; (Ch133) m=11, n=33; (Ch134) m=9, n=39; (Ch135) m=11, n=36; (Ch136) and mixtures thereof.

31. The cell according to any one of claims 29 to 30, wherein the terminal head group includes a triazole moiety.

32. The cell according to any one of claims 29 to 31, wherein the terminal head group comprises a group selected from hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine.

33. The cell according to any one of claims 29 to 32, wherein the terminal head group is selected from the following.

34. The cell according to any one of claims 25 to 33, wherein the triblock copolymer is amphiphilic.

35. The cell according to any one of claims 25 to 33, wherein the phospholipid is selected from DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DSPE, or a mixture thereof.

36. The cell according to any one of claims 25 to 35, wherein the pore-forming molecule connecting both sides of the bilayer is a protein nanopore, and optionally the protein nanopore comprises naturally occurring α-hemolysin and / or a variant of α-hemolysin.

37. The cell according to any one of claims 25 to 36, which exhibits at least a 10% increase in HQMT / min / cell compared to an electrochemical cell having a lipid bilayer that does not contain the triblock copolymer.

38. A method for sequencing a target nucleic acid, (e) an electrochemical cell: (i) Nanopores embedded in a lipid bilayer, wherein the lipid bilayer comprises a mixture of phospholipids and triblock copolymers; (ii) A cis-side chamber and a trans-side chamber separated by the lipid bilayer and operably connected by the nanopore, each containing an electrode and an electrolyte solution; and (iii) Inlet and outlet ports operably connected to the cell; To provide an electrochemical cell containing, (f) Adding a molecule derived from the target nucleic acid to the cis-side chamber, (g) Applying a voltage to the cell that moves at least a portion of the molecules through the nanopore, (h) detecting a change in the voltage flow within the cell as the molecule moves through the nanopore, wherein the change in the voltage flow indicates the sequence of the target nucleic acid. Methods that include...

39. The method according to claim 38, wherein the triblock copolymer accounts for about 2% to about 40% of the solid content mass in the mixture.

40. The method according to any one of claims 38 to 39, wherein the molecular weight of the triblock copolymer is about 3,500 daltons to about 6,500 daltons, and / or the polydispersity of the triblock copolymer is about 1.1 to about 1.

8.

41. The method according to any one of claims 38 to 40, wherein the triblock copolymer is poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline)triblock copolymer.

42. The aforementioned triblock copolymer is given by the following formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (In the formula, R 1 This is the terminal head base, PMOXA is a poly(2-methyl-2-oxazoline) subunit, PDMS is a poly(dimethylsiloxane) subunit, R 2 is -OH or terminal head group, and (m is between 4 and 20, and n is between 20 and 60) The method according to any one of claims 38 to 41, comprising:

43. The method according to claim 42, wherein the triblock copolymer has average m and n values ​​selected from the following: m=12, n=21; (Ch03) m=8, n=34; (Ch05) m=7, n=21; (Ch15) m=9, n=30; (Ch20 / Ch127) m=11, n=40; ​​(Ch42) m=9, n=37; (Ch67) m=5, n=18; (Ch71) m=5, n=17; (Ch72) m=4, n=18; (Ch74) m=11, n=53; (Ch93) m=9, n=37; (Ch103) m=12, n=53; (Ch104) m=9, n=37; (Ch106 / Ch118) m=13, n=59; (Ch108) m=13, n=52; (Ch109) m=11, n=49; (Ch110) m=12, n=50; (Ch111) m=9, n=36; (Ch112) m=9, n=42; (Ch113) m=12, n=55; (Ch114) m=9, n=39; (Ch115 / Ch117) m=11, n=49; (Ch116) m=10, n=38; (Ch119) m=10, n=40; ​​(Ch120) m=12, n=44; (Ch121) m=12, n=41; (Ch122) m=12, n=37; (Ch123) m=12, n=45; (Ch124) m=10, n=31; (Ch125) m=11, n=37; (Ch126) m=12, n=24; (Ch128) m=13, n=31; (Ch129) m=12, n=40; ​​(Ch130) m=9, n=33; (Ch131) m=11, n=34; (Ch132) m=9, n=32; (Ch133) m=11, n=33; (Ch134) m=9, n=39; (Ch135) m=11, n=36; (Ch136) and mixtures thereof.

44. The method according to any one of claims 42 to 43, wherein the terminal head group of the triblock copolymer comprises a triazole moiety.

45. The method according to any one of claims 42 to 44, wherein the terminal head group of the triblock copolymer comprises a group selected from hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine.

46. The method according to any one of claims 42 to 45, wherein the terminal head group of the triblock copolymer is selected from the following.

47. The method according to any one of claims 42 to 46, wherein the triblock copolymer is amphiphilic.

48. The method according to any one of claims 42 to 47, wherein the phospholipid is selected from DPhPE, DOPhPE, DPhPC, DOPhPC, DPPE, DPPC, DOPE, DMPE, DSPE, or a mixture thereof.

49. The method according to any one of claims 42 to 48, wherein the pore-forming molecule connecting both sides of the bilayer is a protein nanopore, and optionally the protein nanopore comprises naturally occurring α-hemolysin and / or a variant of α-hemolysin.

50. The method according to any one of claims 42 to 49, wherein the cell contains a solution volume between about 0.1 femtoliters and about 1,000 femtoliters.

51. The method according to any one of claims 42 to 50, wherein the electrolyte solution contains ferrocyanide ions and ferricyanide ions in a total concentration range between about 25 mM and 250 mM.

52. The method according to any one of claims 42 to 51, wherein the electrolyte solution comprises a buffer composition.

53. The method according to any one of claims 42 to 52, wherein the buffer composition comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM and ammonium chloride at a concentration of less than 2000 mM, and optionally comprises ammonium acetate at a concentration of 0 mM and ammonium chloride at a concentration of less than 1000 mM.

54. The method according to any one of claims 52 to 53, wherein the molecule derived from the target nucleic acid includes Xpandomer.

55. The method according to claim 54, wherein the Xpandomer comprises a plurality of XNTP subunits coupled with sequences corresponding to a sequence of all or part of the target nucleic acid, and each of the XNTP subunits in the chain comprises a reporter construct, a nucleic acid base residue, and a selectively cleavable bond, and the cleavage of the selectively cleavable bond results in an Xpandomer longer than the plurality of XNTP subunits in the chain.

56. The method according to claim 55, further comprising severing the selectively separable bond to obtain an Xpandomer.

57. The method according to any one of claims 42 to 56, wherein the Xpandomer includes a reporter construct for analyzing genetic information in a sequence corresponding to all or part of the continuous nucleotide sequence of the target nucleic acid.

58. The method according to claim 57, wherein detecting the voltage change includes detecting the voltage change caused by the reporter construct of the Xpandomer moving through the nanopore.

59. The method according to claim 58, wherein the reporter construct for analyzing the genetic information comprises a reporter code and a movement control element, the movement control element providing sterically hindered movement control and pausing the movement of the Xpandomer when passing through a nanopore subjected to a baseline voltage, the movement control element engaging with the reporter code within the opening of the nanopore, and the reporter code being sensed by the nanopore.

60. The method according to any one of claims 42 to 59, wherein the applied voltage includes a baseline voltage, and optionally the baseline voltage is approximately 55 mV to approximately 95 mV.

61. The method according to any one of claims 42 to 60, wherein the applied voltage includes a pulse voltage, and optionally the pulse voltage is approximately 320 mV to approximately 550 mV.

62. The method according to any one of claims 42 to 61, wherein the pulse voltage has a duration of about 5 μs to about 15 μs.

63. The method according to any one of claims 42 to 62, wherein the time between pulse voltages is approximately 0.2 ms to approximately 1.7 ms.

64. The method according to any one of claims 42 to 63, wherein the applied voltage provides an alternating current, and optionally the alternating current has a periodicity of about 0.4 seconds to about 6 seconds.

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