A barrier containing a biological nanopore for DNA sequencing, the barrier being made from a copolymer having end groups and / or intermediate groups, and a method for producing the same

Abstract

A nanopore device including a barrier using a polymer having end groups, and a method of manufacturing the same are provided herein. In some examples, a barrier is provided between a first fluid and a second fluid. The barrier can be suspended by a barrier support that defines an opening. The barrier can be suspended across the opening and include one or more layers containing molecules of a block copolymer. Each molecule of the block copolymer can include one or more hydrophilic blocks having an approximate length A and one or more hydrophilic blocks having an approximate length B. The hydrophilic blocks can form the outer surface of the barrier, and the hydrophobic blocks can be located within the barrier. The end groups can be bonded to the ends of the hydrophilic blocks that form the outer surface of the barrier. The end groups can have a hydrophilicity different from that of the hydrophilic blocks.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Patent Application No. 63 / 325,737, filed Mar. 31, 2022, entitled "NANOPORE DEVICES INCLUDING BARRIERS USING POLYMERS WITH END GROUPS, AND METHODS OF MAKING THE SAME", the entire content of which is incorporated herein by reference.

[0002] (Field of the Invention) This application relates to devices including barriers.

Background Art

[0003] A vast amount of academic and corporate time and energy has been spent on using nanopores for polynucleotide sequencing. For example, the dwell time has been measured for a DNA complex with the Klenow fragment (KF) of DNA polymerase I on a nanopore in an applied electric field. Or, for example, a current or flux measurement sensor has been used in experiments on DNA captured within an α - hemolysin nanopore. Or, for example, KF - DNA complexes have been distinguished based on their properties when captured in an electric field above an α - hemolysin nanopore. In yet another example, polynucleotide sequencing is performed using a single polymerase enzyme complex that includes a polymerase enzyme and a template nucleic acid attached proximal to a nanopore, and nucleotide analogs in solution. The nucleotide analogs include a charge - blocking label attached to the polyphosphate portion of the nucleotide analog such that when the nucleotide analog is incorporated into the synthesized polynucleotide, the charge - blocking label is cleaved. The charge - blocking label is detected by the nanopore to determine the presence and identity of the incorporated nucleotide, thereby determining the sequence of the template polynucleotide. In still other examples, constructs include transmembrane protein pore subunits and nucleic acid - handling enzymes.

[0004] However, such previously known devices, systems, and methods are not necessarily sufficiently robust, reproducible, or sensitive, and may not have a throughput that is sufficiently high for practical implementation, for example, in commercial applications such as genomic sequencing in clinical and other settings that require cost - effectiveness and very accurate operation. Thus, what is needed are improved devices, systems, and methods for sequencing polynucleotides, which may include using a membrane having nanopores disposed therein. SUMMARY OF THE INVENTION

[0005] A nanopore device including a barrier using a polymer having end groups, and a method of making the same are provided herein.

[0006] In some examples, a barrier is provided between a first fluid and a second fluid. The barrier may be suspended by a barrier support defining an opening. The barrier may be suspended across the opening and may include one or more layers including molecules of a block copolymer. Each molecule of the block copolymer may include one or more hydrophilic blocks having an approximate length A and one or more hydrophilic blocks having an approximate length B. The hydrophilic blocks may form the outer surface of the barrier, and the hydrophobic blocks may be located within the barrier. The end groups may be attached to the ends of the hydrophilic blocks forming the outer surface of the barrier. The end groups may have a hydrophilicity different from that of the hydrophilic blocks.

[0007] In some examples, the end groups are selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert - butylcarbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3 - ).

[0008] In some examples, the hydrophobic block comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polybutadiene, fluorinated polyethylene, polypeptide, and poly(isobutylene) (PIB).

[0009] In some examples, the block copolymer is a diblock copolymer. In some examples, the hydrophobic block is polybutadiene (PBd). In some examples, the barrier has a thickness of approximately 2A + 2B.

[0010] In some examples, the block copolymer is a triblock copolymer having two hydrophilic blocks and one hydrophobic block. In some examples, the hydrophobic block is poly(isobutylene) (PIB). In some examples, the barrier has a thickness of approximately 2A + B.

[0011] In some examples, the block copolymer is a triblock copolymer having two hydrophobic blocks and one hydrophilic block. In some examples, the barrier has a thickness of approximately A + 2B.

[0012] In some examples, the barrier further comprises nanopores disposed within the barrier to provide contact between the first fluid and the second fluid.

[0013] Some examples of this specification provide a barrier that includes at least one layer containing a plurality of molecules. Each of the molecules can include a first hydrophilic block and a second hydrophilic block, a first end group and a second end group, and a hydrophobic block. The hydrophobic block can be disposed between and bonded to the first hydrophilic block and the second hydrophilic block. The first end group and the second end group can each be bonded to the ends of the first hydrophilic block and the second hydrophilic block, respectively, and can have a hydrophilicity different from that of the first hydrophilic block and the second hydrophilic block. The first end group and the second end group can form the first outer surface and the second outer surface of the barrier. The hydrophobic block can be within the barrier.

[0014] In some examples, each of the first hydrophilic block and the second hydrophilic block includes poly(ethylene oxide) (PEO). In some examples, each of the first hydrophilic block and the second hydrophilic block includes from about 2 to about 100 PEO repeat units. In some examples, each of the first hydrophilic block and the second hydrophilic block includes from about 2 to about 12 PEO repeat units. In some examples, each of the first hydrophilic block and the second hydrophilic block includes from about 2 to about 4 PEO repeat units. In some examples, each of the first hydrophilic block and the second hydrophilic block includes from about 3 to about 9 PEO repeat units. In some examples, each of the first hydrophilic block and the second hydrophilic block includes from about 9 to about 12 PEO repeat units.

[0015] In some examples, the hydrophobic block includes poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB). In some examples, the hydrophobic block includes from about 2 to about 100 PDMS repeat units. In some examples, the hydrophobic block includes from about 13 to about 44 PDMS repeat units, or from about 30 to about 44 PDMS repeat units. In some examples, the hydrophobic block includes from about 2 to about 100 PIB repeat units. In some examples, the hydrophobic block includes from about 13 to about 44 PIB repeat units, or from about 30 to about 44 PIB repeat units.

[0016] In some examples, the hydrophobic block is attached to the first hydrophilic block and the second hydrophilic block via respective sulfide, ether, ester, alkyl, or triazole linkages.

[0017] In some examples, the first end group and the second end group are fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3 - ) independently selected from the group consisting of.

[0018] In some examples, the barrier further includes a nanopore. In some examples, the nanopore includes α-hemolysin or MspA.

[0019] Some examples herein provide a barrier that includes at least one layer containing a plurality of molecules. Each of the molecules can include a first ionic end group, a second ionic end group, and a hydrophobic block. The hydrophobic block is disposed between and can be attached to the first ionic end group and the second ionic end group. The ionic end groups can form the first outer surface and the second outer surface of the barrier. The hydrophobic block can be within the barrier.

[0020] In some examples, each of the first ionic end group and the second ionic end group includes a zwitterion. In some examples, the first ionic end group and the second ionic end group are selected from the group consisting of 2-methacryloyloxyethyl phosphorylcholine, 3-[dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azaniumyl]propane-1-sulfonate (DMAPS), 3-{[3-(acryloylamino)propyl](dimethyl)ammonio}propanoate, and 3-{[3-(acryloylamino)propyl](dimethyl)ammonio}-1-propanesulfonate.

[0021]

[0021] In some examples, each of the first ionic end group and the second ionic end group includes a cation. In some examples, each of the first ionic end group and the second ionic end group includes 2-(trimethylammonio)ethyl methacrylate.

[0022]

[0021] In some examples, each of the first ionic end group and the second ionic end group includes an anion. In some examples, each of the first ionic end group and the second ionic end group includes 3-sulfopropyl acrylate, 2-propene-1-sulfonate, or vinylphosphonic acid.

[0023]

[0021] In some examples, the hydrophobic block includes poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB). In some examples, the PDMS includes about 2 to 100 PDMS repeating units. In some examples, the PDMS includes about 13 to about 44 PDMS repeating units, or about 30 to about 44 PDMS repeating units. In some examples, the PIB includes about 2 to about 100 PIB repeating units. In some examples, the PIB includes about 13 to about 44 PIB repeating units, or about 30 to about 44 PIB repeating units.

[0024]

[0021] In some examples, each of the first ionic end group and the second ionic end group is coupled to the hydrophobic block via a product of hydrosilylation, amine-ester coupling, CuAAC click chemistry, DBCO-azide, thiol-Michael addition, or thiol-ene click reaction.

[0025]

[0021] In some examples, the barrier further includes nanopores. In some examples, the nanopores include α-hemolysin or MspA.

[0026] Some examples of this specification provide a barrier that includes a first layer including a first plurality of molecules and a second layer including a second plurality of molecules. Each of the molecules may include a hydrophilic block, a hydrophobic block, and a terminal group. The hydrophilic block may be bonded to the hydrophobic block. The terminal group may be bonded to the end of the hydrophilic block and may have a hydrophilicity different from that of the hydrophilic block. The terminal group may form the first outer surface and the second outer surface of the barrier. The hydrophobic blocks of the first plurality of molecules and the second plurality of molecules may contact each other within the barrier.

[0027] In some examples, the hydrophilic block includes poly(ethylene oxide) (PEO). In some examples, the hydrophilic block includes about 2 to about 100 PEO repeating units. In some examples, the hydrophilic block includes about 2 to about 12 PEO repeating units, for example, about 2 to about 9 PEO repeating units.

[0028] In some examples, the hydrophobic block includes poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB). In some examples, the hydrophobic block includes about 2 to about 100 PDMS repeating units. In some examples, the hydrophobic block includes about 14 to about 44 PDMS repeating units. In some examples, the hydrophobic block includes about 14 to about 26 PDMS repeating units, or about 26 to about 44 PDMS repeating units. In some examples, the hydrophobic block includes about 2 to about 100 PIB repeating units. In some examples, the hydrophobic block includes about 14 to about 44 PIB repeating units. In some examples, the hydrophobic block includes about 14 to about 26 PIB repeating units, or about 26 to about 44 PIB repeating units.

[0029] In some examples, the terminal group is fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH 3 )), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3- is selected from the group consisting of.

[0030] In some examples, the barrier further includes a nanopore. In some examples, the nanopore includes α-hemolysin or MspA.

[0031] Some examples herein provide a barrier that includes a first layer comprising a first plurality of molecules and a second layer comprising a second plurality of molecules. Each of the molecules can include a first hydrophobic block and a second hydrophobic block, a hydrophilic block, and a first end group and a second end group. The hydrophilic block can be disposed between and bonded to the first hydrophobic block and the second hydrophobic block. Each of the first end group and the second end group can be bonded to the ends of the first hydrophobic block and the second hydrophobic block and can have a hydrophobicity different from that of the first hydrophobic block and the second hydrophobic block. The hydrophilic blocks of the first plurality of molecules can form the first outer surface of the barrier. The hydrophilic blocks of the second plurality of molecules can form the second outer surface of the barrier. The first end groups and the second end groups of the first plurality of molecules and the second plurality of molecules can contact each other within the barrier.

[0032] In some examples, the hydrophilic block includes poly(ethylene oxide) (PEO). In some examples, the hydrophilic block includes from about 2 to about 100 PEO repeat units. In some examples, the hydrophilic block includes from about 2 to about 13 PEO repeat units, such as from about 7 to about 13 PEO repeat units, or from about 2 to about 7 PEO repeat units.

[0033] In some examples, each of the first hydrophobic block and the second hydrophobic block comprises poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB). In some examples, each of the first hydrophobic block and the second hydrophobic block comprises from about 2 to about 100 PDMS repeat units. In some examples, each of the first hydrophobic block and the second hydrophobic block comprises from about 14 to about 44 PDMS repeat units, such as from about 14 to about 26 PDMS repeat units, or from about 26 PDMS repeat units to about 44 PDMS repeat units. In some examples, each of the first hydrophobic block and the second hydrophobic block comprises from about 2 to about 100 PIB repeat units. In some examples, each of the first hydrophobic block and the second hydrophobic block comprises from about 14 to about 44 PIB repeat units, such as from about 14 to about 26 PIB repeat units, or from about 26 PIB repeat units to about 44 PIB repeat units.

[0034] In some examples, the first end group and the second end group comprise a lower alkyl (C 1-4 alkyl) or aryl group, a polycyclic aromatic hydrocarbon, a fluorinated alkyl, or a fluorinated aryl group. In some examples, the lower alkyl comprises a methyl, ethyl, propyl, or n-butyl group.

[0035] In some examples, the barrier further comprises nanopores. In some examples, the nanopores comprise α-hemolysin or MspA.

[0036] To achieve the benefits described herein, it should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented in any suitable combination with any of the features of any of the other aspects described herein. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0061] A nanopore device including a barrier using a polymer having a terminal group, and a method for manufacturing the same are provided herein.

[0062] For example, nanopore sequencing can utilize a nanopore inserted into a barrier and including an aperture through which ions and / or other molecules can flow from one side of the barrier to the other. The circuit can be used, for example, during synthesis-based sequencing (SBS) to detect a sequence, such as a nucleotide sequence, in which, on a first side of the barrier, polymerase adds nucleotides to a growing polynucleotide in an order based on the sequence of a template polynucleotide to which the growing polynucleotide is hybridized. The sensitivity of the circuit can be improved, for example, by using fluids having different compositions on each side of the barrier to provide appropriate electron transport for detection on one side of the barrier while appropriately promoting polymerase activity on the other side of the barrier. The difference in fluid composition can generate an osmotic pressure that can weaken the barrier and thus increase the likelihood that the barrier can break or leak during normal use. However, it can be difficult to insert a nanopore into a barrier that is too strong.

[0063] As provided herein, a barrier for use in a nanopore device can include a polymer that provides stability characteristics suitable for long-term use of the device and facilitates nanopore insertion to increase the number of devices that can be used during manufacture. As described in more detail below, in some examples, the polymers of the invention can include hydrophilic blocks attached to hydrophobic blocks (e.g., can include diblock copolymers). In other examples, the polymer can include a hydrophilic block bonded between two hydrophobic blocks, or a hydrophobic block bonded between two hydrophilic blocks (e.g., can include triblock copolymers). In still other examples, the polymer can include hydrophobic blocks and may not include hydrophilic block(s). As provided herein, through various examples, each end of the hydrophobic block(s), hydrophilic block(s), or both hydrophilic block(s) and hydrophilic block(s) can include end groups having a hydrophilicity different from the block to which they are attached. The end groups, as well as the respective lengths of the hydrophobic and / or hydrophilic blocks in the polymer, can be selected such that the polymer assembles into a barrier having appropriate stability and utility, e.g., in nanopore sequencing.

[0064] First, some of the terms used herein are briefly described. Next, some exemplary barriers using polymers having end groups, methods of making the same, and devices and methods of using the same are described.

[0065] Term Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art. The term "including", as well as the use of other forms such as "include", "includes", and "included", is not limiting. The term "having", as well as the use of other forms such as "have", "has", and "had", is not limiting. As used herein, whether in a transitional phrase or in the body of a claim, the terms "comprise" and "comprising" shall be interpreted as having an open-ended meaning. That is, the above terms shall be interpreted as synonymous with the phrase "having at least" or "including at least". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or system, the term "comprising" means that the compound, composition, or system includes at least the recited features or components, but may also include additional features or components.

[0066] As used herein, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

[0067] The terms "substantially", "approximately", and "about" as used throughout this specification are used to account for and describe minor variations, such as variations in processing. For example, they may refer to ±10% or less, such as ±5% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.2% or less, ±0.1% or less, ±0.05% or less.

[0068] As used herein, the term "nucleotide" is intended to mean a molecule that includes a sugar and at least one phosphate group and, in some instances, also includes a nucleobase. Nucleotides lacking a nucleobase may be referred to as "abasic." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidineIt includes deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0069] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analog that is a type of nucleotide containing a modified nucleobase, sugar, backbone, and / or phosphate moiety as compared to a naturally occurring nucleotide. Nucleotide analogs may also be referred to as "modified nucleic acids". Exemplary modified nucleobases include inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioladenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyladenine or guanine, 5-halo-substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. As is known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, such as nucleotide analogs like adenosine 5'-phosphosulfate. A nucleotide may contain any suitable number of phosphates, e.g., 3, 4, 5, 6, or more than 6 phosphates. Nucleotide analogs include locked nucleic acid (LNA), peptide nucleic acid (PNA), and 5-hydroxylbutynyl-2'-deoxyuridine ("super T").

[0070] As used herein, the term "polynucleotide" refers to a molecule comprising a sequence of nucleotides that are joined to one another. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogs thereof such as locked nucleic acid (LNA) and peptide nucleic acid (PNA). A polynucleotide can be a single-stranded sequence of nucleotides such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides such as double-stranded DNA (dsDNA), or can include a mixture of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. A polynucleotide can include non-naturally occurring DNA such as enantiomeric DNA, LNA, or PNA. The exact sequence of nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, expressed sequence tag (EST), or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer of any of the foregoing, or amplified copy.

[0071] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to a primer and a single-stranded target polynucleotide and can continuously add nucleotides to the growing primer to form a "complementary copy" polynucleotide having a sequence complementary to the sequence of the target polynucleotide. A DNA polymerase can bind to a target polynucleotide and then move the target polynucleotide downstream, continuously adding nucleotides to the free hydroxyl group at the 3' end of the growing polynucleotide strand. A DNA polymerase can synthesize a complementary DNA molecule from a DNA template. An RNA polymerase can synthesize an RNA molecule from a DNA template (transcription). Other RNA polymerases, such as reverse transcriptase, can synthesize a cDNA molecule from an RNA template. Still other RNA polymerases can synthesize an RNA molecule from an RNA template, such as RdRp. A polymerase can initiate strand growth using a short RNA or DNA strand (primer). Some polymerases can displace the strand upstream of the site where they add bases to the strand. Such polymerases can also be said to be strand-displacing, which can be said to have the activity of removing the complementary strand from the template strand read by the polymerase.

[0072] Exemplary DNA polymerases include Bst DNA polymerase, 9°Nm DNA polymerase, Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I (Large), (Klenow) fragment, Klenow fragment (3'-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep VentR™ (exo-) DNA polymerase, Deep VentR™ DNA polymerase, DyNAzyme™ EXT DNA, DyNAzyme™ II Hot Start DNA polymerase, Phusion™ High-Fidelity DNA polymerase, Therminator™ DNA polymerase, Therminator™ II DNA polymerase, VentR® DNA polymerase, VentR™ (exo-) DNA polymerase, RepliPHI™ Phi29 DNA polymerase, rBst DNA polymerase, rBst DNA polymerase (Large), fragment (IsoTherm™ DNA polymerase), MasterAmp™ AmpliTherm™ DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase beta, ThermoPhi DNA polymerase, and Isopol™ SD+ polymerase. In certain non-limiting examples, the polymerase is selected from the group consisting of Bst, Bsu, and Phi29. Some polymerases have the activity of degrading the strand behind them (3' exonuclease activity). Some useful polymerases are modified by mutation or other methods to reduce or eliminate 3' and / or 5' exonuclease activity.

[0073] Exemplary RNA polymerases include RdRp (RNA-dependent, RNA polymerase) that catalyzes the synthesis of an RNA strand complementary to a given RNA template. Exemplary RdRps include poliovirus 3Dpol, vesicular stomatitis virus L, and hepatitis C virus NS5B protein. Exemplary RNA reverse transcriptases. Non-limiting exemplary lists include reverse transcriptases derived from avian myelomatosis virus (AMV), murine moloney leukemia virus (MMLV), and / or human immunodeficiency virus (HIV), telomerase reverse transcriptases such as (hTERT), SuperScript™ III, SuperScript™ IV reverse transcriptase, ProtoScript® II reverse transcriptase.

[0074] As used herein, the term "primer" is defined as a polynucleotide to which nucleotides can be added via a free 3' OH group. The primer may include a 3' block that prevents polymerization until the block is removed. The primer may include a 5' end modification to enable a coupling reaction or to enable the primer to bind to another moiety. The primer may include one or more moieties such as 8-oxo-G that can be cleaved under appropriate conditions such as UV light, chemicals, enzymes, etc. The length of the primer can be any suitable number of bases and can include suitable combinations of natural and / or unnatural nucleotides. The target polynucleotide may include an "amplification adapter" (or more simply an "adapter") that hybridizes to the primer (has a sequence complementary to the primer) and can be amplified to generate a complementary copy polynucleotide by adding nucleotides to the free 3' OH group of the primer.

[0075] As used herein, the term "plurality" is intended to mean a group of two or more different members. The plurality can range from small, medium, large, to very large sizes. A plurality of small size can, for example, range from several members to several tens of members. A plurality of medium size can, for example, range from several tens of members to about 100 members or several hundreds of members. A large plurality can, for example, range from about several hundreds of members to about 1000 members, several thousands of members, and tens of thousands of members. A very large plurality can, for example, range from tens of thousands of members to about several hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, the plurality can range from 2 to well over 100 million members in size, as well as between all sizes measured by the number of members and larger than the exemplary ranges above. Thus, the definition of this term is intended to include all integer values greater than 2.

[0076] As used herein, the term "double-stranded", when used with respect to a polynucleotide, is intended to mean that all or substantially all of the nucleotides in the polynucleotide are hydrogen-bonded to each of the nucleotides in a complementary polynucleotide. A double-stranded polynucleotide can also be referred to as a "duplex".

[0077] As used herein, the term "single-stranded", when used with respect to a polynucleotide, means that none of the nucleotides in the polynucleotide are hydrogen-bonded to each of the nucleotides in a complementary polynucleotide.

[0078] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the subject of an analysis or action, and may also be referred to using terms such as "library polynucleotide", "template polynucleotide", or "library template". The analysis or action includes subjecting the polynucleotide to amplification, sequencing, and / or other procedures. The target polynucleotide may include additional nucleotide sequences to the target sequence being analyzed. For example, the target polynucleotide may include one or more adapters that include amplification adapters that function as primer binding sites, which flank the target polynucleotide sequence being analyzed. In certain examples, multiple target polynucleotides may have different sequences from each other, but may have the same first and second adapters as each other. Two adapters that may flank a particular target polynucleotide sequence may have the same sequence as each other, or complementary sequences to each other, or the two adapters may have different sequences. Thus, the species among multiple target polynucleotides may include known sequence regions flanked by unknown sequence regions that are evaluated, for example, by sequencing (e.g., SBS). In some examples, the target polynucleotide carries an amplification adapter at a single end, and such adapter may be located at either the 3' end or the 5' end of the target polynucleotide. The target polynucleotide may be used without an adapter, in which case the primer binding sequence may directly use the sequence present in the target polynucleotide.

[0079] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in terms is not intended to indicate any particular difference in size, sequence, or other characteristics, unless otherwise specified. To clarify the description, when describing a particular method or composition that includes several polynucleotide species, the terms may be used differently to distinguish one species of polynucleotide from another.

[0080] As used herein, the term "substrate" refers to a material used as a support for the compositions described herein. Exemplary substrate materials can include glass, silica, plastic, quartz, metal, metal oxide, organo-silicates (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of POSS can be that described in Kehagias et al., Microelectronic Engineering 86(2009), pp.776-778, which is hereby incorporated by reference in its entirety. In some examples, the substrates used in the present application include silica-based substrates such as glass, fused silica, or other silica-containing materials. In some examples, the silica-based substrate can include silicon, silicon dioxide, silicon nitride, or hydrogenated silicone. In some examples, the substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Examples of plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material, or a combination thereof. In a particular example, the substrate has at least one surface that includes glass or a silicon-based polymer. In some examples, the substrate can include metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface that includes a metal oxide. In one example, the surface includes tantalum oxide or tin oxide. Acrylamide, enone, or acrylate can also be utilized as a substrate material or component. Other substrate materials can include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramic, polyimide, quartz, resin, polymers, and copolymers.In some examples, the substrate and / or the substrate surface can be quartz or can include quartz. In some other examples, the substrate and / or the substrate surface can be a semiconductor such as GaAs or ITO, or can include such a semiconductor. The above list is intended to illustrate the present application but not to limit it. The substrate can include a single material or a plurality of different materials. The substrate can be a composite or a laminate. In some examples, the substrate includes an organic silicate material.

[0081] The substrate can be horizontal, circular, spherical, rod-shaped, or any other suitable shape. The substrate can be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0082] The substrate may not have a pattern, may have a textured finish, or may have a pattern on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns can include posts, pads, wells, ridges, channels, or other three-dimensional concave or convex structures. The pattern can be regular or irregular across the surface of the substrate. The pattern can be formed, for example, by nanoimprint lithography or, for example, by the use of metal pads that form features on a non-metallic surface.

[0083] In some examples, the substrates described herein form at least a portion of a flow cell, are located within a flow cell, or are coupled to a flow cell. The flow cell can include a flow chamber divided into a plurality of lanes or sectors. Examples of flow cells that can be used in the methods and compositions described herein, as well as examples of substrates for manufacturing a flow cell, include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA).

[0084] As used herein, the term "electrode" is intended to mean a solid structure that conducts electricity. The electrode can include any suitable conductive material such as gold, palladium, silver, or platinum, or combinations thereof. In some examples, the electrode may be disposed on a substrate. In some examples, the electrode may define a substrate.

[0085] As used herein, the term "nanopore" is intended to mean a structure that includes an opening that allows a molecule to pass from a first side of the nanopore to a second side of the nanopore, and a portion of the opening of the nanopore has a width of 100 nm or less, for example, 10 nm or less, or 2 nm or less. The opening extends through the first and second sides of the nanopore. Molecules that can pass through the opening of the nanopore can include, for example, ions, or water-soluble molecules such as amino acids or nucleotides. The nanopore can be disposed within a barrier or provided through a substrate. Optionally, a portion of the opening can be narrower than one or both of the first and second sides of the nanopore, in which case that portion of the opening can be referred to as a "constriction." Alternatively or additionally, the opening of the nanopore, or the constriction of the nanopore (if present), or both can be 0.1 nm, 0.5 nm, 1 nm, 10 nm or more. The nanopore can include a plurality of constrictions, for example, at least two, or 3, or 4, or 5, or more than 5 constrictions, and the nanopore can include a biological nanopore, a solid nanopore, or a biological and solid hybrid nanopore.

[0086] Examples of biological nanopores include, for example, polypeptide nanopores and polynucleotide nanopores. "Polypeptide nanopore" is intended to mean a nanopore made from one or more polypeptides. The one or more polypeptides can include monomers, homopolymers, or heteropolymers. The structure of polypeptide nanopores includes, for example, α-helix bundle nanopores and β-barrel nanopores, as well as all others well known in the art. Exemplary polypeptide nanopores include aerolysin, α-hemolysin, Mycobacterium smegmatis porin A, gramicidin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F-pilus, SP1, mitochondrial porin (VDAC), Tom40, outer membrane phospholipase A, CsgG, aerolysin, and Neisseria autotransporter lipoprotein (NaIP). Mycobacterium smegmatis porin A (MspA) is a membrane porin produced by mycobacteria that allows hydrophilic molecules to enter the bacteria. MspA resembles a goblet and forms a tightly interconnected octamer and a transmembrane beta-barrel that includes a central constriction. For further details regarding α-hemolysin, see U.S. Patent No. 6,015,714, the entire contents of which are incorporated herein by reference. For further details regarding SP1, see Wang et al., Chem. Commun., 49:1741-1743 (2013), the entire contents of which are incorporated herein by reference.For further details regarding MspA, see Butler et al., "Single-molecule DNA detection with an engineered MspA protein nanopore", Proc. Natl. Acad. Sci. 105:20647-20652 (2008) and Derrington et al., "Nanopore DNA sequencing with MspA", Proc. Natl. Acad. Sci. USA, 107:16060-16065 (2010), the entire contents of which are incorporated herein by reference. Other nanopores include, for example, MspA homologs from Norcadia farcinica, and lysenin. For further details regarding lysenin, see International Publication No. WO 2013 / 153359, the entire contents of which are incorporated herein by reference.

[0087] "Polynucleotide nanopore" is intended to mean a nanopore made from one or more nucleic acid polymers. A polynucleotide nanopore can include, for example, polynucleotide origami.

[0088] "Solid nanopore" is intended to mean a nanopore made from one or more materials not of biological origin. Solid nanopores can be formed from inorganic or organic materials. Solid nanopores include, for example, silicon nitride (SiN), silicon dioxide (SiO 2 ), silicon carbide (SiC), hafnium oxide (HfO 2 ), molybdenum disulfide (MoS 2 ), hexagonal boron nitride (h-BN), or graphene. Solid nanopores can include an aperture formed within a solid membrane, for example, a membrane comprising any such material.

[0089] "Biological and solid hybrid nanopores" is intended to mean hybrid nanopores made from materials of both biological and non-biological origin. Materials of biological origin are as defined above and include, for example, polypeptides and polynucleotides. Biological and solid hybrid nanopores include, for example, polypeptide solid hybrid nanopores and polynucleotide solid nanopores.

[0090] As used herein, "barrier" is generally intended to mean a structure that inhibits the passage of molecules from one side of the barrier to the other. Molecules whose passage is inhibited can include, for example, ions or water-soluble molecules such as nucleotides and amino acids. However, when a nanopore is disposed within a barrier, the opening of the nanopore can allow the passage of molecules from one side of the barrier to the other. As one specific example, when a nanopore is disposed within a barrier, the opening of the nanopore can allow the passage of molecules from one side of the barrier to the other. Barriers include membranes of biological origin such as lipid bilayers, and non-biological barriers such as solid membranes or substrates.

[0091] As used herein, "of biological origin" refers to materials derived from or isolated from a biological environment such as an organism or cell, or a synthetically produced version of a biologically available structure.

[0092] As used herein, "solid" refers to materials that are not of biological origin.

[0093] As used herein, "synthetic" refers to membrane materials that are not of biological origin (e.g., polymer materials, synthetic phospholipids, solid membranes, or combinations thereof).

[0094] As used herein, "solution" is intended to refer to a homogeneous mixture containing two or more substances. In such a mixture, the solute is the substance that is dissolved in another substance called the solvent. A solution may contain a single solute or multiple solutes. "Aqueous solution" refers to a solution in which the solvent is water or contains water.

[0095] As used herein, "polymeric membrane" or "polymer membrane" refers to a synthetic barrier mainly composed of polymers not of biological origin. In some examples, the polymeric membrane consists essentially of polymers not of biological origin. Block copolymers are examples of polymers that are not of biological origin and can be included in this barrier. Hydrophobic polymers with ionic end groups are another example of polymers that are not of biological origin and can be included in this barrier. Since the barriers of the present invention are related to polymers not of biological origin, the terms "polymeric membrane", "polymer membrane", "membrane", and "barrier" can be used interchangeably herein when referring to the barriers of the present invention, even though the terms "barrier" and "membrane" may generally also encompass other types of materials as well.

[0096] As used herein, the term "block copolymer" is intended to refer to a polymer having at least a first portion or "block" containing a first type of monomer and at least a second portion or "block" directly or indirectly attached to the first portion and containing a second different type of monomer. The first portion may contain a polymer of the first type of monomer, or the second portion may contain a polymer of the second type of monomer, or the first portion may contain a polymer of the first type of monomer and the second portion may contain a polymer of the second type of monomer. The first portion may optionally contain a terminal group having a hydrophilicity different from that of the first type of monomer, or the second portion may optionally contain a terminal group having a hydrophilicity different from that of the second type of monomer, or the first portion may optionally contain a terminal group having a hydrophilicity different from that of the first type of monomer and the second portion may optionally contain a terminal group having a hydrophilicity different from that of the second type of monomer. The terminal groups of any hydrophilic block may be located on the outer surface of the barrier formed using such hydrophilic blocks. Depending on the particular configuration, the terminal groups of any hydrophobic block may be located on the inner surface of the barrier or on the outer surface of the barrier formed using such hydrophobic blocks.

[0097] Examples of block copolymers include, but are not limited to, diblock copolymers and triblock copolymers.

[0098] "Diblock copolymer" is intended to refer to a block copolymer comprising or consisting essentially of a first block and a second block directly or indirectly bonded to each other. The first block may be hydrophilic and the second block may be hydrophobic, in which case the diblock copolymer may be referred to as an "AB" copolymer, where "A" refers to the hydrophilic block and "B" refers to the hydrophobic block.

[0099] "Triblock copolymer" is intended to refer to a block copolymer that comprises or consists essentially of a first block, a second block, and a third block that are directly or indirectly bonded to one another. The first block and the third block may comprise or consist essentially of the same type of monomer (repeating unit) as one another, and the second block may comprise a different type of monomer (repeating unit). In some examples, the first block may be hydrophobic, the second block may be hydrophilic, the third block may be hydrophobic, and may comprise the same type of monomer as the first block, in which case the triblock copolymer may be referred to as a "BAB" copolymer, where "A" refers to the hydrophilic block and "B" refers to the hydrophobic block. In other examples, the first block may be hydrophilic, the second block may be hydrophobic, the third block may be hydrophilic, and may comprise the same type of monomer as the first block, in which case the triblock copolymer may be referred to as an "ABA" copolymer, where "A" refers to the hydrophilic block and "B" refers to the hydrophobic block.

[0100] The particular arrangement of the molecules of the polymer chains (e.g., block copolymers) within the polymer membrane can depend, inter alia, on the respective block lengths, the type(s) of monomer used in the different blocks, the relative hydrophilicity and hydrophobicity of the blocks, the composition of the fluid(s) in which the membrane is formed, and / or the density of the polymer chains within the membrane. During membrane formation, these and other factors generate forces among the polymer chains that position and reorient the molecules laterally in a manner that substantially minimizes the free energy of the membrane. Once the polymer chains have completed these rearrangements, the membrane can be considered to be substantially "stable", even if the molecules retain some mobility of movement within the membrane.

[0101] As used herein, the term "hydrophobic" is intended to mean having a tendency to exclude water molecules. Hydrophobicity is a relative concept regarding the difference in polarity of molecules with respect to their environment. Non-polar (hydrophobic) molecules in a polar environment tend to associate with each other in a manner that minimizes contact with polar (hydrophilic) molecules and reduces the overall free energy of the system.

[0102] As used herein, the term "hydrophilic" is intended to mean having a tendency to bind to water molecules. Polar (hydrophilic) molecules in a polar environment tend to associate with each other in a manner that minimizes contact with non-polar (hydrophobic) molecules and reduces the overall free energy of the system.

[0103] As used herein, the term "amphiphilic" is intended to mean having both hydrophilic and hydrophobic properties. For example, a block copolymer containing a hydrophobic block and a hydrophilic block can be considered "amphiphilic". Exemplarily, AB copolymers, ABA copolymers, and BAB copolymers can all be considered amphiphilic. Additionally, a molecule containing a hydrophobic polymer bound to an ionic end group can be considered amphiphilic.

[0104] As used herein, "solution" is intended to refer to a homogeneous mixture containing two or more substances. In such a mixture, the solute is the substance that is uniformly dissolved in another substance called the solvent. A solution may contain a single solute or multiple solutes. Additionally or alternatively, a solution may contain a single solute or multiple solutes. "Aqueous solution" refers to a solution in which the solvent is water or contains water.

[0105] As used herein, the term "electroporation" means applying a voltage across a membrane such that nanopores are inserted into the membrane.

[0106] As used herein, "a" and "b" being integers, "C a ~C b " or "C a~b " refers to the number of carbon atoms in a particular group. That is, the group can contain from "a" to "b" carbon atoms (including both ends). Thus, for example, "C 1 ~C 4 alkyl" or "C 1~4 alkyl" or "C 1~4 alkyl" groups refer to all alkyl groups having from 1 to 4 carbon atoms, i.e., CH 3 -, CH 3 CH 2 -, CH 3 CH 2 CH 2 -, (CH 3 ) 2 CH-, CH 3 CH 2 CH 2 CH 2 -, CH 3 CH 2 CH(CH 3 )-, and (CH 3 ) 3 C-.

[0107] As used herein, "alkyl" refers to a straight-chain or branched-chain hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). An alkyl group may have from 1 to 20 carbon atoms (whenever indicated herein, a numerical range such as "1 to 20" refers to each integer within the given range. For example, "from 1 to 20 carbon atoms" means that an alkyl group can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms, but this definition also covers the occurrence of the term "alkyl" (when no numerical range is specified). An alkyl group may also be a medium-sized alkyl having from 1 to 9 carbon atoms. An alkyl group may also be a lower alkyl having from 1 to 4 carbon atoms. An alkyl group may be designated as "C 1~4 alkyl" or in a similar notation. By way of example only, "C 1~4 alkyl" or "C 1~4"Alkyl" indicates that 1 to 4 carbon atoms are present in the alkyl chain, that is, the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0108] As used herein, the term "linker" is intended to mean a molecule (singular or plural) by which one element is attached to another element. For example, a linker can attach a first reactive moiety to a second reactive moiety. The linker can be a covalent bond or a non-covalent bond. Non-limiting examples of covalent linkers include alkyl chains, sulfides, polyethers, amides, esters, aryl groups, polyaryls, and the like. Non-limiting examples of non-covalent linkers include host-guest complexation, cyclodextrin / norbornene, adamantane encapsulation with β-CD, complexation, DNA hybridization interactions, streptavidin / biotin, and the like.

[0109] As used herein, the term "terminal group" is intended to mean a portion located at the end of an extended molecule, such as a polymer. For example, a terminal group can be attached to the end of a polymer and thus can form the end of the molecule containing the polymer and the terminal group.

[0110] As used herein, the term "ionic group" is intended to mean a moiety that contains at least one charged atom. An ionic group may contain a cation (a positively charged atom), an anion (a negatively charged atom), or both a cation and an anion, in which case the ionic group may be referred to as a "zwitterion". An ionic group may be associated with a counterion that balances the charge of the ionic group, but may not be covalently bonded. For example, an ionic group containing a cation may associate with a negatively charged counterion that is not covalently bonded to the ionic group. Alternatively, for example, an ionic group containing an anion may associate with a positively charged counterion that is not covalently bonded to the ionic group. The positively charged atom and the negatively charged atom of a zwitterionic group may be charge-balanced with each other such that the entire zwitterionic group is electrically balanced, i.e., charge-neutral.

[0111] As used herein, the term "linker" is intended to mean a moiety, molecule(s) by which one element is attached to another element. A linker may be a covalent bond or a non-covalent bond. Non-limiting examples of covalent linkers include moieties such as alkyl chains, polyethers, amides, esters, aryl groups, polyaryls, etc. Non-limiting examples of non-covalent linkers include host-guest complexation, cyclodextrin / norbornene, adamantane inclusion with β-CD, complexation, DNA hybridization interactions, streptavidin / biotin, etc.

[0112] As used herein, the terms "PEO", "PEG", "poly(ethylene oxide)", and "poly(ethylene glycol)" are used interchangeably and are intended to refer to a polymer containing -[CH 2 -CH 2 -O] n -. In some examples, n is from about 2 to about 100.

[0113] As used herein, the term "barrier support" is intended to refer to a structure capable of suspending a barrier. When the barrier includes a polymer membrane, the barrier support may be referred to as a "membrane support". The barrier support may define an opening such that a first portion of the barrier is suspended across the opening and a second portion of the barrier is disposed on and supported by the barrier. The barrier support may include any suitable arrangement of elements for defining an opening and suspending the barrier across the opening. In some examples, the barrier support may include a substrate having an opening defined therethrough, across which the barrier may be suspended. Additionally or alternatively, the barrier support may include one or more first features (such as one or more lips or ledges of a well within a substrate) that are raised relative to one or more second features (such as the bottom of the well), and the height difference between (a) the one or more first features and (b) the one or more second features defines an opening across which the barrier may be suspended. The opening may have any suitable shape, such as circular, elliptical, polygonal, or irregular. The barrier support may include any suitable material, or combination of materials. For example, the barrier support may be biogenic or in a solid state. In some examples, the barrier support may include or consist essentially of a curable resin such as an organic material, such as SU-8, polytetrafluoroethylene (PTFE), polymethyl methacrylate (PMMA), parylene. Additionally or alternatively, in various embodiments, the barrier support may include or consist essentially of an inorganic material such as silicon nitride, silicon oxide, or molybdenum disulfide.

[0114] As used herein, the term "annular portion" is intended to refer to a liquid that adheres to the barrier support, is located within the barrier, and extends partially within the opening defined by the barrier support. Thus, it will be understood that the annular portion may conform to the shape of the opening of the barrier and may have a shape such as circular, elliptical, polygonal, or irregular.

[0115] Nanopore device comprising a barrier using a polymer having end groups, and method of making the same Several exemplary devices including barriers using polymers having end groups, and methods of making the same, will be described with reference to FIGS. 1, 2A-2B, 3, 4, 5, 6, 7A-7C, and 8A-8C.

[0116] FIG. 1 schematically shows a cross-sectional view of an exemplary nanopore composition and device 100 including a barrier using a polymer having an end group. Device 100 includes a fluid well 100' including a polymer membrane (barrier) 101 having a first (trans) side 111 and a second (cis) side 112, a first fluid 120 within the fluid well 100' and in contact with the first side 111 of the membrane, and a second fluid 120' within the fluid well and in contact with the second side 112 of the membrane. The polymer membrane 101 can have any suitable structure that generally prevents the passage of molecules from one side of the membrane to the other side of the membrane, e.g., generally prevents contact between the fluids 120 and 120'. Exemplarily, the polymer membrane 101 can include a diblock or triblock copolymer including one or more end groups, or a hydrophobic polymer bonded to an ionic end group, and can have a structure as described in more detail below with reference to FIGS. 2A-2B, 3, 4, 5, 6, or 7A-7C. As provided herein, the end group(s) of the polymer in the polymer membrane can improve stability and utility as compared to a membrane that does not include such end groups, and in some examples, can be selected to provide a membrane having a reduced length of hydrophobic and / or hydrophilic block(s).

[0117] The first fluid 120 can have a first composition including a first concentration of salt 160, which may be represented as a cation for simplicity, although it will be understood that a counterion may also be present. The second fluid 120' can have a second composition including a second concentration of salt 160, which may be the same as or different from the first concentration. Any suitable salt 160 can be used in the first fluid 120 and the second fluid 120', e.g., ranging from common salts to ionic crystals, metal complexes, ionic liquids, or water-soluble organic ions. For example, the salt can be a cation (H, Li, Na, K, NH 4, Ag, Ca, Ba, and / or Mg, etc., but not limited thereto) and anions (OH, Cl, Br, I, NO 3 , ClO 4 , F, SO 4 , and / or CO 3 2- ... etc., but not limited thereto) and may include any suitable combination thereof. In one non-limiting example, the salt includes potassium chloride (KCl). It will also be understood that the first fluid and the second fluid may optionally include any suitable combination of other solutes. Exemplarily, the first fluid 120 and the second fluid 120' may include an aqueous buffer (e.g., N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) commercially available from Fisher BioReagents).

[0118] Continuing to refer to FIG. 1, in some examples provided herein, device 100 may optionally be disposed within barrier 101 and further include a nanopore that provides an opening 113 fluidly coupling a first side 111 to a second side 112. Thus, opening 113 of nanopore 110 may provide a path for fluid 120 and / or fluid 120' to flow through barrier 101. For example, a portion of salt 160 may move through opening 113 from the second side 112 of barrier 101 to the first side 111 of the barrier. Nanopore 110 may include a solid nanopore, a biological nanopore (e.g., MspA as illustrated in FIG. 1), or a biological and solid hybrid nanopore. Non-limiting examples and characteristics of barriers and nanopores are described elsewhere herein as well as in U.S. Patent No. 9,708,655, the entire content of which is incorporated herein by reference. In the manner shown in FIG. 1, device 100 may optionally include a first electrode 102 in contact with a first fluid 120, a second electrode 103 in contact with a second fluid 120', and a circuit 180 operably communicating with the first and second electrodes and configured to detect changes in the electrical characteristics of the opening. Such changes may respond to, for example, any suitable stimulus. Indeed, it is understood that the methods, compositions, and devices of the present invention may be used in any suitable application or situation, including any suitable method or device for sequencing, e.g., polynucleotide sequencing. As provided herein, the polymers and end groups used in barrier 101 may be selected to provide a barrier having sufficient stability for use over a desired period, e.g., in the manner described with reference to FIGS. 9-12 or 17, for use over the course of a process for sequencing, e.g., polynucleotides.

[0119] In some examples, the polymer membrane 101 between the first fluid 120 and the second fluid 120' includes a block copolymer having end groups. For example, FIGS. 2A-2B schematically show a plan view and a cross-sectional view of further details of the nanopore composition and device of FIG. 1. As shown in FIG. 2A, the membrane 101 can include a first layer 201 that includes a first plurality of block copolymer molecules 221 and a second layer 202 that includes a second plurality of block copolymer molecules. In the non-limiting example shown in FIG. 2A, the copolymer is a diblock copolymer (AB), and each molecule 221 includes a hydrophobic "B" block 231 (the dark filled circle 241 represents a hydrophobic monomer) and a hydrophilic "A" block 232 (the light filled circle 242 represents a hydrophilic monomer) directly or indirectly bonded thereto. One end of the hydrophilic A block 232 may optionally be bonded to an end group 250 (represented by the white filled circle), and the other end of the A block may be bonded to the hydrophobic block 231. The end group 250 can have a hydrophilicity different from that of the hydrophilic block 232. For example, the end group 250 can include a different type of molecule from the hydrophilic monomer 242, i.e., a molecule different from the molecule that would occur at the end of the hydrophilic A block 232 in the absence of the end group.

[0120] Additionally or alternatively, one end of the hydrophobic block 231 may optionally be bonded to an end group 260 (represented by the black filled circle), and the other end of the B block may be bonded to the hydrophilic block 232. The end group 260 can have a hydrophilicity different from that of the hydrophobic block 231. For example, the end group 260 can include a different type of molecule from the hydrophobic monomer 241, i.e., a molecule different from the molecule that would occur at the end of the hydrophilic A block 232 in the absence of the end group. In other examples, such as those described with reference to FIGS. 4 and 6, the polymer can instead include a triblock copolymer (e.g., ABA or BAB, respectively), and the ends thereof can each be bonded to an end group. In still other embodiments, such as those described with reference to FIG. 5, the polymer can instead include a hydrophobic polymer, and the ends thereof are each bonded to an ionic group.

[0121] In the example shown in FIG. 2A, the end group 250 bonded to the hydrophilic block 232 of the first plurality of molecules 221 may form the surface of the membrane 101 that contacts the fluid 120 on the first outer surface of the membrane 101, for example, the first side surface 111. The end group 250 bonded to the hydrophilic block 232 of the second plurality of molecules 221 may form the surface of the membrane 101 that contacts the fluid 120' on the second outer surface of the membrane 101, for example, the second side surface 112. Additionally or alternatively, the end groups 260 bonded to the hydrophobic blocks 231 of the first plurality of molecules and the second plurality of molecules 221 may contact each other within the membrane, or otherwise, the ends of the hydrophobic blocks 231 of the first plurality of molecules and the second plurality of molecules 221 may contact each other within the membrane.

[0122] In the example shown in FIGS. 2A-2B, the membrane 101 can be suspended using a barrier support that defines the opening 230, such as the membrane support 200. For example, the membrane support 200 can include a substrate having an opening 230 defined therethrough, such as a substantially circular opening, or an opening having another shape. Additionally or alternatively, the membrane support can include one or more features of the well in which the nanopore device is formed, such as a lip or ledge on either side of the well. Non-limiting examples of materials that can be included in the barrier support are provided further above. The annular portion 210, which can include a hydrophobic (non-polar) solvent and can also include other compound(s), can adhere to the membrane support 200, support a part of the barrier 101, and can be located, for example, within the barrier 101 (here, between the layer 201 and the layer 202). Additionally, the annular portion 210 can be tapered inwardly as shown in FIG. 2A. The outer portion of the molecules 221 of the membrane 101 can be disposed on the support 200 (e.g., the portion extending between the opening 230 and the barrier periphery 220), while the inner portion of the molecules can form a self-standing portion of the membrane 101 (e.g., the portion within the opening 210 that is partially supported by the annular portion 210). Using the operations as described elsewhere herein, the barrier 101 can be prepared and the nanopore 110 can be inserted into the self-standing portion of the barrier 101. FIGS. 2A-2B show the nanopore 110 within the barrier 101, but it should be understood that the nanopore can be omitted and the barrier 101 can be used for any suitable purpose. More generally, the barriers described herein are particularly suitable for use with nanopores (e.g., for nanopore sequencing as described with reference to FIGS. 9-12 and 17), but it should be understood that the barrier does not necessarily have to have a nanopore inserted therein.

[0123] Figure 3 schematically shows a cross-sectional view of a suspension barrier including an exemplary diblock copolymer having end groups. As shown in Figure 3, the diblock copolymer film 301 can be configured in the same manner as the film 101 described with reference to Figures 2A - 2B. For example, it can include a layer in which the molecules of the diblock copolymer are oriented such that the hydrophobic "B" portions of the AB diblock copolymer face each other and are arranged within the film, and the hydrophilic "A" portions form the outer surfaces of the film. Suitable methods for forming a suspension film on a membrane support are known in the art and include, for example, "painting" (e.g., brush painting (manual), mechanical painting (e.g., using a stir bar), and bubble painting (e.g., using a flow through a device)). The nanopores 110 can be inserted into the film 301 after the film is formed. Non-limiting examples of techniques for inserting the nanopores 110 into the film 301 include electroporation, pipette pump cycling, and surfactant-assisted nanopore insertion. Tools for forming a suspension barrier using synthetic polymers and inserting nanopores into the suspension barrier are commercially available, such as the Orbit 16 TC platform available from Nanion Technologies Inc. (California, USA).

[0124] Figures 2A and 3 show a device and a barrier that include a diblock copolymer, but it will be understood that such devices and barriers may include other types of polymers and that nanopores may optionally be inserted into such barriers. For example, FIG. 4 schematically shows a cross-sectional view of a barrier that includes an exemplary triblock copolymer having end groups. FIG. 4 shows a membrane 401 that includes molecules 421, 422 of an ABA triblock copolymer, which can be suspended using a barrier support 200 and an annular portion as described with reference to FIGS. 2A-2B. Each of the molecules 421, 422 includes a first hydrophilic A block and a second hydrophilic A block 442, a first end group and a second end group 450, and a hydrophobic B block 441 that is bonded between and between the first hydrophilic A block and the second hydrophilic A block 442. The first end group and the second end group 450 are each bonded to the ends of the first hydrophilic block and the second hydrophilic block 442. The first end group and the second end group 450 may have a hydrophilicity different from that of the first hydrophilic block and the second hydrophilic block 442. For example, the end group 450 may include a different type of molecule from the repeating unit of the hydrophilic monomer that forms the hydrophilic block 442, that is, a molecule different from the molecule that occurs at the end of the hydrophilic A block 442 in the absence of the end group 450. In the example shown in FIG. 4, each individual ABA molecule can be in one of two arrangements. For example, the ABA molecule 421 can have an "A" block on each side of the membrane and a "B" block in the center of the membrane and can extend linearly through the layer. Alternatively, for example, the ABA molecule 422 can extend to the center of the membrane such that both "A" blocks are on the same side of the membrane and the "B" block is in the center of the membrane and can then fold back on itself. For example, in a region where the ABA molecules are inhibited from fully extending in the annular portion 210 or the barrier 200, the ABA molecules can take a folded configuration 422. In either arrangement, the end groups 250 form the first outer surface and the second outer surface of the barrier 401, and the hydrophobic blocks 441 contact each other within the barrier. Thus, in this example, the barrier 401 can be considered to be partially a monolayer and partially a bilayer.In another example (not specifically shown) where the barrier 401 substantially comprises a molecule 421 that extends linearly through the barrier, the barrier 401 can be substantially a monolayer. In yet another embodiment (not specifically illustrated) where the barrier 401 substantially comprises a molecule 422 that extends to approximately the center of the barrier and then folds back on itself, the barrier 401 can be substantially a bilayer. Although not specifically shown, the nanopore can optionally be inserted into any of such alternatives for the barrier 1501 as described elsewhere herein, for example, as shown in FIGS. 2A - 2B.

[0125] In another example, FIG. 5 schematically shows a cross - sectional view of a barrier comprising an exemplary polymer having ionic end groups. FIG. 5 shows a membrane 501 comprising a molecule 521 including a first ionic end group and a second ionic end group 550 and a hydrophobic block 541, which can be suspended using a barrier support 200 and an annulus as described with reference to FIGS. 2A - 2B. The hydrophobic block 541 is disposed between and can be bonded to the first ionic end group and the second ionic end group. Such bonding can be direct or indirect. In the non - limiting example shown in FIG. 5, the end groups 550 are bonded to respective ends of the hydrophobic block 541 via linkers 570. The first end group and the second end group 550 can have a different hydrophilicity than the hydrophobic block 541. For example, the end groups 550 can include a different type of molecule than the repeating unit of the hydrophobic monomer forming the hydrophobic block 541, i.e., a molecule different from the molecule that would occur at the ends of the hydrophobic block 541 in the absence of the end groups 550.

[0126] In the example shown in FIG. 5, each individual molecule can be in one of two configurations. For example, in the suspension portion of membrane 501 (within aperture 230), molecule 521 can have ionic end groups 550 on each side of the membrane and a hydrophobic "B" block in the center of the membrane and extend linearly through the layer. Alternatively, for example, in a region where the extension of molecule 521 is inhibited in the annular portion 210 or barrier 200 (not shown), the molecule can adopt a folded configuration similar to that described with reference to FIG. 4. In such an example, barrier 501 can be considered to be substantially a single layer since the suspension portion is substantially a single layer. In either configuration, the ionic end groups 550 form the first and second outer surfaces of barrier 501, and the hydrophobic blocks 541 contact each other within the barrier. In other examples (not specifically shown), the suspension portion of barrier 501 may include a mixture of molecules 521 that extend linearly through the barrier and molecules that extend to approximately the center of the barrier and then fold back onto themselves, such as molecule 422 described with reference to FIG. 4. In that case, barrier 501 can be considered to be partially a single layer and partially a bilayer. In yet other examples, barrier 501 can substantially include molecules that extend to approximately the center of the barrier and then fold back onto themselves, such as molecule 422 described with reference to FIG. 4. In this case, barrier 501 can be considered to be substantially a bilayer. Although not specifically shown, the nanopore can optionally be inserted into any of such alternatives for barrier 501, similar to those described elsewhere in this specification, for example, as shown in FIGS. 2A - 2B.

[0127] FIG. 6 schematically shows a cross-sectional view of a barrier including another exemplary triblock copolymer having end groups. FIG. 6 shows a membrane 601 including a molecule 621 of a BAB triblock copolymer including a hydrophilic "A" block 642, a first hydrophobic "B" block and a second hydrophobic "B" block 641, and a first end group and a second end group 660, which can be suspended using a barrier support 200 and an annular portion as described with reference to FIGS. 2A-2B. The hydrophilic block 642 is disposed between the first hydrophobic block and the second hydrophobic block 641 and is bonded to the first hydrophobic block and the second hydrophobic block 641s. The first end group and the second end group 660 are each bonded to the ends of the first hydrophobic block and the second hydrophobic block 641, respectively. The first end group and the second end group 660 can have a hydrophilicity different from that of the hydrophobic block 641. For example, the end group 660 can include a different type of molecule from the repeating unit of the hydrophobic monomer forming the hydrophobic block 641, i.e., a molecule different from the molecule that would occur at the end of the hydrophobic block 641 in the absence of the end group 660. In this example, the membrane 601 can have a bilayer structure in which the "B" blocks 641 are oriented towards each other. The end groups 660 bonded to the hydrophobic blocks of the BAB molecule can generally be located approximately in the center of the barrier 601, then the molecule extends towards one of the outer surfaces of the barrier and then folds back on itself. Thus, both "B" blocks are located in the center of the membrane and the "A" block forms the first and second outer surfaces of the membrane. Although not specifically shown, nanopores can optionally be inserted into the barrier 601 in any of such options for the barrier as described elsewhere herein, e.g., as shown in FIGS. 2A-2B.

[0128] FIGS. 7A-7C schematically show further details of a barrier using a polymer that can be included in the nanopore composition and device of FIG. 1 and can be used for barriers as described with reference to FIGS. 2A-2B and 3-6. It is understood that such barriers can be suitably adapted for use in any other composition or device and are not limited to use with nanopores.

[0129] Referring now to FIG. 7A, the barrier 721 uses a triblock "ABA" copolymer. The barrier 721 includes a layer 729 that may include end groups 450 (not specifically shown) that contact the fluid 120 or 120', respectively. The layer 729 includes a plurality of molecules 722 of the triblock ABA copolymer. As shown in FIG. 7A, each molecule 722 of the triblock copolymer includes a first hydrophilic block and a second hydrophilic block, each indicated by "A" and having an approximate length "A", and a hydrophobic block, indicated by "B" and having an approximate length "B", disposed between the first hydrophilic block and the second hydrophilic block. The hydrophilic A block at the first end of the molecule 722 (the molecule forming the layer 729) may be bonded to an end group 450 that forms the first outer surface of the barrier 721 and contacts, for example, the fluid 120. The hydrophilic A block at the second end of the molecule 722 may be bonded to an end group 450 that forms the second outer surface of the barrier 721 and contacts, for example, the fluid 120'. The hydrophobic B block of the molecule 722 is within the barrier 711, as shown in FIG. 7C.

[0130] In the example illustrated in FIG. 7B, most of the molecules 722 within layer 729 can extend substantially linearly and in the same orientation relative to one another. Optionally, as shown in FIG. 7A, some of the molecules 722' can be folded at the B block such that both hydrophilic A blocks of such molecules can contact the same fluid relative to one another. Thus, the example shown in FIG. 7A can be considered to be partially a monolayer and partially a bilayer. In other examples (not specifically shown), layer 729 can be, for example, a completely monolayer or a completely bilayer, as described with reference to FIG. 4. As shown in FIG. 7A, regardless of whether the film includes molecules 722 that extend substantially linearly and / or folded molecules 722', layer 729 can have a thickness of approximately 2A + B. In some examples, length A is from about 1 RU to about 100 RU, such as from about 2 RU to about 100 RU, or from about 10 RU to about 80 RU, or from about 20 RU to about 50 RU, or from about 50 RU to about 80 RU. Additionally or alternatively, in some examples, length B is from about 2 RU to about 100 RU, or from about 5 RU to about 100 RU, such as from about 10 RU to about 80 RU, or from about 20 RU to about 50 RU, or from about 50 RU to about 80 RU. It will be understood that any end groups attached to the hydrophilic blocks contribute to the overall thickness of the barrier. Optionally, barrier 721, described with reference to FIG. 7A, can be suspended across the opening as described with reference to FIGS. 2A - 2B and 4.

[0131] Referring now to FIG. 7B, the barrier 701 uses a diblock "AB" copolymer. The barrier 701 includes a first layer 707 that can contact the fluid 120 and a second layer 708 that can contact the fluid 120'. The first layer 707 includes a first plurality of molecules 702 of the diblock AB copolymer, and the second layer 708 includes a second plurality of molecules 702 of the diblock AB copolymer. As shown in FIG. 7B, each molecule 702 of the diblock copolymer includes a hydrophilic block, denoted by "A" and having an approximate length "A", and a hydrophobic block, denoted by "B" and having an approximate length "B", which is bonded to the hydrophilic block. The hydrophilic A-blocks of the first plurality of molecules 702 (the molecules forming the layer 707) may include, for example, terminal groups 250 (not specifically shown) that contact the fluid 120 and form the first outer surface of the barrier 701. The hydrophilic A-blocks of the second plurality of molecules 702 (the molecules forming the layer 708) may include, for example, terminal groups 250 (not specifically shown) that contact the fluid 120 and form the second outer surface of the barrier 702. The respective ends of the hydrophobic B-blocks of the first plurality of molecules and the second plurality of molecules (optionally including terminal groups 260 not specifically shown) contact each other within the barrier 701 as shown in FIG. 7B.

[0132] As shown in the example of FIG. 7B, substantially all of the molecules 702 within layer 707 may extend substantially linearly and in the same orientation relative to each other, and similarly, substantially all of the molecules 702 within layer 708 may extend substantially linearly and in the same orientation relative to each other (which is opposite to the orientation of the molecules within layer 707). Thus, each of the first layer 707 and the second layer 708 may have a thickness of approximately A + B, and the barrier 701 may have a thickness of approximately 2A + 2B. In some examples, length A is from about 1 repeat unit (RU) to about 100 RU, such as from about 2 RU to about 100 RU, or from about 5 RU to about 40 RU, or from about 10 RU to about 30 RU, or from about 10 RU to about 20 RU, or from about 20 RU to about 40 RU, or from about 13 RU to about 44 RU, or from about 30 RU to about 44 RU. Additionally or alternatively, in some examples, length B is from about 2 RU to about 100 RU, or from about 5 RU to about 100 RU, such as from about 10 RU to about 80 RU, or from about 20 RU to about 50 RU, or from about 50 RU to about 80 RU, or from about 13 RU to about 44 RU, or from about 30 RU to about 44 RU. It will be appreciated that any end groups attached to the hydrophilic or hydrophobic blocks contribute to the overall thickness of the barrier. Optionally, the barrier 701 described with reference to FIG. 7B may be suspended across the opening as described with reference to FIGS. 2A - 2B and 3.

[0133] Referring now to FIG. 7C, the barrier 711 uses a triblock "BAB" copolymer. The barrier 711 includes a first layer 717 that can contact the fluid 120 and a second layer 718 that can contact the fluid 120'. The first layer 717 includes a first plurality of molecules 712 of the triblock copolymer, and the second layer 718 includes a second plurality of molecules 712 of the triblock copolymer. As shown in FIG. 7C, each molecule 712 of the triblock copolymer is represented by "B" and is approximately of length "B", and includes a first hydrophobic block and a second hydrophobic block that are each optionally bonded to a terminal group 660 (not specifically shown), and a hydrophilic block that is disposed between the first hydrophobic block and the second hydrophobic block, is represented by "A", and is approximately of length "A". The hydrophilic A blocks of the first plurality of molecules 712 (the molecules forming the layer 717) form the first outer surface of the barrier 711 and contact, for example, the fluid 120. The hydrophilic A blocks of the second plurality of molecules 712 (the molecules forming the layer 718) form the second outer surface of the barrier 711 and contact, for example, the fluid 120'. The respective ends of the hydrophobic B blocks of the first plurality of molecules and the second plurality of molecules (or the terminal groups 660 bonded thereto, not specifically shown in the figure) contact each other within the barrier 711 as shown in FIG. 7C.

[0134] In the example shown in FIG. 7C, substantially all of the molecules 712 within layer 717 can extend in the same orientation relative to one another and can be folded in the A-block such that the B-block is within the barrier 711 while the A-block can be in contact with the fluid. Similarly, substantially all of the molecules 712 within layer 718 can extend in the same orientation relative to one another (opposite to the orientation of the molecules within layer 717) and can be folded in the A-block such that the A-block is in contact with the fluid while the B-block is within the barrier 711. Thus, each of the first layer 717 and the second layer 718 can have a thickness of approximately A / 2 + B, and the barrier 711 can have a thickness of approximately A + 2B. In some examples, the length A is from about 1 RU to about 100 RU, or from 2 RU to about 100 RU, such as from about 10 RU to about 80 RU, or from about 20 RU to about 50 RU, or from about 50 RU to about 80 RU, or from about 13 RU to about 44 RU, or from about 30 RU to about 44 RU. Additionally or alternatively, in some examples, the length B is from about 2 RU to about 100 RU, or from about 5 RU to about 100 RU, such as from about 10 RU to about 80 RU, or from about 20 RU to about 50 RU, or from about 50 RU to about 80 RU, or from about 13 RU to about 44 RU, or from about 30 RU to about 44 RU. It will be appreciated that any end groups attached to the hydrophobic block contribute to the overall thickness of the barrier. Optionally, the barrier 711 described with reference to FIG. 7C can be suspended across the opening as described with reference to FIGS. 2A - 2B and 6.

[0135] Referring further to FIG. 5 described above, the barrier can have a thickness of approximately B. In some examples, the length B is from about 2 RU to about 100 RU, or from about 5 RU to about 100 RU, such as from about 10 RU to about 80 RU, or from about 20 RU to about 50 RU, or from about 50 RU to about 80 RU, or from about 13 RU to about 44 RU, or from about 30 RU to about 44 RU. It will be appreciated that any ionic groups attached to the hydrophobic block contribute to the overall thickness of the barrier. In this regard, the ionic group can be considered to correspond to "A", but can have a relatively small number of RUs, such as from about 1 to about 5 RUs, or from about 1 to about 2 RUs, or about 1 RU.

[0136] It will be understood that the various barrier layers provided herein may be configured to have any suitable dimensions. Exemplarily, to form barriers of similar dimensions to each other,

[0137] The A-B-A triblock copolymer (Figure 7A) can have two hydrophilic blocks each of length A (each A block has M w =x) and one hydrophobic block of length B (M w =y). When self-assembled, those A-B-A triblock copolymers form a film having an upper hydrophilic layer of length A, a core hydrophobic layer of length B, and a bottom hydrophilic layer of length A.

[0138] The A-B diblock copolymer (Figure 7B) can have one hydrophilic block of length A (M w =x) and one hydrophobic block of length B (M w =y / 2). When self-assembled, those A-B diblock copolymers form a film having an upper hydrophilic layer of length A, a core hydrophobic layer of length 2B, and a bottom hydrophilic layer of length A.

[0139] The B-A-B triblock copolymer (Figure 7C) can have one hydrophilic block of length A (M w =x) and two hydrophobic blocks each of length B (each B block has M w =y / 2). When self-assembled, these B-A-B triblock copolymers form a film having an upper hydrophilic layer of length A / 2, a core hydrophobic layer of length 2B, and a bottom hydrophilic layer of length A / 2.

[0140] The B block having an ionic end group (Figure 5) can be considered an ABA block copolymer, where A corresponds to the ionic end group.

[0141] Additionally or alternatively, polymer filling into the layer(s) of the film can affect the hydrophilicity ratio for each of the barriers, and the hydrophilicity ratio is the molecular weight of the hydrophilic block and the total molecular weight of the block copolymer (BCP) (MW or M w) can be defined as the ratio to (hydrophilic ratio = M w hydrophilic block / M w BCP). For example, A - B - A triblock copolymer (Figure 7A or Figure 5), hydrophilic ratio = 2x / (2x + y); A - B diblock copolymer (Figure 7B), hydrophilic ratio = x / (x + y / 2); and B - A - B triblock copolymer (Figure 7C), hydrophilic ratio = x / (x + y).

[0142] The polymers of the present invention can include any suitable combination of hydrophobic blocks and hydrophilic blocks. In some examples, the hydrophilic A block can include polymers selected from the group consisting of N - vinylpyrrolidone, polyacrylamide, zwitterionic polymers, hydrophilic polypeptides, nitrogen - containing units, and poly(ethylene oxide) (PEO). Exemplarily, polyacrylamide can be selected from the group consisting of poly(N - isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate - functionalized polyacrylamide. Non - limiting examples of zwitterionic monomers that can polymerize to form zwitterionic polymers include the following.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0143] In some examples, the hydrophobic B block may include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated poly-diene, fluorinated polyethylene, polypeptide, and poly(isobutylene) (PIB). Non-limiting examples of hydrogenated poly-diene include saturated polybutadiene (PBu), saturated polyisoprene (PI), and saturated polymyrcene.

Chemical formula

Chemical formula

Chemical formula

[0144] Various suitable end groups for attaching to hydrophobic blocks and / or hydrophilic blocks can be envisioned. (For example, as described with reference to FIGS. 2A, 3, or 4) Non-limiting examples of end groups that can be attached to the ends of hydrophilic blocks include fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3 - ), but are not limited thereto.

[0145] (For example, as described with reference to FIGS. 2A or 6) Non-limiting examples of end groups that can be attached to the ends of hydrophobic blocks are lower alkyl (C 1-4 alkyl) such as methyl, ethyl, propyl, or n-butyl groups, or aryl groups, polycyclic aromatic hydrocarbons, or fluorinated alkyl or fluorinated aryl groups. (For example, as described with reference to FIG. 5) Non-limiting examples of ionic end groups that can be attached to hydrophobic polymers are those containing zwitterions, cations, and anions. Examples of end groups containing zwitterions include 2-methacryloyloxyethyl phosphorylcholine, 3-[dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azaniumyl]propane-1-sulfonate (DMAPS), 3-{[3-(acryloylamino)propyl](dimethyl)ammonio}propanoate, and 3-{[3-(acryloylamino)propyl](dimethyl)ammonio}-1-propanesulfonate, but are not limited thereto. Zwitterionic end groups can be attached to the polymer using any suitable type of linker such as sulfide, ether, ester, alkyl, triazole, etc.

[0146] Examples of polymers containing ionic end groups are shown below. [Chemical formula] (PDMS-phosphorylcholine), where n is from about 2 to about 100, [Chemical formula] (PDMS sulfonic acid ammonium ester linker), where n is from about 2 to about 100, [Chemical formula] (PDMS ammonium carboxylate; note that the carboxylate can be substantially deprotonated at neutral pH as follows. [Chemical formula] ) where n is from about 2 to about 100, [Chemical formula] (PIB-phosphorylcholine), where n is from 2 to about 100, [Chemical formula] (PDMS sulfonic acid ammonium, amide linker), where n is from about 2 to about 100.

[0147] Examples of end groups containing cations include, but are not limited to, 2-(trimethylammonio)ethyl methacrylate. Examples of polymers containing end groups with cations are shown below. [Chemical formula] (PDMS ethyl(trimethylammonium)), where n is from about 2 to about 100.

[0148] Examples of end groups containing anions include, but are not limited to, 3-sulfopropyl acrylate, 2-propene-1-sulfonate, or vinylphosphonic acid. Examples of polymers containing end groups having anions are shown below. [Chemical formula] (PDMS propyl sulfonate), wherein n is from about 2 to about 100, [Chemical formula] (PDMS sulfonate), wherein n is from about 2 to about 100), and [Chemical formula] (PDMS phosphonic acid), wherein n is from about 2 to about 100.

[0149] The end groups can be attached to the hydrophobic block and / or the hydrophilic block in any suitable manner. In some examples, the end groups are attached to the hydrophobic or hydrophilic block via an amide bond or, for example, but not limited to, via the product of hydrosilylation, amine-ester coupling, CuAAC click chemistry, DBCO-azide, thiol-Michael addition, or thiol-ene click reaction. Exemplary reaction schemes are further provided below.

[0150] In one non-limiting example, the AB diblock copolymer includes PDMS-b-PEO, where "-b-" indicates that the polymer is a block copolymer. In another non-limiting example, the AB diblock copolymer includes PBd-b-PEO. In another non-limiting example, the AB diblock copolymer includes PIB-b-PEO. In another non-limiting example, the BAB triblock copolymer includes PDMS-b-PEO-b-PDMS. In another non-limiting example, the BAB triblock copolymer includes PBd-b-PEO-b-PBd. In another non-limiting example, the BAB triblock copolymer includes PIB-b-PEO-b-PIB. In another non-limiting example, the ABA triblock copolymer includes PEO-b-PBd-b-PEO. In another non-limiting example, the ABA triblock copolymer includes PEO-b-PDMS-b-PEO. In another non-limiting example, the ABA triblock copolymer includes PEO-b-PIB-b-PEO. In some examples, the hydrophobic polymer includes PDMS or PIB.

[0151] The hydrophobic block(s) can be attached to the hydrophilic block(s) in any suitable manner, for example, via each amide bond or via the product of a polymerization reaction. It will be understood that any suitable hydrophilic block(s) can be used with any suitable hydrophobic block(s), and any suitable end group(s) can be attached to one or both ends of the block copolymer. Additionally, in examples including two hydrophilic blocks, these blocks may or may not contain the same polymer as each other, and may or may not be attached to the same end group as each other. Similarly, in examples including two hydrophobic blocks, these blocks may or may not contain the same polymer as each other, and may or may not be attached to the same end group as each other.

[0152] The molecular weight, glass transition temperature, and chemical structure of each of the hydrophobic block and the hydrophilic block, and of the end groups attached to the hydrophobic block and / or the hydrophilic block can be appropriately selected to provide a barrier having suitable stability and the ability to insert nanopores for use. For example, the molecular weight of each of the hydrophobic block and the hydrophilic block can affect the thickness of each of the blocks (and thus the layer of the barrier), and can affect stability and the ability to insert nanopores, for example, by electroporation, pipette pump cycling, or surfactant-assisted pore insertion. Additionally or alternatively, the ratio of the molecular weights of the hydrophilic block and the hydrophobic block can affect the self-assembly of these blocks into the layer of the barrier. Additionally or alternatively, the glass transition temperature (T g ) of each of the hydrophobic block and the hydrophilic block can affect the lateral fluidity of the layer of the barrier. Thus, in some examples, it can be useful for the hydrophobic block and / or the hydrophilic block to have a T g that is below the operating temperature of the device, for example, below room temperature, and in some examples below about 0 °C. Additionally or alternatively, the chemical structure of the hydrophobic block and the hydrophilic block can affect the way the chains are packed into the layer and the stability of those layers. Additionally or alternatively, the chemical structure of the end groups can affect the way the chains are packed into the layer and the stability of those layers.

[0153] In the non-limiting examples provided herein, a barrier 401 as described with reference to FIG. 4 may include molecules 421 of an ABA triblock copolymer, where the first hydrophilic block and the second hydrophilic block 442 may include PEO, or other suitable hydrophilic polymers such as polyacrylamide, polyalcohol, polypeptide, polyoxazoline, or poly-N-vinylpyrrolidone. The hydrophilic blocks may have any suitable length. For example, each of the first hydrophilic block and the second hydrophilic block may include from about 2 to about 12 PEO repeat units, illustratively from about 2 to about 12 PEO repeat units, for example, from about 2 to about 4 PEO repeat units, or from about 3 to about 9 PEO repeat units, or from about 9 to about 12 PEO repeat units. Additionally or alternatively, in some examples, the hydrophobic block 441 may include PDMS or PIB. The hydrophobic block may have any suitable length. For example, the hydrophobic block may include from about 2 to about 100 PDMS repeat units, for example, from about 13 to about 44 PDMS repeat units, for example, from about 30 to about 44 PDMS repeat units. Or, for example, the hydrophobic block may include from about 2 to about 100 PIB repeat units, for example, from about 13 to about 44 PIB repeat units, for example, from about 30 to about 44 PIB repeat units. The hydrophobic block (e.g., PDMS or PIB) may be bonded to the first hydrophilic block and the second hydrophilic block (e.g., PEO) via respective amide, sulfide, ether, ester, alkyl, or triazole linkages. The first end group and the second end group may be independently selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3 - ). The end groups may be bonded to PEO (or other hydrophilic polymer) directly or via an amide or other suitable linkage as shown below.

Chemical Structure

[0154] In one exemplary example, molecule 421 includes a methyl end group 250 and may have the following structure. [Chemical formula] Wherein, m is from about 2 to about 100, and n is from about 2 to about 100. Non-limiting examples of molecule 421 with a methyl end group, which may be different from those in which the numbers of PDMS and PEO RU are shown, are shown below. [Chemical formula] Another non-limiting example of molecule 421 with a COOH end group, which may be different from those in which the numbers of PDMS and PEO RU are shown, is shown below. [Chemical formula] Another non-limiting example of molecule 421 with a methyl end group, which may be different from those in which the numbers of PDMS and PEO RU are shown, is shown below. [Chemical formula]

[0155] In other non-limiting examples of this specification, a barrier 501, as described with reference to FIG. 5, may include a molecule 521 that includes a first ionic end group and a second ionic end group 550, and a hydrophobic block 541 disposed between and bonded to the first ionic end group and the second ionic end group. Each of the first ionic end group and the second ionic end group 550 may include a zwitterion, a cation, or an anion. Non-limiting examples of end groups that include a zwitterion, a cation, or an anion are provided elsewhere in this specification. In one non-limiting example, the hydrophobic block includes poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB). The hydrophobic block may have any suitable length. For example, the hydrophobic block may include from about 2 to about 100 PDMS repeating units, such as from about 13 to about 44 PDMS repeating units, such as from about 30 to about 44 PDMS repeating units. Or, for example, the hydrophobic block may include from about 2 to about 100 PIB repeating units, such as from about 13 to about 44 PIB repeating units, such as from about 30 to about 44 PIB repeating units. The first ionic end group and the second ionic end group may be bonded to the PDMS or PIB in any suitable manner, such as via the product of hydrosilylation, amine-ester coupling, CuAAC click chemistry, DBCO-azide, thiol-Michael addition, or thiol-ene click reaction. Non-limiting examples of molecule 521 where the end group includes a zwitterionic end group and may differ from those where the number of PDMS RUs is indicated are shown below.

Chemical formula

[0156] In yet other non-limiting examples herein, the barrier 301, as described with reference to FIG. 3, may include molecules 221 of an AB diblock copolymer where the hydrophilic block 232 may include PEO. The hydrophilic block may have any suitable length. For example, the hydrophilic block may include from about 2 to about 100 PEO repeat units, such as from about 2 to about 9 PEO repeat units. Additionally or alternatively, the hydrophobic block 231 may include PDMS or PIB. The hydrophobic block may have any suitable length. For example, the hydrophobic block may include from about 2 to about 100 PDMS repeat units, such as from about 14 to about 44 PDMS repeat units, such as from about 14 to about 26 PDMS repeat units. Or, for example, the hydrophobic block may include from about 2 to about 100 PIB repeat units, such as from about 14 to about 44 PIB repeat units, such as from about 14 to about 26 PIB repeat units. In some examples, the end group 250 may include fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), or sulfonate (SO 3 - ). In some examples, the end group 260 may include a lower alkyl (C 1-4 alkyl), such as a methyl, ethyl, propyl, or n-butyl group, or an aryl group, polycyclic aromatic hydrocarbon, or fluorinated alkyl or fluorinated aryl group. A non-limiting example of a molecule 221 where the end group 250 is methyl and the end group 260 is n-butyl is shown below.

Chemical formula

[0157] In a further non-limiting example herein, the barrier 601, as described with reference to FIG. 6, may include molecules 621 of a BAB triblock copolymer in which the hydrophilic block includes PEO. The hydrophilic block 642 may have any suitable length. For example, the hydrophilic block may include from about 7 to about 13 PEO repeat units. Additionally or alternatively, the first hydrophobic block and the second hydrophobic block 641 may include PDMS or PIB. The hydrophobic blocks may have any suitable length. For example, each of the first hydrophobic block and the second hydrophobic block may include from about 14 to about 26 PDMS repeat units. Or, for example, the first hydrophobic block and the second hydrophobic block may include from about 2 to about 100 PIB repeat units, such as from about 14 to about 44 PIB repeat units, such as from about 14 to about 26 PIB repeat units. The end groups 660 may include lower alkyl (C 1-4 alkyl), such as methyl, ethyl, propyl, or n-butyl groups, or aryl groups, polycyclic aromatic hydrocarbons, or fluorinated alkyl or fluorinated aryl groups. A non-limiting example of a molecule 621 in which the end group 660 is propyl is shown below.

Chemical formula

[0158] It will be understood that the diblock and triblock copolymers of the present invention can be made using any suitable combination of operations. FIGS. 8A-8C schematically illustrate an exemplary scheme for preparing a triblock copolymer for use in the nanopore compositions and devices of FIG. 1. In some examples, the diblock and triblock copolymers of the present invention can be made using a "macroinitiator" approach as illustrated in FIG. 8A, where one polymer block is made first and then used as an initiator (X in FIG. 8A) to grow one or more additional blocks using monomers ([M] in FIG. 8A). Exemplarily, the operations for making a diblock copolymer can include polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer, forming an initiator at the end of the hydrophilic polymer, and using the initiator to polymerize a plurality of hydrophobic monomers to form a hydrophobic polymer attached to the hydrophilic polymer. Alternatively, the operations for making a diblock copolymer can include polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer, forming an initiator at the end of the hydrophobic polymer, and using the initiator to polymerize a plurality of hydrophilic monomers to form a hydrophilic polymer attached to the hydrophobic polymer. Similarly, the operations for making a triblock BAB copolymer can include polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer, forming an initiator at each end of the hydrophilic polymer, and using the initiator to polymerize a plurality of hydrophobic monomers to form a hydrophobic polymer attached to each end of the hydrophilic polymer. Similarly, the operations for making a triblock ABA copolymer can include polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer, forming an initiator at each end of the hydrophobic polymer, and using the initiator to polymerize a plurality of hydrophilic monomers to form a hydrophilic polymer attached to each end of the hydrophobic polymer. In such a "macroinitiator" approach, the initiator (X in FIG. 8A) can suitably be selected based on the particular monomers used and the particular type of polymerization being carried out.For example, in the case of atom transfer radical polymerization (ATRP), the initiator may contain bromine or chlorine. Alternatively, for example, in the case of reversible addition fragmentation chain transfer (RAFT) polymerization, the initiator may contain a chain transfer agent. After the polymerization is completed, the end group(s) (X in FIG. 8A) may be modified or removed (e.g., to provide the end group(s) Y in FIG. 8A).

[0159] In other examples, the diblock and triblock copolymers of the present invention can be made using a "coupling" approach as shown in FIG. 8B, where the polymer blocks are made separately and then coupled together using reactive moieties (X and Y in FIG. 8B). Exemplarily, the operations for making a diblock copolymer can include polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer, polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer, and coupling the hydrophilic polymer to the hydrophobic polymer. The operations for making a triblock copolymer can include polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer having ends, polymerizing a plurality of hydrophobic monomers to form a first hydrophobic polymer and a second hydrophobic polymer, and coupling the first hydrophobic polymer and the second hydrophobic polymer to respective ends of the hydrophilic polymer. Alternatively, the operations for making a triblock copolymer can include polymerizing a plurality of hydrophilic monomers to form a first hydrophilic polymer and a second hydrophilic polymer, polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer having ends, and coupling the first hydrophilic polymer and the second hydrophilic polymer to respective ends of the hydrophobic polymer. In such a "coupling" approach, the ends of the hydrophobic polymer can include a first reactive moiety (Y in FIG. 8B), and the ends of the hydrophilic polymer can include a second reactive moiety (X in FIG. 8B) that reacts with the first reactive moiety to couple the hydrophilic polymer to the hydrophobic polymer. The reactive moieties (X and Y in FIG. 8B) can be appropriately selected based on the specific polymers being coupled and the type of coupling being performed. For example, "click" chemistry moieties can be used. Exemplarily, one of the first reactive moiety and the second reactive moiety can include an azide, the other of the first reactive moiety and the second reactive moiety can include an alkyne, or one of the first reactive moiety and the second reactive moiety can include a thiol, the other of the first reactive moiety and the second reactive moiety can include an alkene, or one of the first reactive moiety and the second reactive moiety can include a thiol, and the other of the first reactive moiety and the second reactive moiety can include an alkyne. Or, for example, an amide linker can be formed.Exemplarily, one of the first reactive moiety and the second reactive moiety may contain an amine, and the other of the first reactive moiety and the second reactive moiety may contain N-hydroxysuccinimide (NHS).

[0160] FIG. 8C shows a non-limiting example, where the hydrophobic polymer is PDMS having an amine (NH 2 ) group at one end and a 3-carbon alkyl group at the other end, the hydrophilic polymer is PEO having NHS at its end, and the amine group and the NHS group react with each other in the presence of triisopropylamine to provide a BAB triblock copolymer. This particular example includes a 3-carbon alkyl group 660 at the end of the PDMS block, but it will be understood that other end groups as described elsewhere herein may be used.

[0161] Another non-limiting example of forming a PEO-PDMS-PEO ABA triblock copolymer having methyl end groups is shown below. In this example, PDMS-bisallyl is reacted with PEG-thiol by thiol-ene click chemistry. To allow the reaction to proceed, the reaction is carried out in a degassed solvent (e.g., chloroform) under an inert atmosphere in the presence of a photoinitiator (e.g., irgacure 2959) under UV exposure for 5 to 30 minutes. [Chemical formula]

[0162] Another non-limiting example of forming a triblock copolymer is shown below. In this example, PIB-bisallyl is reacted with PEG-thiol by thiol-ene click chemistry. To allow the reaction to proceed, a degassed solvent (e.g., chloroform) is used, and the reaction is carried out under an inert atmosphere (dry argon or dry nitrogen) in the presence of a photoinitiator (e.g., irgacure 2959) under UV exposure for 5 to 60 minutes. In some examples, the reaction is carried out at 1 mW / cm 2 ~100 mW / cm 2It is carried out under UV exposure for 2 to 180 minutes with a UV output in the range of. In some examples, the UV wavelength used is 365 nm.

Chemical formula

[0163] In nanopore sequencing applications, membrane fluidity can be considered beneficial. Without wishing to be bound by any theory, the fluidity of the block copolymer membrane is thought to be mainly imparted by the physical properties of the hydrophobic "B" block. More specifically, a membrane with higher fluidity can be generated using a B block containing a "low T" hydrophobic polymer (e.g., having a T below about 0 °C) than a membrane having a B block containing a "high T" polymer (e.g., having a T higher than room temperature). For example, in certain instances, the hydrophobic B block of the copolymer has a T of less than about 20 °C, less than about 0 °C, or less than about -20 °C. g A B block containing a "low T" hydrophobic polymer (e.g., having a T below about 0 °C) is used to g generate a membrane with higher fluidity than a membrane having a B block containing a "high T" polymer (e.g., having a T higher than room temperature). For example, in certain instances, the hydrophobic B block of the copolymer has a T of less than about 20 °C, less than about 0 °C, or less than about -20 °C. g A B block containing a "high T" polymer (e.g., having a T higher than room temperature). g For example, in certain instances, the hydrophobic B block of the copolymer has a T of less than about 20 °C, less than about 0 °C, or less than about -20 °C. g has.

[0164] Low T g Hydrophobic B blocks having can be used, for example, in a manner as described with reference to FIGS. 9 - 12 or 17, to help maintain membrane flexibility under conditions suitable for performing nanopore sequencing. In some examples, hydrophobic B blocks having a T sufficiently low for use in nanopore sequencing can include or consist essentially of PIB having a T in the range of about -75 °C to about -25 °C. In other examples, hydrophobic B blocks having a T sufficiently low for use in nanopore sequencing can include or consist essentially of PDMS having a T that can be expected to be in the range of about -135 °C (or lower) to about -115 °C. In yet other examples, hydrophobic B blocks having a T sufficiently low for use in nanopore sequencing g has. g For example, in certain instances, the hydrophobic B block of the copolymer has a T of less than about 20 °C, less than about 0 °C, or less than about -20 °C. g Hydrophobic B blocks having can be used, for example, in a manner as described with reference to FIGS. 9 - 12 or 17, to help maintain membrane flexibility under conditions suitable for performing nanopore sequencing. In some examples, hydrophobic B blocks having a T sufficiently low for use in nanopore sequencing can include or consist essentially of PIB having a T in the range of about -75 °C to about -25 °C. In other examples, hydrophobic B blocks having a T sufficiently low for use in nanopore sequencing can include or consist essentially of PDMS having a T that can be expected to be in the range of about -135 °C (or lower) to about -115 °C. In yet other examples, hydrophobic B blocks having a T sufficiently low for use in nanopore sequencing g has. gThe hydrophobic B block having [it] may contain PBd or may consist essentially of PBd. Different forms of PBd can be used as the B block in this barrier. For example, the cis-1,4 form of PBd is expected to have a T in the range of about -105 °C to about -85 °C g It may be expected to have. Alternatively, for example, the cis-1,2 form of PBd is expected to have a T in the range of about -25 °C to about 0 °C g It may be expected to have. Alternatively, for example, the trans-1,4 form of PBd is expected to have a T in the range of about -95 °C to about -5 °C g It may be expected to have. In yet another example, the hydrophobic B block having a T low enough for use in nanopore sequencing is expected to have a T in the range of about -75 °C to about -45 °C g It may contain or may consist essentially of polymyrcene (PMyr) which may be expected to have a T in the range of about -75 °C to about -45 °C g In yet another example, the hydrophobic B block having a T low enough for use in nanopore sequencing may contain or may consist essentially of polyisoprene (PIP). Different forms of PIP can be used as the B block in the barrier of the present invention. For example, the cis-1,4 form of PIP is expected to have a T in the range of about -85 °C to about -55 °C g It may be expected to have. Alternatively, for example, the trans-1,4 form of PIP is expected to have a T in the range of about -75 °C to about -45 °C g It may be expected to have. g It may be expected to have.

[0165] A hydrophobic B-block having a fully saturated carbon skeleton (e.g., PIB) may also be expected to enhance the chemical stability of the block copolymer membrane. Additionally or alternatively, a branched structure within a hydrophobic B-block such as PIB may be expected to induce chain entanglement, which may be expected to enhance the stability of the block copolymer membrane. This allows for the use of smaller hydrophobic blocks and improves the disadvantage of hydrophobic mismatch for the inserted nanopores. Additionally or alternatively, a hydrophobic B-block having a relatively low polarity is a better electrical insulator and may thus be expected to improve the electrical performance of the device for nanopore sequencing (e.g., as described with reference to FIGS. 9-12 or 17).

[0166] In some examples of the following AB copolymers containing PBd as the B-block and PEO as the A-block, R is selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH 3 ), carboxyl (COOH), propargyl, azide group (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), biotin, or sulfonate (SO 3 - ). m is from about 2 to about 100, and n is from about 2 to about 100. [Chemical formula]

[0167] In some non-limiting examples, R = OH, n is from about 8 to about 50, and m is from about 1 to about 20. In some non-limiting examples, R = OH, n is from about 10 to about 15, and m is from about 5 to about 15.

[0168] In some examples of the following ABA copolymers containing one or more PIB blocks as the B-block and PEO as the A-block, R 1 and R 2is, independently, a moiety selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butylcarbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3 - ). V is an optional group corresponding to a bifunctional initiator from which isobutylene can propagate, and can be tert-butylbenzene, phenyl, naphthalene, another aromatic group, an alkane chain having about 2 to about 20 carbons, or another aliphatic group bonded to the hydrophobic block via the para, meta, or ortho position, and m = about 2 to about 100, and n = about 2 to about 100. V can optionally be flanked by a functional group selected from the group consisting of carboxylic acid, carboxyl group, methyl group, hydroxyl group, primary amine, secondary amine, tertiary amine, biotin, thiol, azide, propargyl group, allyl group, acrylate group, zwitterionic group, sulfate, sulfonate, alkyl group, aryl group, orthogonal functional group, and hydrogen. In the absence of V, only one PIB block is present and n is about 2 to about 100. L 1 and L 2 are independently linkers that can contain at least one moiety selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, alkyl group (e.g., methylene), ether, and the product of a click reaction.

Chemical formula

[0169] In some non-limiting examples of the above structure, n = about 2 to about 50, and m = about 1 to about 50, R 1 = R 2 = COOH, V = tert-butylbenzene, and L 1 = L 2 = ethyl sulfide. In other non-limiting examples, n = about 5 to about 20, m = about 2 to about 15, R 1=R 2 = COOH, V = tert-butylbenzene, and L 1 = L 2 = ethyl sulfide. In other non-limiting examples, n = about 13 to about 19, m = about 2 to about 5, R 1 = R 2 = COOH, V = tert-butylbenzene, and L 1 = L 2 = ethyl sulfide. In other non-limiting examples, n = about 7 to about 13, m = about 7 to about 13, R 1 = R 2 = COOH, V = tert-butylbenzene, and L 1 = L 2 = ethyl sulfide. In particular, in one non-limiting example (the structure of which is shown below), n = 16, m = 3, R 1 = R 2 = COOH, V = tert-butylbenzene, and L 1 = L 2 = ethyl sulfide. In another non-limiting example (the structure of which is shown below), n = 10, m = 10, and R 1 = R 2 = COOH, V = tert-butylbenzene, and L 1 = L 2 = ethyl sulfide. In another non-limiting example (the structure of which is shown below), n = 16, m = 8, R 1 = R 2 = CH 3 、V = tert-butylbenzene, and L 1 = L 2 = ethyl sulfide.

Chemical Structure

[0170] In another non-limiting example of the more general structure shown above, the end groups can be, for example, zwitterionic as shown below.

Chemical Structure

[0171] In some examples, a polyfunctional precursor is supplied, and in the above example, V can be used as a precursor for the synthesis of a corresponding bifunctional initiator. For example, the polyfunctional precursor can be 5-tert-butylisophthalic acid (TBIPA), which can be synthesized to 1-(tert-butyl)-3,5-bis(2-methoxypropan-2-yl)benzene (TBDMPB) using reactions known in the art. In another example, TBIPA can be synthesized to 1-tert-butyl-3,5-bis(2-chloropropan-2-yl)benzene using reactions known in the art. The use of such a bifunctional initiator enables cationic polymerization on both sides of the initiator, generating a bifunctional PIB such as allyl-PIB-allyl, which can then be coupled to a hydrophilic A block to produce an ABA block copolymer containing PIB as the B block. Here, the bifunctional initiator can be located between the first PIB polymer and the second PIB polymer, but it should be understood that the first PIB polymer, the second PIB polymer, and the bifunctional initiator (V) can together form, for example, the B block of an ABA triblock copolymer.

[0172] In another non-limiting example, the ABA triblock copolymer

Chemical formula

[0173] In particular, as shown below, in one non-limiting example, m = 3, n = 34, p = 3, and R 1 = R 2 = COOH. In another non-limiting example shown below, m = 9, n = 37, p = 9, and R 1 = R 2 = COOH.

Chemical formula

[0174] In some examples of the following AB copolymers containing a PIB block as the B block and PEO as the A block, R is fluorenylmethoxycarbonyl (Fmoc), tert-butylcarbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3 - ) is a moiety selected from the group consisting of. m is from about 2 to about 100, n is from about 2 to about 100, and L is a linker selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, alkyl group (e.g., methylene), ether, or the product of a click reaction.

Chemical formula

[0175] In particular, as shown below, in a non-limiting example, n = 13, m = 8, R is methyl, and L is ethyl sulfide. In another non-limiting example shown below, n = 13, m = 3, R is a carboxyl group, and L is ethyl sulfide. In another non-limiting example shown below, n = 30, m = 8, R is methyl, and L is ethyl sulfide. In another non-limiting example shown below, n = 30, m = 3, R is a carboxyl group, and L is ethyl sulfide.

Chemical formula

[0176] The following scheme shows an exemplary synthetic route for ionically (e.g., zwitterionically) functionalized PDMS via hydroxylation, and a zwitterionic monomer having an acrylate, methacrylate (here), acrylamide, or methacrylamide functional group (here, a zwitterionic monomer containing both a phosphate and a quaternary ammonium chloride moiety) can be reacted with hydride-terminated PDMS.

Chemical formula

[0177] The following scheme shows an exemplary synthetic route for zwitterion-functionalized PDMS via thiolation, and a thiol-functionalized zwitterion monomer (here, an SH-terminated 2-methacryloyloxyethyl phosphorylcholine derivative) is reacted with allyl-functionalized PDMS in a thiol-ene click reaction.

Chemical formula

[0178] Ionic monomers having acrylate, methacrylate, acrylamide, or methacrylamide functional groups (e.g., those useful in hydroxide reactions), such as zwitterionic, anionic, or cationic monomers, can be functionalized to contain thiol functional groups (e.g., those useful in thiol-ene click reactions) using thiol-Michael addition in the presence of a secondary base. For example, the following scheme shows the reaction of 1,4-butanedithiol with zwitterionic 2-methacryloyloxyethyl phosphorylcholine. [Chemical formula] It is understood that any suitable dithiol can be used to attach a thiol-terminated linker to an ionic monomer having an acrylate, methacrylate, acrylamide, or methacrylamide functional group via thiol-Michael addition as exemplified in the above scheme.

[0179] In some examples, the membrane can have a survival rate of 70% or more, 80% or more, 90% or more, or 95% or more when subjected to a voltage of 450 mV across the barrier. In some examples, the membrane has an open pore current of 95 pA or more, or 100 pA or more at 100 mV. In some examples, the membrane has an open pore current of 32 pA or more, 34 pA or more, or 36 pA or more at 50 mV. In some examples, the membrane pore RMS noise is 2.2 pA or less, 2.0 pA or less, 1.8 pA or less, 1.6 pA or less, or 1.5 pA or less. In some examples, the membrane has a signal-to-noise ratio of 40 or more, 50 or more, 60 or more, or 70 or more. In some examples, the membrane has a membrane coating yield of 90% or more or 95% or more.

[0180] In some examples, the membrane has a pore insertion voltage of about 300 mV to about 1100 mV. In some examples, the membrane has a single pore percentage of 85% or more, 90% or more, or 95% or more after insertion. In some examples, the membrane has a single pore survival rate of 90% or more or 95% or more. In some examples, the membrane has a single pore current standard deviation of 2 pA or less, 1 pA or less, or 0.5 pA or less.

[0181] In some examples, a waveform consisting of a series of positive voltage micropulses separated only by a negative voltage period of 100 ms at -100 mV is applied to the barrier. The series of positive voltage pulses has a duration of 10 μs and a total of 20 pulses. The interval between them has a set duration value of 30 ms and a voltage held at +50 mV. During the first cycle, the waveform can be applied continuously for 5 minutes, and the pulse magnitude is maintained at +700 mV. In further applied cycles (each applied for 5 minutes again), the pulse intensity is increased from +700 mV to +1200 mV in steps of 100 mV over a total of 6 cycles. In some examples, the membrane survival rate under such a waveform is 60% or more, 80% or more, 90% or more, or 95% or more. In some examples, the voltage at a membrane survival rate of 50% is 1000 mV or more, or 1200 mV or more. In some examples, the voltage at 50% membrane and single pore survival rate is 900 mV or more, or 1000 mV or more.

[0182] It is understood that the compositions and devices described with reference to FIGS. 1 - 12 and 17 can be prepared in any suitable manner. FIG. 18 shows a flow of operations for forming a device as shown in FIG. 1. The method 1800 shown in FIG. 18 includes forming a barrier between a first fluid and a second fluid, the barrier being suspended by a barrier support that defines an opening, the barrier being suspended across the opening and including one or more layers containing molecules of a block copolymer, and the end groups attached to the hydrophilic blocks having a hydrophilicity different from that of the hydrophilic blocks (operation 1810). The barrier can be formed using any suitable combination of operations provided herein or otherwise known in the art. For example, forming the barrier can include what is known in the art as "coating". Known techniques for coating a barrier suspended by a barrier support include brush coating (manual), mechanical coating (e.g., using a stir bar), and bubble coating (e.g., using a flow through the device).

[0183] Each molecule of the block copolymer can include one or more hydrophilic blocks having an approximate length A and one or more hydrophobic blocks having an approximate length B. The one or more hydrophilic blocks can form the outer surface of the barrier, and the hydrophobic blocks can be located within the barrier. For example, the barrier can include any AB, ABA, or copolymer provided herein. In some examples, the one or more hydrophobic blocks can include polymers selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polybutadiene, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). For reasons as described elsewhere herein, such polymers can be expected to produce suspension membranes of particularly useful quality for use in nanopore sequencing operations as described, for example, with reference to FIGS. 9-12 and 17. Any suitable end group can be attached to the hydrophilic block. Exemplarily, the end group can be selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH 3 ), biotin, carboxyl (COOH), propargyl, azide (N 3 ), amino (NH 2 ), hydroxyl (OH), thiol (SH), and sulfonate (SO 3 - ).

[0184] Method 1800 also optionally includes inserting a nanopore into the barrier (operation 1820). The nanopore can provide contact between the first fluid and the second fluid. The nanopore can be inserted into the barrier using operations as described elsewhere in this specification or otherwise known in the art. Known techniques for inserting a nanopore into a suspended barrier include electroporation, pipette pump cycling, and surfactant-assisted pore insertion. Tools for forming a suspended barrier using synthetic polymers and inserting a nanopore into the suspended barrier, such as the Orbit 16 TC platform available from Nanion Technologies Inc. (California, USA), are commercially available. It will be appreciated that if it is desirable to use the barrier without a nanopore, operation 820 need not necessarily be performed after operation 810.

[0185] Exemplarily, the block copolymer of FIG. 18 is an AB diblock copolymer as described with reference to FIGS. 2A-2B and 3. Thus, the barrier can have a thickness of approximately 2A + 2B. In one non-limiting example of such a diblock copolymer, the hydrophobic block can be polybutadiene (PBd). Alternatively, the block copolymer can be an ABA triblock copolymer having two hydrophilic blocks and one hydrophobic block. Thus, the barrier can have a thickness of approximately 2A + B. In one non-limiting example of such a triblock copolymer, the hydrophobic block is poly(isobutylene) (PIB) or PDMS.

[0186] Although not specifically shown in FIG. 18, in other examples contemplated herein, the block copolymer may be a BAB triblock copolymer having two hydrophobic blocks and one hydrophilic block, where the end groups are attached to the hydrophobic blocks in a manner as described with reference to FIG. 6. Thus, the barrier may have a thickness of approximately A + 2B. Although not specifically shown in FIG. 18, in still other examples contemplated herein, the block copolymer may include a hydrophobic block attached to a first ionic group and a second ionic group. Thus, the barrier may have a thickness of approximately B.

[0187] Devices and methods using barriers for nanopore sequencing It will be further understood that this barrier can be used in any suitable device or application. For example, FIG. 9 schematically shows a cross-sectional view of an exemplary use of the composition and device of FIG. 1. The device 900 shown in FIG. 9 can have a configuration as described with reference to the fluid well 100', FIGS. 2A-2B, 3, 4, 5, 6, or 7A-7C, and can be configured to include a barrier 901, a first fluid 120 and a second fluid 120', and nanopores 110 in a manner as described with reference to FIG. 1. In the non-limiting example shown in FIG. 9, the second fluid 120' can optionally include a plurality of respective nucleotides 921, 922, 923, 924, for example, G, T, A, and C, respectively. Each of the nucleotides 921, 922, 923, 924 in the second fluid 120' can optionally be coupled to respective labels 931, 932, 933, 934 coupled to the nucleotides via an extender (especially an unlabeled extender). Optionally, the device 900 can further include a polymerase 905. As shown in FIG. 9, the polymerase 905 can be within the second composition of the second fluid 120'. Optionally, the polymerase 905 can be coupled to the nanopore 110 or the barrier 901 via, for example, a suitable extender (not specifically shown). The device 900 can optionally further include a first polynucleotide 940 and a second polynucleotide 950 as shown in FIG. 9. The polymerase 905 can be for sequentially adding a plurality of nucleotides to the first polynucleotide 940 using the sequence of the second polynucleotide 950. For example, at a particular point in time shown in FIG. 9, the polymerase 905 incorporates the nucleotide 922 (T) into the first polynucleotide 940, which hybridizes to the second polynucleotide 950 to form a double strand. At other times (not specifically shown), the polymerase 905 can sequentially incorporate other ones of the nucleotides 921, 922, 923, 924 into the first polynucleotide 940 using the sequence of the second polynucleotide 950.

[0188] The circuit 180 shown in FIG. 9 can be configured to detect a change in the electrical characteristics of the aperture in response to the polymerase sequentially adding a plurality of nucleotides to the first polynucleotide 940 using the sequence of the second polynucleotide 950. In the non-limiting example shown in FIG. 9, the nanopore 110 can be coupled to a permanent tether 910 that can include a head region 911, a tail region 912, an elongator 913, a reporter region 914 (e.g., abasic nucleotide), and a portion 915. The head region 911 of the tether 910 is coupled to the nanopore 910 via any suitable chemical bond, protein-protein interaction, or any other suitable attachment that is typically irreversible. The head region 911 positions the reporter region 914 within the aperture 913 and positions the portion 915 in sufficient proximity to the polymerase 905 to interact with the respective labels 931, 932, 933, 934 of the nucleotides 921, 922, 923, 924 acted upon by the polymerase 905. The portion 915 can be attached to any suitable portion of the nanopore 910 that interacts with the labels 931, 932, 933, 934, respectively, to move the reporter region 914 within the aperture 913, thereby changing the rate at which the salt 160 moves through the aperture 113 and thereby detectably changing the conductivity of the aperture 113 as detected by the circuit 180. For further details regarding the use of a permanent tether coupled to a nanopore for polynucleotide sequencing, see U.S. Patent No. 9,708,655, the entire contents of which are incorporated herein by reference.

[0189] FIG. 10 schematically shows a cross-sectional view of another exemplary use of the compositions and devices of FIG. 1. As shown in FIG. 10, device 1000 can include a fluid well 100', a barrier 1001 that can have a configuration as described with reference to FIGS. 2A-2B, 3, 4, 5, 6, or 7A-7C, a first fluid 120 and a second fluid 120', nanopores 110, and a first polynucleotide 1040 and a second polynucleotide 1050, all of which can be similarly configured as described with reference to FIG. 9. However, in the non-limiting example shown in FIG. 10, nucleotides 1021, 1022, 1023, 1024 need not necessarily be attached to their respective labels. Polymerase 1005 can be attached to nanopore 110 and can be attached to a permanent tether 1010 that can include a head region 1011, a tail region 1012, an elongator 1013, and a reporter region 1014 (e.g., abasic nucleotide). The head region 1011 of tether 1010 is attached to polymerase 1005 via any suitable chemical bond, protein-protein interaction, or any other suitable attachment that is typically irreversible. The head region 1011 can be attached to any suitable portion of polymerase 1005 that positions the reporter region 1014 within the aperture 113. When polymerase 1005 interacts with nucleotides 1021, 1022, 1023, 1024, such interaction can cause a conformational change in polymerase 1005. Such a conformational change can move the reporter region 1014 within the aperture 113, thereby changing the rate at which salt 160 moves through the aperture 113 and thereby detectably changing the conductivity of the aperture 113 in a manner detectable by circuit 180. For further details regarding the use of a permanent tether attached to a polymerase for polynucleotide sequencing, see U.S. Patent No. 9,708,655, the entire contents of which are incorporated herein by reference.

[0190] FIG. 11 schematically shows a cross-sectional view of another exemplary use of the composition and device of FIG. 1. As shown in FIG. 11, device 1100 can include fluid well 100', barrier 1101 which can have a configuration as described with reference to FIGS. 2A-2B, 3, 4, 5, 6, or 7A-7C, first fluid 120 and second fluid 120', and nanopore 110, all of which can be similarly configured as described with reference to FIG. 9. However, in the non-limiting example shown in FIG. 11, polynucleotide 1150 moves through nanopore 110 under an applied force, such as a bias voltage applied by circuit 180 between electrodes 102 and 103. As the bases in polynucleotide 1150 pass through nanopore 110, such bases can change the rate at which salt 160 moves through opening 113, thereby detectably changing the conductivity of opening 113 in a manner detectable by circuit 180. For further details regarding the use of nanopores for sequencing polynucleotides moved therethrough, see U.S. Patent No. 5,795,782, the entire contents of which are incorporated herein by reference.

[0191] FIG. 12 schematically shows a cross-sectional view of another exemplary use of the compositions and devices of FIG. 1. As shown in FIG. 12, device 1200 can include a fluid well 100', a barrier 1201 that can have a configuration as described with reference to FIGS. 2A-2B, 3, 4, 5, 6, or 7A-7C, a first fluid 120 and a second fluid 120', and nanopores 110, all of which can be similarly configured as described with reference to FIG. 9. In the non-limiting example shown in FIG. 12, alternative polymer 1250 moves through nanopore 110 under an applied force, such as a bias voltage applied by circuit 180 between electrodes 102 and 103. As used herein, "alternative polymer" is intended to mean an elongated chain of labels having a sequence corresponding to the sequence of nucleotides in a polynucleotide. In the example shown in FIG. 12, alternative polymer 1250 includes labels 1251 coupled to each other via linker 1252. XPANDOMER™ is a particular type of alternative polymer developed by Roche Sequencing, Inc. (Pleasanton, CA). XPANDOMERS™ can be prepared using Sequencing By eXpansion™ (SBX™, Roche Sequencing, Pleasanton CA). In sequencing by Expansion™, the engineered polymerase uses the sequence of the target polynucleotide to polymerize xNTPs that include nucleobases coupled to labels via linkers. The polymerized nucleotides are then processed to generate an extended chain of labels that are separated from each other by linkers coupled between the labels and have a sequence complementary to the sequence of the target polynucleotide.For example, for descriptions of methods for XPANDOMERS (trademark), linker (tether), label, engineered polymerase, and SBX (trademark), see U.S. Patent Nos. 7,939,249, 8,324,360, 8,349,565, 8,586,301, 8,592,182, 9,670,526, 9,771,614, 9,920,386, 10,301,345, 10,457,979, 10,676,782, 10,745,685, 10,774,105, and 10,851,405, the entire contents of each of which are incorporated herein by reference.

[0192] FIG. 17 schematically shows a cross-sectional view of another exemplary use of the compositions and devices of FIG. 1. As shown in FIG. 17, device 100 can include a fluid well 100’, a barrier 1701 that can have a configuration as described with reference to FIGS. 2A-2B, 3, 4, 5, 6, or 7A-7C, a first fluid 120 and a second fluid 120’, and a nanopore 110, all of which can be similarly configured as described with reference to FIG. 9. In the non-limiting example shown in FIG. 17, the double strand between polynucleotide 140 and polynucleotide 150 is located within nanopore 110 under an applied force (e.g., the bias voltage applied by circuit 180 between electrode 102 and electrode 103). The combination of the bases in the double-stranded portion (here, base pairs GC121, 124 at the ends of the double strand) and the bases in the single-stranded portion of polynucleotide 150 (here, bases A and T123, 122) can change the rate at which salt 160 moves through opening 113, and thus can detectably change the conductivity of opening 113 as detected by circuit 180. For further details regarding the use of nanopores for sequencing polynucleotides that are moved through the nanopore, see U.S. Patent Application Publication No. 2023 / 0090867 to Mandell et al., the entire contents of which are incorporated herein by reference.

Examples

[0193] The following examples are intended purely for illustration and do not limit the present invention unless specifically recited in the claims.

[0194] Example 1.

[0195] The suspension barriers were prepared using a support with a circular opening and different materials as described below. All materials were tested on a NanION Orbit-16 instrument. This tool enables mechanical coating by rotation of a Teflon® stir bar at the top of the chip cavity, as well as electrical testing of the membrane / pore construct (membrane capacitance measurement, nanopore I / V curve). The barriers were generated under standard buffer conditions (1 M KCl, 50 mM HEPES, pH = 7.4), and the materials were diluted with octane (5 mg / mL) and then tested by membrane formation (also called membrane coating). Figure 13 shows the dielectric breakdown voltage waveform used to evaluate barrier stability. Membrane stability was quantified as the percentage of the membrane remaining at the end of each step of the indicated voltage ramp. The voltage ramp was stepped in 50 mV steps from 150 mV to 500 mV as shown in Figure 13. Each step lasted for 10 seconds. Nanopore insertion was represented as the number of successful single nanopore insertions during each individual experiment with a maximum of 16 nanopores per experiment.

[0196] The following ABA polymer (ABA1) with methyl end groups was used to form the barriers.

Chemical formula

Chemical formula

[0197] Figure 14A shows a plot of the measured breakdown voltages of barriers formed using exemplary barriers, namely ABA1 and DPhPC. This test is used as a stability measurement criterion, and higher breakdown voltages are associated with higher mechanical stability of the membrane, and thus are better for use when the membrane is exposed to extreme conditions (such as sequencing) over long periods of time. As can be understood from Figure 14A, the barrier formed using ABA1 had significantly higher stability than the barrier using DPhPC as the voltage increased. Figure 14B shows a plot of the respective currents through the barriers of Figure 14A with MspA nanopores inserted. Figure 14C shows a plot of the respective noise in the currents through the barriers of Figure 14A with MspA nanopores inserted. From Figures 14A - 14C, it can be understood that DPhPC provides relatively high pore current values and low noise values, but shows a relatively low breakdown voltage, which limits its widespread use and shortens the useful lifetime for sequencing. In comparison, ABA1 shows lower pore current values and higher noise values, but shows a significantly higher breakdown voltage, with approximately 80% of the membrane remaining at 450 mV.

[0198] Additional barriers were formed using the following ABA polymers having COOH terminal groups (ABA2 and ABA3, respectively).

Chemical formula

[0199] As shown in FIGS. 15A - 15C, the performance of barriers formed using ABA2 and ABA3 polymers was evaluated and compared with that of ABA1. More specifically, FIG. 15A shows a plot of the measured breakdown voltages of barriers formed using additional exemplary barriers, namely ABA1, ABA2, and ABA3. FIG. 15B shows a plot of the respective currents through the barriers of FIG. 15A into which MspA nanopores were inserted. FIG. 15C shows a plot of the respective noises in the currents through the barriers of FIG. 15A into which MspA nanopores were inserted. Both ABA2 and ABA3 were able to form stable membranes. ABA3 showed an increased breakdown voltage compared to DPhPC but a lower breakdown voltage than ABA1. However, the MspA pore current values seen for the ABA3 membrane were high and the noise values were low, comparable to DPhPC in these measurement criteria. ABA2 showed a remarkable breakdown voltage, with approximately 84% of the membrane remaining at 450 mV, showing high pore current values and low noise values comparable to DPhPC.

[0200] From FIGS. 14A - 14C and 15A - 15C, it can be understood that by decreasing the size of the hydrophilic block, the nanopore current value can increase significantly and the nanopore noise value can decrease. The polymers used as hydrophilic blocks and / or hydrophobic blocks, their respective lengths, and the end groups can be selected simultaneously to provide a barrier with similar stability to a barrier having a larger hydrophilic block while having appropriate fluidity to enable easy nanopore insertion. FIG. 16 shows a plot of barrier noise and half - decay voltage as a function of the number of repeating units (RUs) in the hydrophobic A block. More specifically, FIG. 16 shows measured values from titrations of ABA polymers having the same hydrophobic block (PDMS) size but varying hydrophilic block (PEO) units, with respect to stability for the breakdown voltage test and the signal - to - noise (current / noise) ratio. The blue line shows an increase in stability with respect to the breakdown voltage reported as the half - decay of the membrane (the voltage at which more than 50% of the membrane breaks down). The red line shows a decrease in the signal - to - noise (current / noise) ratio with the number of hydrophilic repeating units. Similar results are thought to be interpretable for other hydrophobic - hydrophilic block chemistries.

[0201] Example 2. The performance of different copolymers was evaluated in terms of ease of membrane preparation, controlled single to multiple nanopore insertion, and stability against osmotic pressure.

[0202] All materials were tested on a NanION Orbit-16 instrument. This tool enables mechanical coating by rotation of a Teflon® stir bar at the top of the chip cavity, as well as electrical testing of the membrane / pore construct (membrane capacitance measurement, nanopore I / V curve).

[0203] Membrane Coating and Nanopore Insertion The copolymers listed in Table 1 below were dissolved in an octane:butanol (95:5 volume) solvent mixture at a concentration of 5 mg / mL prior to testing by suspension membrane formation (also referred to as membrane coating), using a support containing circular openings such as those described with reference to FIGS. 2A-2B, 3-6, and 18. [Table 1]

[0204] Characterization tests were used to extract measurement criteria relevant to nanopore sensing applications of such membranes. These measurement criteria are classified into categories such as stability (e.g., elasticity of the membrane / membrane-pore system against stress tests including accelerated tests, sequencing conditions), throughput (e.g., membrane coating yield, pore insertion and retention yield), and quality (e.g., membrane-pore current and noise level and consistency, SNR, electrical insulation / leakage of the membrane).

[0205] The first characterization test performed focused on the success rate in membrane formation (membrane coating yield), membrane resistance against breakdown voltage, biological pore insertion (MspA pore), and the resulting current and noise of the pores inside the block copolymer membrane. These helped to evaluate the performance of the PIB-PEO-based membranes against each other and against membranes formed using other polymers.

[0206] Regarding the coating quality, all PIB-b-PEO block copolymers (AB1, AB2, AB3, AB4) and PEO-b-PIB-b-PEO block copolymers (ABA4, ABA5, ABA6) were coated to form a suspension film. Particularly satisfactory performance was achieved by the PEO-b-PIB-b-PEO block copolymer having an ABA structure. For example, FIG. 19 shows a plot depicting the breakdown voltage measured for the films formed using P5, ABA4, AB1, AB2, AB3, AB4, and ABA5. For the film formed using P5, in FIG. 19, at voltages below about 300 mV, the normalized number of films that remained substantially intact ranged from about 1.0 at 0 V to about 0.95 at 300 mV, and at voltages above about 350 mV, it can be seen that the normalized number of films decreased from about 0.9 at 350 mV to about 0.22 at 500 mV. For the film formed using ABA4, in FIG. 19, at voltages below about 300 mV, the normalized number of films that remained substantially intact ranged from about 1.0 at 0 V to about 0.95 at 300 mV, and at voltages above about 350 mV, it can be seen that the normalized number of films decreased from about 0.9 at 350 mV to about 0.5 at 500 mV. For the film formed using ABA5, in FIG. 19, at voltages below about 300 mV, the normalized number of films that remained substantially intact ranged from about 1.0 at 0 V to about 0.95 at 300 mV, and at voltages above about 350 mV, it can be seen that the normalized number of films decreased from about 0.9 at 350 mV to about 0.5 at 500 mV.

[0207] Regarding the film formed using AB1, in Fig. 19, at voltages below about 300 mV, the normalized number of the film that remained substantially intact ranged from about 1.0 at 0 V to about 0.16 at 300 mV, and at voltages of about 350 mV or higher, it can be seen that the normalized number of the film decreased from about 0.16 at 350 mV to about 0.04 at 500 mV. Regarding the film formed using AB2, in Fig. 19, at voltages below about 300 mV, the normalized number of the film that remained substantially intact ranged from about 1.0 at 0 V to about 0.75 at 300 mV, and at voltages of about 350 mV or higher, it can be seen that the normalized number of the film decreased from about 0.45 at 350 mV to about 0.01 at 500 mV. Regarding the film formed using AB3, in Fig. 19, at voltages below about 300 mV, the normalized number of the film that remained substantially intact ranged from about 1.0 at 0 V to about 0.9 at 300 mV, and at voltages of about 350 mV or higher, it can be seen that the normalized number of the film decreased from about 0.85 at 350 mV to about 0.035 at 500 mV. Regarding the film formed using AB4, in Fig. 19, at voltages below about 300 mV, the normalized number of the film that remained substantially intact ranged from about 1.0 at 0 V to about 0.97 at 300 mV, and at voltages of about 350 mV or higher, it can be seen that the normalized number of the film decreased from about 0.9 at 350 mV to about 0.65 at 500 mV.

[0208] As can be understood from the plots shown in Fig. 19, the ABA - type block copolymers ABA4 and ABA5, and the AB4 - type block copolymer showed particularly high resistance to the dielectric breakdown voltage stress test.

[0209] Another difference between the films was how difficult it was to insert a single nanopore into the film. For nanopore sequencing as described with reference to Figs. 9 - 12 and 17, it is useful to insert a single nanopore into each film.

[0210] Figure 20 shows the plot of the stability of the MspA nanopore / membrane construct in 1 M KCl + 50 mM HEPES buffer. For the membrane formed using P5 into which the MspA nanopore was inserted, it can be seen from Figure 20 that at a voltage of 100 mV, the membrane-pore construct had a current in the range of about 92 pA to about 97 pA. For the membrane formed using ABA4 into which the MspA nanopore was inserted, it can be seen from Figure 20 that at a voltage of 100 mV, the membrane-pore construct had a current in the range of about 89 pA to about 104 pA. For the membrane formed using ABA5 into which the MspA nanopore was inserted, it can be seen from Figure 20 that at a voltage of 100 mV, the membrane-pore construct had a current in the range of about 87 pA to about 110 pA. For the membrane formed using AB1 into which the MspA nanopore was inserted, it can be seen from Figure 20 that at a voltage of 100 mV, the membrane-pore construct had a current in the range of about 104 pA to about 135 pA. For the membrane formed using AB2 into which the MspA nanopore was inserted, it can be seen from Figure 20 that at a voltage of 100 mV, the membrane-pore construct had a current in the range of about 100 pA to about 105 pA. For the membrane formed using AB3 into which the MspA nanopore was inserted, it can be seen from Figure 20 that at a voltage of 100 mV, the membrane-pore construct had a current in the range of about 94 pA to about 107 pA. For the membrane formed using AB4 into which the MspA nanopore was inserted, it can be seen from Figure 20 that at a voltage of 100 mV, the membrane-pore construct had a current in the range of about 96 pA to about 110 pA.

[0211] ABA5 and ABA6 membranes were identified as having particularly good performance. The significant improvement shown in ABA5 is the enhancement of membrane elasticity. The significant improvement shown in ABA6 is the enhancement of the insertion and retention of a single MspA nanopore into the membrane with lower variability. The various properties of the membrane are shown in Table 2 below.

Table 2

[0212] The film quality was measured by the film retention rate at a current of 450 mV across the barrier. Both ABA5 and ABA6 had a retention rate of 100%, and ABA7 had a retention rate of approximately 95%.

[0213] The current passing through the film when the MspA nanopore was inserted was also measured. The open pore current was measured at a voltage across a 100 mV barrier. ABA5 and ABA6 had similar currents of 103 pA and 104 pA respectively, and ABA7 had a current of 104 pA. After inserting the MspA nanopore into the film, the root mean square (RMS) average of the current noise across the barrier was also measured in the same way. ABA5 and ABA6 had similar RMS current noise averages of 1.46 pA and 1.62 pA respectively, and ABA7 had an RNS current noise of 2.13 pA. The signal-to-noise ratio (SNR) of the current across the barrier was also measured in the same way. ABA5 and ABA6 had SNRs of 71 and 64 respectively, and ABA7 had an SNR of 49.

[0214] Various yield percentages were also measured. ABA5, ABA6, and ABA7 all had a film coating yield of over 95%. The voltage required to insert the MspA nanopore into the film was also measured in the same way. ABA5 required a voltage of approximately 800 mV to approximately 1000 mV, while ABA6 required a voltage of approximately 350 mV to 450 mV, and ABA7 required a voltage of approximately 500 mV to approximately 850 mV. After performing the nanopore insertion process, the percentage of the film containing the MspA nanopore was approximately 90% for ABA5, over 95% for ABA6, and over 95% for ABA7. The percentage of single pore membranes that withstood washing was also measured in the same way. Specifically, after washing the film three times with 250 μL of fluid, the retention rate was determined. Both ABA5 and ABA6, like ABA7, had a retention rate of over 95%.

[0215] The tightness of the open pore current diffusion of MspA nanopores within the membrane was also measured. Specifically, the current was measured in 400 mM KCl, and a current of 50 mV was applied to the membrane. ABA5 and ABA6 had open pore currents of 35.02 pA and 36.04 pA, respectively, and ABA7 had an open pore current of 36.18 pA. The standard deviation of the current was measured similarly. ABA5 and ABA6 had standard deviations of 0.8 pA and 0.4 pA, respectively, and ABA7 had a standard deviation of 1.93 pA.

[0216] The elasticity of the barrier was also measured. The membrane survival rate was measured after subjecting the barrier to a waveform consisting of a series of positive voltage micropulses spaced by a negative voltage period of 100 ms at -100 mV. The positive voltage pulse train had a duration of 10 μs and a total of 20 pulses. The interval between them had a set duration value of 30 ms and a voltage held at +50 mV. During the first cycle, the waveform was applied continuously for 5 minutes, and the pulse magnitude was maintained at +700 mV. In further applied cycles (each applied for 5 minutes again), the pulse intensity was increased from +700 mV to +1200 mV in 100 mV increments over a total of 6 cycles. Both ABA5 and ABA6 had a survival rate of approximately 95% after application of the described waveform, while ABA7 had a survival rate of approximately 65%. In the same experiment / test, the voltage at which 50% of the membrane remained after being exposed to the waveform was determined. For ABA5, ABA6, and AB4, the voltage was determined to be greater than 1200 mV. In another test / experiment, the same waveform cycle / test was repeated, but using a membrane after insertion of a single MspA pore. For ABA5 and ABA7, the voltage was determined to be approximately 1000 mV, while for ABA6, the voltage was determined to be approximately 900 mV.

[0217] Based on different measurement criteria selected to evaluate the suitability for use in a polymer membrane for nanopore sequencing applications, these results demonstrate that the performance of membranes containing PIB as a hydrophobic B block is particularly suitable for use in such applications. For example, different measurement criteria indicate that ABA and AB copolymers using PIB as a hydrophobic B block, especially those containing -COOH as a terminal group, can form membranes with useful properties for nanopore sequencing applications, including a relatively high film-forming yield, a relatively high pore insertion and retention yield, relatively high stability under sequencing conditions, and relatively good electrical characteristics for high read quality. The inventors believe that such properties may correlate with the ease of flow cell manufacturing, overall instrument, instrument / chip reliability, and high read accuracy, which can be important for the commercial manufacture and use of nanopore sequencing devices as described, for example, with reference to FIGS. 9-12 and 17.

[0218] Further comments Although various exemplary examples are described above, it will be apparent to those skilled in the art that various changes and modifications can be made herein without departing from the spirit of the invention. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention.

[0219] To realize the benefits described herein, it should be understood that each and every feature / example of each aspect of the disclosure described herein may be implemented together in any suitable combination, and any feature / example from any one or more of these aspects may be implemented in any suitable combination with any of the features of any of the other aspects described herein.

Claims

1. A barrier between a first fluid and a second fluid, the barrier being suspended by a barrier support defining an opening, the barrier being suspended across the opening and comprising one or more layers containing molecules of a block copolymer, each molecule of the block copolymer comprising one or more hydrophilic blocks having an approximate length A and one or more hydrophobic blocks having an approximate length B, one or more layers in which the hydrophilic blocks form the outer surface of the barrier and the hydrophobic blocks are located within the barrier; and end groups attached to the ends of the hydrophilic blocks forming the outer surface of the barrier and having a hydrophilicity different from that of the hydrophilic blocks.

2. The terminal group is selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH ３ ), biotin, carboxyl (COOH), propargyl, azide (N ３ ), amino (NH ２ ), hydroxyl (OH), thiol (SH), and sulfonate (SO ３ － ), the barrier according to claim 1.

3. The barrier according to claim 1 or claim 2, wherein the hydrophobic block comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polybutadiene, fluorinated polyethylene, polypeptide, and poly(isobutylene) (PIB).

4. The barrier according to any one of claims 1 to 3, wherein the block copolymer is a diblock copolymer.

5. The barrier according to claim 4, wherein the hydrophobic block is polybutadiene (PBd).

6. The barrier according to claim 4 or 5, wherein the barrier has a thickness of approximately 2A + 2B.

7. The barrier according to any one of claims 1 to 3, wherein the block copolymer is a triblock copolymer having two hydrophilic blocks and one hydrophobic block.

8. The barrier according to claim 5, wherein the hydrophobic block is poly(isobutylene) (PIB).

9. The barrier according to claim 5 or 6, wherein the barrier has a thickness of approximately 2A + B.

10. The barrier according to any one of claims 1 to 9, wherein the block copolymer is a triblock copolymer having two hydrophobic blocks and one hydrophilic block.

11. The barrier according to claim 10, wherein the barrier has a thickness of approximately A + 2B.

12. The barrier according to any one of claims 1 to 11, further comprising nanopores disposed within the barrier to provide contact between the first fluid and the second fluid.

13. A barrier comprising: at least one layer containing a plurality of molecules; each of said molecules comprising a first hydrophilic block, a second hydrophilic block, a first end group, a second end group, and a hydrophobic block; said hydrophobic block being disposed between said first hydrophilic block and said second hydrophilic block and bonded to said first hydrophilic block and said second hydrophilic block; said first end group and said second end group being bonded to the ends of said first hydrophilic block and said second hydrophilic block respectively, having a hydrophilicity different from that of said first hydrophilic block and said second hydrophilic block; said first end group and said second end group forming the outer surface of said barrier; said hydrophobic block being within said barrier. **Claim 14** The barrier according to claim 13, wherein each of said first hydrophilic block and said second hydrophilic block contains poly(ethylene oxide) (PEO). **Claim 15** The barrier according to claim 14, wherein each of said first hydrophilic block and said second hydrophilic block contains about 2 to about 12 PEO repeating units. **Claim 16** The barrier according to any one of claims 13 to 15, wherein said hydrophobic block contains poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB). **Claim 17** The barrier according to claim 16, wherein said hydrophobic block contains about 13 to about 44 PDMS repeating units. **Claim 18** The barrier according to claim 16, wherein said hydrophobic block contains about 13 to about 44 PIB repeating units. **Claim 19** The first terminal group and the second terminal group are selected independently from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH ３ ), biotin, carboxyl (COOH), propargyl, azide (N ３ ), amino (NH ２ ), hydroxyl (OH), thiol (SH), and sulfonate (SO ３ － ), the barrier according to any one of claims 13 to 18. **Claim 20** The barrier according to any one of claims 13 to 19, further comprising nanopores. **Claim 21** The barrier according to claim 20, wherein said nanopores contain α-hemolysin or MspA. **Claim 22** A barrier comprising: at least one layer containing a plurality of molecules; each of said molecules comprising a first ionic end group, a second ionic end group, and a hydrophobic block, said hydrophobic block being disposed between said first ionic end group and said second ionic end group and bonded to said first ionic end group and said second ionic end group; said ionic end groups forming the first outer surface and the second outer surface of said barrier; said hydrophobic block being within said barrier. **Claim 23** The barrier according to claim 22, wherein each of the first ionic end group and the second ionic end group contains a zwitterion.

24. The barrier according to claim 23, wherein the first ionic end group and the second ionic end group are selected from the group consisting of 2-methacryloyloxyethyl phosphorylcholine, 3-[dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azaniumyl]propane-1-sulfonate (DMAPS), 3-{[3-(acryloylamino)propyl](dimethyl)ammonio}propanoate, and 3-{[3-(acryloylamino)propyl](dimethyl)ammonio}-1-propanesulfonate.

25. The barrier according to claim 22, wherein each of the first ionic end group and the second ionic end group contains a cation.

26. The barrier according to claim 25, wherein each of the first ionic end group and the second ionic end group contains 2-(trimethylammonio)ethyl methacrylate.

27. The barrier according to claim 22, wherein each of the first ionic end group and the second ionic end group contains an anion.

28. The barrier according to claim 27, wherein each of the first ionic end group and the second ionic end group contains 3-sulfopropyl acrylate, 2-propene-1-sulfonate, or vinylphosphonic acid.

29. The barrier according to any one of claims 22 to 28, wherein the hydrophobic block contains poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB).

30. The barrier according to claim 29, wherein the PDMS contains about 13 to about 44 PDMS repeating units.

31. The barrier according to claim 29, wherein the PIB contains about 13 to about 44 PIB repeating units.

32. The barrier according to any one of claims 22 to 31, wherein each of the first ionic end group and the second ionic end group is bonded to the hydrophobic block via a product of hydrosilylation, amine-ester coupling, CuAAC click chemistry, DBCO-azide, thiol-Michael addition, or thiol-ene click reaction.

33. The barrier according to any one of claims 22 to 32, further comprising nanopores.

34. The barrier according to claim 33, wherein the nanopore comprises α-hemolysin or MspA.

35. A barrier comprising: A first layer comprising a first plurality of molecules, Each of the molecules includes a hydrophilic block, a hydrophobic block, and a terminal group, The hydrophilic block is bonded to the hydrophobic block, The terminal group is bonded to the end of the hydrophilic block and has a hydrophilicity different from that of the hydrophilic block, a first layer; A second layer comprising a second plurality of molecules; The terminal groups form a first outer surface and a second outer surface of the barrier, A barrier in which the hydrophobic blocks of the first plurality of molecules and the second plurality of molecules are in contact with each other within the barrier.

36. The barrier according to claim 35, wherein the hydrophilic block is poly(ethylene oxide) (PEO).

37. The barrier according to claim 35, wherein the hydrophilic block includes about 2 to about 12 PEO repeating units.

38. The barrier according to any one of claims 35 to 37, wherein the hydrophobic block includes poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB).

39. The barrier according to claim 38, wherein the hydrophobic block includes about 14 to about 44 PDMS repeating units.

40. The barrier according to claim 38, wherein the hydrophobic block includes about 14 to about 44 PIB repeating units.

41. The terminal group is selected from the group consisting of fluorenylmethoxycarbonyl (Fmoc), tert-butyl carbamate (NHBoc), methyl (CH ３ ), biotin, carboxyl (COOH), propargyl, azide (N ３ ), amino (NH ２ ), hydroxyl (OH), thiol (SH), and sulfonate (SO ３ － ), and the barrier according to any one of claims 35 to 40.

42. The barrier according to any one of claims 35 to 41, further comprising a nanopore.

43. The barrier according to claim 42, wherein the nanopore comprises α-hemolysin or MspA.

44. A barrier comprising: A first layer comprising a first plurality of molecules, Each of the molecules includes a first hydrophobic block, a second hydrophobic block, a hydrophilic block, and a first terminal group and a second terminal group, The hydrophilic block is disposed between the first hydrophobic block and the second hydrophobic block and is bonded to the first hydrophobic block and the second hydrophobic block, The first terminal group and the second terminal group are bonded to the ends of the first hydrophobic block and the second hydrophobic block, respectively, and have a hydrophobicity different from that of the first hydrophobic block and the second hydrophobic block, a first layer; A second layer comprising a second plurality of molecules; The hydrophilic blocks of the first plurality of molecules form a first outer surface of the barrier, the hydrophilic blocks of the second plurality of molecules form the second outer surface of the barrier, a barrier, wherein the first terminal groups and the second terminal groups of the first plurality of molecules and the second plurality of molecules are in contact with each other within the barrier. **Claim 45** The barrier according to claim 44, wherein the hydrophilic block is poly(ethylene oxide) (PEO). **Claim 46** The barrier according to claim 45, wherein the hydrophilic block comprises from about 2 to about 13 PEO repeating units. **Claim 47** The barrier according to any one of claims 44 to 46, wherein each of the first hydrophobic block and the second hydrophobic block comprises poly(dimethylsiloxane) (PDMS) or poly(isobutylene) (PIB). **Claim 48** The barrier according to claim 47, wherein each of the first hydrophobic block and the second hydrophobic block comprises from about 14 to about 44 PDMS repeating units. **Claim 49** The barrier according to claim 47, wherein each of the first hydrophobic block and the second hydrophobic block comprises from about 14 to about 44 PIB repeating units. **Claim 50** wherein the first end group and the second end group are lower alkyl (C １－４ alkyl), an aryl group, a polycyclic aromatic hydrocarbon, a fluorinated alkyl, or a fluorinated aryl group, the barrier according to any one of claims 44 to 49. **Claim 51** The barrier according to claim 50, wherein the lower alkyl comprises a methyl, ethyl, propyl, or n-butyl group. **Claim 52** The barrier according to any one of claims 44 to 51, further comprising a nanopore. **Claim 53** The barrier according to claim 52, wherein the nanopore comprises α-hemolysin or MspA.

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