Nanopore devices including barriers using diblock or triblock copolymers and methods for making same

Abstract

Provided herein are nanopore devices including a barrier using a diblock or triblock copolymer, and methods for making the same. In some embodiments, a barrier between a first fluid and a second fluid is suspended by a barrier support that defines an opening. The barrier is 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 approximately a length A and one or more hydrophobic blocks having approximately a length B. The hydrophilic blocks may form an outer surface of the barrier, and the hydrophobic blocks may be located within the barrier. The hydrophobic blocks may include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB).

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Patent Application No. 63 / 325,726, filed March 31, 2022, entitled “NANOPORE DEVICES INCLUDING BARRIERS USING DIBLOCK OR TRIBLOCK COPOLYMERS, AND METHODS OF MAKING THE SAME,” the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present application relates to barriers comprising block copolymers. [Background technology]

[0003] A huge amount of time and energy has been spent by academics and companies on using nanopores for polynucleotide sequencing. For example, residence times have been measured for complexes of DNA with Klenow fragment (KF) of DNA polymerase I on nanopores in an applied electric field. Or, for example, current or flux measuring sensors have been used in experiments on DNA trapped in an α-hemolysin nanopore. Or, for example, KF-DNA complexes have been differentiated based on their properties when trapped 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 the charge blocking label is cleaved when the nucleotide analog is incorporated into the polynucleotide being synthesized. 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 yet another example, the construct comprises a transmembrane protein pore subunit and a nucleic acid handling enzyme.

[0004] However, such previously known devices, systems, and methods are not always sufficiently robust, reproducible, or sensitive, and may not have a sufficiently high throughput for practical implementation, requiring commercial applications such as, for example, genome sequencing in clinical and other settings that demand cost-effective and highly accurate operation. Thus, what is needed are improved devices, systems, and methods for sequencing polynucleotides, which may include using membranes having nanopores disposed therein. Summary of the Invention

[0005] Provided herein are nanopore devices that include barriers that use diblock or triblock copolymers, and methods for making the same.

[0006] Some examples herein provide a barrier between a first fluid and a second fluid. The barrier may be suspended by a barrier support that defines an opening. The barrier may be suspended across the opening and include one or more layers that include molecules of a block copolymer. Each molecule of the block copolymer includes one or more hydrophilic blocks having approximately a length A and one or more hydrophobic blocks having approximately a length B. The hydrophilic blocks may form an outer surface of the barrier, and the hydrophobic blocks may be located within the barrier. In some examples, the hydrophobic blocks include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB).

[0007] In some examples, the block copolymer is a diblock copolymer. In some examples, the hydrophobic block is polybutadiene (PBd). Additionally or alternatively, in some examples, the barrier has a thickness of approximately 2A+2B.

[0008] 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). Additionally or alternatively, in some examples, the barrier has a thickness of approximately 2A+B.

[0009] In some examples, the block copolymer is a triblock copolymer having two hydrophobic blocks and one hydrophilic block, hi some examples, the barrier has a thickness of approximately A+2B.

[0010] In some examples, the method further includes including a nanopore disposed within the barrier, the nanopore providing contact between the first fluid and the second fluid.

[0011] Some examples herein provide a barrier between a first fluid and a second fluid. The barrier may be suspended by a barrier support that defines an opening. The barrier may include one or more layers suspended across the opening and including molecules of a block copolymer. Each molecule of the block copolymer may include a first hydrophilic block and a second hydrophilic block, and a hydrophobic block disposed between the first hydrophilic block and the second hydrophilic block. The first hydrophilic block and the second hydrophilic block may form an outer surface of the barrier, and the hydrophobic block may be located within the barrier. The barrier may further include a nanopore disposed within the barrier and providing contact between the first fluid and the second fluid.

[0012] In some examples, the first and second hydrophilic blocks are about length A, the hydrophobic block is about length B, at least a portion of one or more layers has a thickness of about 2A+B, and the barrier has a thickness of about 2A+B. In some examples, the length A is from about 2 repeat units to about 100 repeat units. Additionally or alternatively, the length B is from about 2 repeat units to about 100 repeat units.

[0013] In some examples, the hydrophobic block comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB).

[0014] In some examples, the first hydrophilic block and the second hydrophilic block comprise a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, a zwitterionic polymer, a polypeptide, and poly(ethylene oxide) (PEO).

[0015] In some examples, the hydrophobic block has a glass transition temperature (T g ) is included.

[0016] In some examples, at least one of the first and second hydrophilic blocks comprises a moiety selected from the group consisting of a carboxylic acid, a carboxyl group, a methyl group, a hydroxyl group, a primary amine, a secondary amine, a tertiary amine, biotin, a thiol, an azide, a propargyl group, an allyl group, an acrylate group, a zwitterionic group, a sulfate, a sulfonate, an alkyl group, an aryl group, an orthogonal functional group, and hydrogen.

[0017] In some examples, a portion of the first hydrophilic block comprises a methyl group or a carboxyl group, and a portion of the second hydrophilic block comprises a methyl group or a carboxyl group.

[0018] In some examples, the molecule further comprises a first linker attaching the first hydrophilic block to the first end of the hydrophobic block and a second linker attaching the second hydrophilic block to the second end of the hydrophobic block.

[0019] Some examples herein provide a barrier between a first fluid and a second fluid. The barrier may include a first layer including a first plurality of molecules of a diblock copolymer. Each molecule of the diblock copolymer may include a hydrophobic block bonded to a hydrophilic block. The barrier may further include a second layer including a second plurality of molecules of the diblock copolymer. The hydrophilic block of the first plurality of molecules may form a first outer surface of the barrier, the hydrophilic block of the second plurality of molecules may form a second outer surface of the barrier, and the hydrophobic blocks of the first plurality of molecules and the second plurality of molecules may contact each other within the barrier.

[0020] In some examples, the hydrophilic block is about length A, the hydrophobic block is about length B, the first and second layers each have a thickness of about A+B, and the barrier has a thickness of about 2A+2B. In some examples, the length A is from about 2 repeat units to about 100 repeat units. Additionally or alternatively, the length B is from about 2 repeat units to about 100 repeat units.

[0021] In some examples, the hydrophilic block comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, a zwitterionic polymer, a polypeptide, and poly(ethylene oxide) (PEO).

[0022] In some examples, the hydrophobic block comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB).

[0023] In some instances, each of the molecules further comprises a linker connecting the hydrophobic block to the hydrophilic block.

[0024] In some examples, the linker comprises at least one moiety selected from the group consisting of an amide, a thioether (sulfide), a succinate group, a maleate group, a methylene, an ether, and a product of a click reaction.

[0025] In some examples, the first and second hydrophilic blocks each comprise a moiety independently selected from the group consisting of a carboxylic acid, a carboxyl group, a methyl group, a hydroxyl group, a primary amine, a secondary amine, a tertiary amine, biotin, a thiol, an azide, a propargyl group, an allyl group, an acrylate group, a zwitterionic group, a sulfate, a sulfonate, an alkyl group, an aryl group, an orthogonal functional group, and hydrogen.

[0026] In some examples, the barrier further comprises a nanopore disposed within the barrier and providing an opening that fluidly couples the first fluid to the second fluid.

[0027] Some examples herein provide a barrier between a first fluid and a second fluid. The barrier may include a first layer including a first plurality of molecules of a triblock copolymer. Each molecule of the triblock copolymer may include a first hydrophobic block and a second hydrophobic block, and a hydrophilic block disposed between the first hydrophobic block and the second hydrophobic block. The barrier may further include a second layer including a second plurality of molecules of the triblock copolymer. The hydrophilic block of the first plurality of molecules may form a first outer surface of the barrier, the hydrophilic block of the second plurality of molecules may form a second outer surface of the barrier, and the hydrophobic blocks of the first plurality of molecules and the second plurality of molecules may contact each other within the barrier.

[0028] In some examples, the hydrophilic blocks are about length A, each hydrophobic block is about length B, the first and second layers each have a thickness of about A / 2+B, and the barrier has a thickness of about A+2B. In some examples, the length A is from about 2 repeat units to about 100 repeat units. Additionally or alternatively, the length B is from about 2 repeat units to about 100 repeat units.

[0029] In some examples, the hydrophilic block comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, a zwitterionic polymer, a polypeptide, and poly(ethylene oxide) (PEO).

[0030] In some examples, each hydrophobic block comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB).

[0031] In some examples, each molecule of the first plurality of molecules and the second plurality of molecules further comprises a linker connecting each hydrophobic block to the hydrophilic block, hi some examples, the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction.

[0032] In some examples, the hydrophilic block comprises a moiety selected from the group consisting of a carboxylic acid, a carboxyl group, a methyl group, a hydroxyl group, a primary amine, a secondary amine, a tertiary amine, biotin, a thiol, an azide, a propargyl group, an allyl group, an acrylate group, a zwitterionic group, a sulfate, a sulfonate, an alkyl group, an aryl group, an orthogonal functional group, and hydrogen.

[0033] In some examples, the barrier further comprises a nanopore disposed within the barrier and providing an opening that fluidly couples the first fluid to the second fluid.

[0034] Some examples herein provide a method of making a barrier. The method may include 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 may be suspended across the opening and 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 approximately a length A and one or more hydrophobic blocks having approximately a length B. The hydrophilic blocks may form an outer surface of the barrier, and the hydrophobic blocks may be located within the barrier. In some examples, the one or more hydrophobic blocks include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB).

[0035] In some examples, the barrier is formed using a coating, hi some examples, the coating is selected from the group consisting of brush coating, machine coating, and bubble coating.

[0036] In some examples, the one or more hydrophilic blocks comprise a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, a zwitterionic polymer, a polypeptide, and poly(ethylene oxide) (PEO).

[0037] In some examples, the method further includes inserting a nanopore into the barrier. In some examples, the nanopore provides contact between the first fluid and the second fluid. Additionally or alternatively, in some examples, the nanopore can be inserted into the barrier by electroporation, pipette pump cycles, or surfactant-assisted pore insertion.

[0038] In some examples, the block copolymer is a diblock copolymer.

[0039] In some examples, the block copolymer is a triblock copolymer having two hydrophilic blocks and one hydrophobic block.

[0040] In some examples, the block copolymer is a triblock copolymer having two hydrophobic blocks and one hydrophilic block.

[0041] 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 together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein. [Brief description of the drawings]

[0042] [Figure 1] 1A-1D show schematic cross-sectional views of exemplary nanopore compositions and devices including barriers using diblock or triblock copolymers. [Figure 2A] 2A-2C show schematic diagrams of barriers using diblock or triblock copolymers that may be included in the nanopore compositions and devices of FIG. [Figure 2B] 2A-2C show schematic diagrams of barriers using diblock or triblock copolymers that may be included in the nanopore compositions and devices of FIG. [Figure 2C] 2A-2C show schematic diagrams of barriers using diblock or triblock copolymers that may be included in the nanopore compositions and devices of FIG. [Figure 3A] 2A-2C show schematic diagrams of exemplary schemes for preparing triblock copolymers for use in the nanopore compositions and devices of FIG. [Figure 3B] 2A-2C show schematic diagrams of exemplary schemes for preparing triblock copolymers for use in the nanopore compositions and devices of FIG. [Figure 3C] 2A-2C show schematic diagrams of exemplary schemes for preparing triblock copolymers for use in the nanopore compositions and devices of FIG. [Figure 4]2A-2C are schematic cross-sectional views of exemplary uses of the compositions and devices of FIG. [Diagram 5] 2A-2C are schematic cross-sectional views of another exemplary use of the compositions and devices of FIG. 1; [Figure 6] 2A-2C are schematic cross-sectional views of another exemplary use of the compositions and devices of FIG. 1; [Figure 7] 2A-2C are schematic cross-sectional views of another exemplary use of the compositions and devices of FIG. 1; [Figure 8] 2 illustrates a flow of operations for forming a device such as that shown in FIG. [Figure 9] 13 shows plots and tables describing the capacitance and noise measured for films formed from DPhPC, PEO500-b-PBd700, and PDMS500-b-PEO420-b-PDMS500. [Figure 10] 1 shows plots of the normalized number of membranes remaining as a function of measured voltage for DPhPC, PEO500-b-PBd700 and PDMS500-b-PEO420-b-PDMS500 membranes. [Figure 11] 13 shows a plot of MspA nanopore / PEO500-b-PBd700 membrane construct stability in 1 M KCl+50 mM HEPES buffer. [Figure 12] 1 shows a plot illustrating the breakdown voltage measured for films formed according to examples herein. [Figure 13] 1 shows plots of MspA nanopore / membrane construct stability under certain conditions. [Figure 14A] 2A-2C are schematic diagrams showing plan and cross-sectional views of further details of one non-limiting example of the nanopore composition and device of FIG. [Figure 14B] 2A-2C are schematic diagrams showing plan and cross-sectional views of further details of one non-limiting example of the nanopore composition and device of FIG. [Figure 15] 14A-14B depict schematic diagrams of alternative barriers that may be used in the examples described with reference to FIGS. [Figure 16] 14A-14B depict schematic diagrams of another alternative barrier that may be used in the examples described with reference to FIGS. [Figure 17] 2A-2C are schematic cross-sectional views of another exemplary use of the compositions and devices of FIG. 1; DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Provided herein are nanopore devices that include barriers that use diblock or triblock copolymers, and methods for making the same.

[0044] For example, nanopore sequencing may utilize a nanopore that is inserted into a barrier and includes an opening through which ions and / or other molecules can flow from one side of the barrier to the other. The circuit may be used to detect the sequence of nucleotides, for example, during sequencing by synthesis (SBS), where a polymerase on a first side of the barrier 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 may be improved by using fluids with different compositions on each side of the barrier, for example, to provide adequate electron transport for detection on one side of the barrier while adequately facilitating the activity of the polymerase on the other side of the barrier. Differences in fluid composition may generate osmotic pressure that may weaken the barrier, thus increasing the likelihood that the barrier may break or leak during normal use. However, it may be difficult to insert a nanopore into a barrier that is too strong.

[0045] As provided herein, barriers for use in nanopore devices can include diblock or triblock copolymers that provide stability characteristics suitable for long-term use of the device and also facilitate nanopore insertion to increase the number of devices available during manufacturing. As described in more detail below, in some examples, the diblock copolymers of the invention can include a hydrophilic block bonded to a hydrophobic block. The respective lengths of the hydrophobic and hydrophilic blocks in the diblock copolymer can be selected such that the copolymer assembles into a bilayer structure in which the hydrophilic block forms the outer surface of each layer (and thus the outer surface of each of the barriers) and the hydrophobic block forms the inner surface of that layer and contacts the hydrophobic block of the other layer. In other examples, the triblock copolymers of the invention can include a hydrophilic block bonded between two hydrophobic blocks. The respective lengths of the hydrophobic and hydrophilic blocks in the diblock copolymer can be selected such that the copolymer assembles into a bilayer structure in which the hydrophilic block forms the outer surface of each layer (and thus the outer surface of each of the barriers) and the hydrophobic block forms the inner surface of that layer and contacts the hydrophobic block of the other layer. In yet another example, the triblock copolymer of the present invention may include a hydrophobic block linked between two hydrophilic blocks. The respective lengths of the hydrophobic and hydrophilic blocks in the triblock copolymer may be selected such that the copolymer assembles into a structure in which the hydrophilic block forms the outer surface of the barrier and the hydrophobic block forms the inner surface of the barrier.

[0046] We will first provide a brief overview of some of the terms used herein, then we will describe some exemplary devices that include barriers using diblock or triblock copolymers, and methods of making and using the same.

[0047] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The use of the term "including" and other forms such as "include", "includes" and "included" is not limiting. The use of the term "having" and 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" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrase "having at least" or "comprising 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 include additional features or components.

[0048] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0049] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to describe and take into account small variations due to processing variations, etc. For example, they can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0050] 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. A nucleotide that lacks 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 (GT ... deoxyadenosine monophosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), and deoxythymidine monophosphate (DTMP).These include deoxythymidine diphosphate (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).

[0051] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analog, which is a type of nucleotide that contains a modified nucleobase, sugar, backbone and / or phosphate moiety 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 ... These include cytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine 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 known in the art, certain nucleotide analogs cannot become incorporated into polynucleotides, such as nucleotide analogs such as adenosine 5'-phosphosulfate. A nucleotide can include any suitable number of phosphates, such as 3, 4, 5, 6, or more than 6 phosphates. Nucleotide analogs also include locked nucleic acids (LNA), peptide nucleic acids (PNA), and 5-hydroxybutynyl-2'-deoxyuridine ("Super T").

[0052] As used herein, the term "polynucleotide" refers to a molecule that comprises a sequence of nucleotides linked together. A polynucleotide is a non-limiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogs, 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, or 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, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing.

[0053] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles a polynucleotide by polymerizing nucleotides into a polynucleotide. A polymerase can bind to a primer and a single-stranded target polynucleotide and can sequentially add nucleotides to the growing primer to form a "complementary copy" polynucleotide with a sequence complementary to that of the target polynucleotide. A DNA polymerase can bind to a target polynucleotide and then move downstream the target polynucleotide sequentially adding nucleotides to the free hydroxyl group at the 3' end of the growing polynucleotide chain. 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 cDNA molecules from an RNA template. Still other RNA polymerases can synthesize RNA molecules from an RNA template, such as RdRp. A polymerase can use a short RNA or DNA strand (primer) to initiate strand growth. Some polymerases can displace the strand upstream of the site where they add a base to the strand. Such polymerases can also be said to be strand displacing, that is, have the activity of removing the complementary strand from the template strand that is read by the polymerase.

[0054] 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 Examples of suitable polymerases include 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 specific, non-limiting examples, the polymerase is selected from the group consisting of Bst, Bsu, and Phi29. Some polymerases have the activity of degrading their trailing strand (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0055] Exemplary RNA polymerases include RdRps (RNA-dependent, RNA polymerases), which catalyze 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 proteins. Exemplary RNA reverse transcriptases. A non-limiting exemplary list includes reverse transcriptases 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.

[0056] As used herein, the term "primer" is defined as a polynucleotide to which nucleotides can be added via a free 3'OH group. A primer may include a 3' block that prevents polymerization until the block is removed. A primer may include a modification at the 5' end to allow a coupling reaction or to allow the primer to be attached to another moiety. A 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 a primer may be any suitable number of bases long and may include a suitable combination of natural and / or non-natural nucleotides. A target polynucleotide may include an "amplification adaptor" or more simply an "adaptor" that hybridizes to the primer (having a sequence complementary to the primer) and can be amplified to generate a complementary copy polynucleotide by adding a nucleotide to the free 3'OH group of the primer.

[0057] As used herein, the term "plurality" is intended to mean a population of two or more distinct members. A plurality may range in size from small, medium, large, to very large. A small size plurality may range, for example, from a few members to tens of members. A medium size plurality may range, for example, from tens of members to about 100 members or hundreds of members. A large plurality may range, for example, from about hundreds of members to about 1000 members, thousands of members, and tens of thousands of members. A very large plurality may range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, a plurality may range in size from 2 to well over 100 million members, as well as between all sizes measured by number of members and larger than the exemplary ranges listed above. Thus, the definition of this term is intended to include all integer values ​​greater than 2.

[0058] As used herein, the term "double-stranded," when used in reference to a polynucleotide, is intended to mean that all or substantially all of the nucleotides in a polynucleotide are hydrogen bonded to each nucleotide in a complementary polynucleotide. A double-stranded polynucleotide may also be referred to as a "duplex."

[0059] As used herein, the term "single-stranded" when used in reference to a polynucleotide means that none of the nucleotides in the polynucleotide are hydrogen bonded to each nucleotide in a complementary polynucleotide.

[0060] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the subject of 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, including amplification adapters that function as primer binding sites, that flank the target polynucleotide sequence being analyzed. In certain examples, the multiple target polynucleotides may have first and second adapters that are the same as each other, although they may have different sequences from each other. The 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, a species in the multiple target polynucleotides may include a region of known sequence flanked by a region of unknown sequence that is evaluated, for example, by sequencing (e.g., SBS). In some instances, the target polynucleotide carries an amplification adapter at a single end, and such adapter may be located at either the 3' or 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.

[0061] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in the terminology is not intended to indicate any particular difference in size, sequence, or other properties, unless otherwise specified. For clarity of explanation, when describing a particular method or composition that includes several polynucleotide species, different terms may be used to distinguish one species of polynucleotide from another species.

[0062] 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 a POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some examples, the substrate used in this application includes a silica-based substrate, 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 this 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 certain examples, the substrate has at least one surface that includes glass or a silicon-based polymer. In some examples, the substrate can include a 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. Acrylamides, enones, or acrylates can also be utilized as substrate materials or components. Other substrate materials include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimides, quartz, resins, polymers, and copolymers.In some examples, the substrate and / or substrate surface can be or include quartz. In some other examples, the substrate and / or substrate surface can be or include a semiconductor, such as GaAs or ITO. The above list is intended to illustrate, but not limit, the present application. The substrate can include a single material or multiple different materials. The substrate can be a composite or laminate. In some examples, the substrate includes an organosilicate material.

[0063] 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.

[0064] The substrate may be unpatterned, textured, or patterned on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns may include posts, pads, wells, ridges, channels, or other three-dimensional concave or convex structures. The pattern may be regular or irregular across the surface of the substrate. The pattern may be formed, for example, by nanoimprint lithography or by using, for example, metal pads to form features on a non-metallic surface.

[0065] 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. A flow cell may include a flow chamber that is divided into multiple lanes or multiple sectors. Examples of flow cells that can be used in the methods and compositions described herein, as well as examples of substrates for manufacturing flow cells, include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, Calif.).

[0066] As used herein, the term "electrode" is intended to mean a solid structure that conducts electricity. The electrode may 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 the substrate.

[0067] 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, where a portion of the opening of the nanopore has a width of 100 nm or less, e.g., 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 in a barrier or can be provided through a substrate. Optionally, the 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 multiple constrictions, e.g., at least two, or three, or four, or five, or more than four constrictions, and the nanopore includes a biological nanopore, a solid-state nanopore, or a hybrid biological and solid-state nanopore.

[0068] 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 may include monomers, homopolymers, or heteropolymers. Polypeptide nanopore structures include, for example, α-helical bundle nanopores and β-barrel nanopores, as well as all others known in the art. Exemplary polypeptide nanopores include erolysin, 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, 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 forms a tightly interconnected octamer and transmembrane beta barrel that resembles a goblet and contains a central constriction. For further details regarding α-hemolysin, see U.S. Pat. No. 6,015,714, the contents of which are incorporated herein by reference in their entirety. For further details regarding SP1, see Wang et al., Chem. Commun., 49:1741-1743 (2013), the contents of which are incorporated herein by reference in their entirety.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), both of which are incorporated herein by reference in their entireties. Other nanopores include, for example, the MspA homologue from Norcadia farcinica, and lysenin. For further details regarding lysenin, see WO 2013 / 153359, which is incorporated herein by reference in its entirety.

[0069] "Polynucleotide nanopore" is intended to mean a nanopore made from one or more nucleic acid polymers. A polynucleotide nanopore can include, for example, a polynucleotide origami.

[0070] "Solid-state nanopore" is intended to mean a nanopore made from one or more materials that are not of biological origin. Solid-state nanopores can be formed from inorganic or organic materials. Solid-state nanopores include, for example, silicon nitride (SiN), silicon dioxide (SiO2), silicon carbide (SiC), hafnium oxide (HfO2), molybdenum disulfide (MoS2), hexagonal boron nitride (h-BN), or graphene. Solid-state nanopores may include an opening formed in a solid-state membrane, for example, a membrane that includes any such material.

[0071] "Biological and solid-state hybrid nanopore" is intended to mean a hybrid nanopore made from materials of both biological and non-biological origin. Materials of biological origin are defined above and include, for example, polypeptides and polynucleotides. Biological and solid-state hybrid nanopores include, for example, polypeptide solid-state hybrid nanopores and polynucleotide solid-state nanopores.

[0072] As used herein, a "barrier" is generally intended to mean a structure that inhibits the passage of molecules from one side of the barrier to the other side of the barrier. Molecules that are inhibited from passing may include, for example, ions or water-soluble molecules such as nucleotides and amino acids. However, when a nanopore is disposed within the barrier, the opening of the nanopore may allow the passage of molecules from one side of the barrier to the other side of the barrier. As one specific example, when a nanopore is disposed within the barrier, the opening of the nanopore may allow the passage of molecules from one side of the barrier to the other side of the barrier. Barriers include membranes of biological origin, such as lipid bilayers, and non-biological barriers, such as solid membranes or substrates.

[0073] As used herein, "biologically derived" refers to material that is derived from or isolated from a biological environment, such as an organism or cell, or a synthetically produced version of a biologically available structure.

[0074] As used herein, "solid" refers to a material that is not of biological origin.

[0075] As used herein, "synthetic" refers to membrane materials that are not of biological origin (e.g., polymeric materials, synthetic phospholipids, solid membranes, or combinations thereof).

[0076] As used herein, a "solution" is intended to refer to a homogenous mixture containing two or more substances. In such a mixture, a solute is a substance that is dissolved in another substance, called a solvent. A solution may contain a single solute or may contain multiple solutes. An "aqueous solution" refers to a solution in which the solvent is or includes water.

[0077] As used herein, "polymeric membrane" or "polymer membrane" refers to a synthetic barrier that is primarily composed of polymers that are not of biological origin. In some examples, the polymeric membrane consists essentially of polymers that are not of biological origin. Block copolymers are an example of polymers that are not of biological origin and can be included in the barrier. Hydrophobic polymers with ionic end groups are another example of polymers that are not of biological origin and can be included in the barrier. Since the barrier of the present invention relates to polymers that are not of biological origin, the terms "polymeric membrane", "polymeric membrane", "membrane" and "barrier" can be used interchangeably herein when referring to the barrier of the present invention, even though the terms "barrier" and "membrane" in general can include other types of materials as well.

[0078] As used herein, the term "block copolymer" is intended to refer to a polymer having at least a first portion or "block" that comprises a first type of monomer and at least a second portion or "block" that is directly or indirectly bonded to the first portion and comprises a second, different type of monomer. The first portion may comprise a polymer of the first type of monomer, or the second portion may comprise a polymer of the second type of monomer, or the first portion may comprise a polymer of the first type of monomer and the second portion may comprise a polymer of the second type of monomer. The first portion may optionally include end groups that have a hydrophilicity that is different from the hydrophilicity of the first type of monomer, or the second portion may optionally include end groups that have a hydrophilicity that is different from the hydrophilicity of the second type of monomer, or the first portion may optionally include end groups that have a hydrophilicity that is different from the hydrophilicity of the first type of monomer and the second portion may optionally include end groups that have a hydrophilicity that is different from the hydrophilicity of the second type of monomer. The end groups of any hydrophilic block may be located on the outer surface of a barrier formed using such hydrophilic block, and depending on the particular configuration, the end groups of any hydrophobic block may be located on the inner surface of a barrier or on the outer surface of a barrier formed using such hydrophobic block.

[0079] Block copolymers include, but are not limited to, diblock copolymers and triblock copolymers.

[0080] "Diblock copolymer" is intended to refer to a block copolymer comprising, or consisting essentially of, a first block and a second block bonded directly or indirectly to one another. 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.

[0081] A "triblock copolymer" is intended to refer to a block copolymer that includes or consists essentially of a first block, a second block, and a third block that are directly or indirectly linked to each other. The first block and the third block may include or consist essentially of the same type of monomer (repeat unit) as each other, and the second block may include a different type of monomer (repeat unit). In some examples, the first block may be hydrophobic, the second block may be hydrophilic, and the third block may be hydrophobic and include the same type of monomer as the first block, in which case the triblock copolymer may be referred to as a "BAB" copolymer, with "A" referring to the hydrophilic block and "B" referring to the hydrophobic block. In other examples, the first block can be hydrophilic, the second block can be hydrophobic, and the third block can be hydrophilic and contain the same type of monomers 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.

[0082] The particular arrangement of the molecules of a polymer chain (e.g., a block copolymer) within a polymer membrane may depend, among other things, on the length of each block, the type of monomers used in the different blocks, the relative hydrophilicity and hydrophobicity of the blocks, the composition of the fluid 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 between the molecules of 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 may be considered substantially "stable", even though the molecules may retain some mobility of motion within the membrane.

[0083] As used herein, the term "hydrophobic" is intended to mean tending to exclude water molecules. Hydrophobicity is a relative concept that relates to the difference in polarity of molecules relative 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, lowering the overall free energy of the system.

[0084] As used herein, the term "hydrophilic" is intended to mean tending to bind to water molecules. Polar (hydrophilic) molecules in a polar environment tend to associate with each other in such a way as to minimize contact with non-polar (hydrophobic) molecules, lowering the overall free energy of the system.

[0085] As used herein, the term "amphiphilic" is intended to mean having both hydrophilic and hydrophobic properties. For example, a block copolymer that includes a hydrophobic block and a hydrophilic block may be considered to be "amphiphilic." Illustratively, AB copolymers, ABA copolymers, and BAB copolymers may all be considered to be amphiphilic.

[0086] As used herein, the term "linker" is intended to mean a moiety, molecule or molecules that attach one element to another. A linker may be a covalent or non-covalent bond. Non-limiting examples of covalent linkers include alkyl chains, polyethers, amides, esters, aryl groups, polyaryls, and other moieties. Non-limiting examples of non-covalent linkers include host-guest complexation, cyclodextrin / norbornene, adamantane ring with β-CD, complexation, DNA hybridization interactions, streptavidin / biotin, and the like.

[0087] As used herein, the terms “PEO”, “PEG”, “poly(ethylene oxide)”, and “poly(ethylene glycol)” are intended to be used interchangeably and refer to —[CH—CH—O]n In some examples, n is from about 2 to about 100.

[0088] As used herein, the term "barrier support" is intended to refer to a structure over which a barrier can be suspended. The barrier support may define an opening such that a first portion of the barrier is suspended over 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 over the opening. In some examples, the barrier support may include a substrate having an opening defined therethrough over which the barrier can 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 in the substrate) elevated relative to one or more second features (such as a bottom surface of the well), with the height difference between (a) the one or more first features and (b) the one or more second features defining an opening over which the barrier can be suspended. The opening may have any suitable shape, such as a circular, elliptical, polygonal, or irregular shape. The barrier support may include any suitable material, or combination of materials. For example, the barrier support may be biologically derived or may be solid-state. In some examples, the barrier support may include or consist essentially of an organic material, such as a curable resin, such as SU-8, polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), parylene, etc. 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.

[0089] As used herein, the term "annulus" is intended to refer to a liquid that is attached to a barrier support, is located within the barrier, and extends partially into an opening defined by the barrier support. It will thus be understood that the annulus may follow the shape of the opening in the barrier, for example having a circular, elliptical, polygonal, or irregular shape.

[0090] Nanopore devices including barriers using diblock or triblock copolymers and methods for making same - Patents.com Some exemplary devices including barriers using diblock or triblock copolymers and methods of making the same are described in Figures 1, 2A-2C, 3A-3C, 4, 5, 6, 7, 8, 14A-14B, 15, 16, and 17.

[0091] FIG. 1 shows a schematic cross-sectional view of an exemplary nanopore composition and device 100 including a barrier using a diblock or triblock copolymer. The device 100 includes a fluid well 100' including a 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 barrier, and a second fluid 120' within the fluid well and in contact with the second side 112 of the barrier. The barrier 101 may have any suitable structure that typically prevents the passage of molecules from one side of the barrier to the other side of the barrier, e.g., typically prevents contact between the fluids 120 and 120'. For example, as illustrated in FIG. 1, the barrier 101 may optionally include a first layer 107 and a second layer 108, one or both of which inhibit the flow of molecules across the layer. Illustratively, the barrier 101 may include a bilayer including layers 107 and 108, which may be formed using the AB diblock copolymers provided herein, or the BAB triblock copolymers provided herein, or the specific ABA triblock copolymers provided herein, respectively, and may have a structure as described in more detail below with reference to Figures 2A-2C, 14A-14B, 15, or 16. Alternatively, the barrier 101 may include only a single layer that inhibits the flow of molecules across that layer. Illustratively, the barrier 101 may include a single layer that may be formed using the specific ABA triblock copolymers provided herein, and may have a structure as described in more detail below with reference to Figures 2A and 15. In other examples, the barrier 101 may be partially single layer and partially bilayer formed using the specific ABA triblock copolymers provided herein, and may have a structure as described in more detail below with reference to Figures 2A and 15.

[0092] The first fluid 120 may 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' may have a second composition including a second concentration of salt 160, which may be the same or different than the first concentration. Any suitable salt 160 may be used in the first fluid 120 and the second fluid 120', ranging from, for example, common salts to ionic crystals, metal complexes, ionic liquids, or water-soluble organic ions. For example, a salt may be a mixture of cations (such as, but not limited to, H, Li, Na, K, NH4, Ag, Ca, Ba, and / or Mg) and anions (OH, Cl, Br, I, NO3, ClO4, F, SO4, and / or CO3). 2- ...etc. In one non-limiting example, the salt comprises potassium chloride (KCl). It will also be appreciated that the first and second fluids may optionally comprise any suitable combination of other solutes. Illustratively, the first and second fluids 120 and 120' may comprise an aqueous buffer (e.g., N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), available from Fisher BioReagents). Any difference between the first and second concentrations of salt 160 and / or between other components of the first and second fluids may generate an osmotic pressure 191 across the barrier 101. As provided herein, the diblock or triblock copolymers used in the barrier 101 may be selected to provide a barrier with sufficient stability for use over a desired period of time, for example, for use over the course of sequencing a polynucleotide in a manner as described with reference to Figures 4-7 or 17.

[0093] 1, in some examples provided herein, the device 100 may optionally further include a nanopore disposed within the barrier 101 and providing an opening 113 fluidically coupling the first side 111 to the second side 112. The opening 113 of the nanopore 110 may thus provide a path for the fluid 120 and / or the fluid 120′ to flow through the barrier 101. For example, a portion of the salt 160 may migrate through the opening 113 from the second side 112 of the barrier 101 to the first side 111 of the barrier. The nanopore 110 may include a solid-state nanopore, a biological nanopore (e.g., MspA as illustrated in FIG. 1), or a hybrid biological and solid-state nanopore. Non-limiting examples and characteristics of barriers and nanopores are described elsewhere herein, as well as in U.S. Pat. No. 9,708,655, the entire contents of which are incorporated herein by reference. In the manner shown in Figure 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 circuitry 180 in operative communication with the first and second electrodes and configured to detect a change in an electrical property of the aperture. Such a change may be in response to, for example, any suitable stimulus. Indeed, it will be understood that the methods, compositions, and devices of the present invention may be used in any suitable application or context, including any suitable method or device for polynucleotide sequencing.

[0094] In some examples, the first layer 107 of the barrier 101 between the first fluid 120 and the second fluid 120' comprises a first plurality of molecules of a diblock or triblock copolymer, and the second layer 108 of the barrier 101 comprises a second plurality of molecules of the copolymer. In examples where the copolymer is a diblock copolymer (which may be referred to as AB), each molecule may comprise a hydrophobic block bound to a hydrophilic block. In some examples where the copolymer is a triblock copolymer, each molecule may comprise a first hydrophobic block and a second hydrophobic block with a hydrophilic block disposed therebetween (the polymer may be referred to as BAB). Regardless of whether the copolymer is diblock (AB) or triblock (BAB), the hydrophilic blocks of the first plurality of molecules may form a first outer surface of the barrier 101, e.g., the surface of the layer 107 that contacts the fluid 120 at the first side 111. The hydrophilic blocks of the second plurality of molecules may form a surface of layer 108 that contacts fluid 120′ on a second exterior surface of barrier 101, e.g., second side 112. The hydrophobic blocks of the first and second plurality of molecules may contact each other within the barrier.

[0095] In another example, the barrier 101 between the first fluid 120 and the second fluid 120' includes a plurality of molecules of a triblock copolymer including a first and a second hydrophilic block and a hydrophobic block disposed therebetween (this polymer may be referred to as ABA). The first hydrophilic block of the plurality of molecules may form a first outer surface of the barrier 101, e.g., a surface of the barrier 101 that contacts the fluid 120 on the first side 111. The second hydrophilic block of the plurality of molecules may form a second outer surface of the barrier 101, e.g., that contacts the fluid 120' on the second side 112. The hydrophobic blocks of the plurality of molecules may contact each other within the barrier. In some examples, the ABA molecules forming the barrier 101 may be in a single layer rather than the two layers shown in FIG. 1. In other examples, the ABA molecules forming the barrier 101 may be in a bilayer similar to that shown in FIG. 1, since the molecules of the B block of the molecules fold such that both A blocks of a given molecule contact each other with the same fluid. In yet another example, a portion of the barrier 101 may be a single layer (because the ABA molecules in that portion extend from one fluid to the other) and a different portion of the barrier 101 may be a bilayer (because the ABA molecules in that portion fold over and the A blocks in that portion contact the same fluid as each other). Figure 2A shows this example.

[0096] For example, Figures 2A-2C illustrate schematic diagrams of barriers using diblock or triblock copolymers that may be included in the nanopore compositions and devices of Figure 1. It will be understood that such barriers may be suitably adapted for use in any other compositions or devices and are not limited to use with nanopores.

[0097] 2A, the barrier 221 uses a triblock "ABA" copolymer. The barrier 221 includes a layer 229 that can contact both fluids 120 and 120'. The layer 229 includes a plurality of molecules 222 of the triblock ABA copolymer. As shown in FIG. 2A, each molecule 222 of the triblock copolymer includes a first hydrophilic block and a second hydrophilic block, each designated "A" and approximately of length "A", and a hydrophobic block, designated "B", and approximately of length "B", disposed between the first and second hydrophilic blocks. The hydrophilic A block at a first end of the molecule 222 (the molecules that form the layer 229) forms a first outer surface of the barrier 221, e.g., the contacting fluid 120. The hydrophilic A block at a second end of the molecule 222 forms a second outer surface of the barrier 221, e.g., the contacting fluid 120'. The hydrophobic B blocks of the molecules 222 are within the barrier 211, as shown in FIG. 2C. As shown, most of the molecules 222 in the layer 229 may extend substantially linearly and in the same orientation as each other. Optionally, as shown in FIG. 2A, some of the molecules 222' may be folded in the B blocks such that both of the hydrophilic A blocks of such molecules may contact the same fluid as each other. Thus, the example shown in FIG. 2A may be considered to be partially monolayer and partially bilayer. In other examples (not specifically shown), the layer 229 may be fully monolayer or fully bilayer, for example, as described with reference to FIG. 1. As shown in FIG. 2A, regardless of whether the membrane includes substantially linearly extending molecules 222 and / or folded molecules 222', the layer 229 may have a thickness of approximately 2A+B. In some examples, length A is from about 1 RU to about 100 RU, e.g., 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, e.g., 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 appreciated that any end groups attached to the hydrophilic or hydrophobic blocks contribute to the overall thickness of the barrier.Optionally, a barrier 221 as described with reference to FIG. 2A may be suspended across the opening as described with reference to FIG.

[0098] 2B, the barrier 201 uses a diblock "AB" copolymer. The barrier 201 includes a first layer 207 that may contact the fluid 120, similar to the layers 107 and 108 described with reference to FIG. 1, and a second layer 208 that may contact the fluid 120'. The first layer 207 includes a first plurality of molecules 202 of the diblock AB copolymer, and the second layer 208 includes a second plurality of molecules 202 of the diblock AB copolymer. As shown in FIG. 2B, each molecule 202 of the diblock copolymer includes a hydrophobic block, designated "B", approximately the length "B", bonded to a hydrophilic block, designated "A", approximately the length "A". The hydrophilic A block of the first plurality of molecules 202 (the molecules forming the layer 207) forms a first outer surface of the barrier 201, e.g., contacting fluid 120. The hydrophilic A blocks of the second plurality of molecules 202 (molecules forming layer 208) form a second outer surface of the barrier 202, e.g., contact fluid 120'. The respective ends of the hydrophobic B blocks of the first and second plurality of molecules contact each other within the barrier 201, as shown in FIG. 2B. As shown, substantially all of the molecules 202 within layer 207 may extend substantially linearly and in the same orientation to each other, and similarly, substantially all of the molecules 202 within layer 207 may extend substantially linearly and in the same orientation to each other (which is opposite to the orientation of the molecules within layer 208). Thus, the first and second layers 207 and 208 may each have a thickness of approximately A+B, and the barrier 201 may have a thickness of approximately 2A+2B. In some examples, length A is from about 2 repeating units (RU) to about 100 RU, or from about 1 repeating unit (RU) to about 50 RU, e.g., 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. Additionally or alternatively, in some examples, length B is from about 2 RU to about 100 RU, 5 RU to about 100 RU, e.g., 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. Optionally, a barrier 201 as described with reference to FIG. 2B may be suspended across the opening as described with reference to FIGS. 14A-14B.

[0099] 2C, the barrier 211 uses a triblock "BAB" copolymer. The barrier 211 includes a first layer 217 that may contact the fluid 120, and a second layer 218 that may contact the fluid 120', similar to the layers 107 and 108 described with reference to FIG. 1. The first layer 217 includes a first plurality of molecules 212 of a triblock copolymer, and the second layer 218 includes a second plurality of molecules 212 of a triblock copolymer. As shown in FIG. 2C, each molecule 212 of the triblock copolymer includes a first hydrophobic block and a second hydrophobic block, each designated "B" and approximately of length "B", and a hydrophilic block, designated "A", approximately of length "A", disposed between the first and second hydrophobic blocks. The hydrophilic A blocks of the first plurality of molecules 212 (molecules forming layer 217) form a first outer surface of the barrier 211, e.g., the contacting fluid 120. The hydrophilic A blocks of the second plurality of molecules 212 (molecules forming layer 218) form a second outer surface of the barrier 211, e.g., the contacting fluid 120'. The respective ends of the hydrophobic B blocks of the first and second plurality of molecules contact each other within the barrier 211 as shown in FIG. 2C. As shown, substantially all of the molecules 212 in layer 217 may extend in the same orientation as each other and may be folded at the A blocks such that the A blocks can contact the fluid while the B blocks are in the interior of the barrier 211. Similarly, substantially all of the molecules 212 in layer 217 may extend in the same orientation as each other (opposite to the orientation of the molecules in layer 218) and may be folded at the A blocks such that the A blocks contact the fluid while the B blocks are in the interior of the barrier 211. Thus, first layer 217 and second layer 218 may each have a thickness of approximately A / 2+B, and barrier 211 may have a thickness of approximately A+2B. In some examples, length A is between about 2 RU and about 100 RU, e.g., between about 10 RU and about 80 RU, or between about 20 RU and about 50 RU, or between about 50 RU and about 80 RU. Additionally or alternatively, in some examples, length B is between about 2 RU and about 100 RU, or between about 5 RU and about 100 RU, e.g., between about 10 RU and about 80 RU, or between about 20 RU and about 50 RU, or between about 50 RU and about 80 RU.Optionally, the barrier 211 described with reference to FIG. 2C may be suspended across the opening in the manner described with reference to FIG.

[0100] It will be understood that the various barrier layers provided herein can be configured to have any suitable dimensions. Illustratively, to form barriers of similar dimensions to one another, ABA triblock copolymers (Figure 2A) consist of two hydrophilic blocks, each of length A (each A block is M w = x) and one hydrophobic block of length B (M w Upon self-assembly, these ABA triblock copolymers form a membrane with a top hydrophilic layer of length A, a core hydrophobic layer of length B, and a bottom hydrophilic layer of length A. The AB diblock copolymer (Figure 2B) consists of one hydrophilic block (M w = x) and one hydrophobic block of length B (M w =y / 2). Upon self-assembly, these AB diblock copolymers form a membrane with a top hydrophilic layer of length A, a core hydrophobic layer of length 2B, and a bottom hydrophilic layer of length A. The BAB triblock copolymer (Figure 2C) consists of one hydrophilic block (M w = x) and two hydrophobic blocks, each of length B (each B block is M w = y / 2. Upon self-assembly, these BAB triblock copolymers form membranes with a top hydrophilic layer of length A / 2, a core hydrophobic layer of length 2B, and a bottom hydrophilic layer of length A / 2.

[0101] Additionally or alternatively, polymer loading into the layers of the membrane can affect the hydrophilicity ratio for each of the barriers, which is determined by the molecular weight of the hydrophilic block and the total molecular weight (MW or M) of the block copolymer (BCP). w ) (hydrophilicity ratio = M w Hydrophilic Block / M w BCP). For example, ABA triblock copolymer ( Figure 2 A), hydrophilic ratio = 2x / (2x+y); AB diblock copolymer (Figure 2B), hydrophilic ratio = x / (x+y / 2); and BAB triblock copolymer (Figure 2C), hydrophilicity ratio = x / (x+y).

[0102] 14A-14B show schematic plan and cross-sectional views of further details of one non-limiting example of the nanopore composition and device of FIG. 1. More specifically, in the example shown in FIGS. 14A-14B, the barrier 101 can be suspended using a barrier support 1400 that defines an opening 1430. For example, the barrier support 1400 can include a substrate having an opening 1430 defined therethrough, e.g., a substantially circular opening, or an opening having another shape. Additionally or alternatively, the barrier 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. An annular portion 1410, which includes a hydrophobic (non-polar) solvent and may also include other compounds, can be attached to the barrier support 1400 and can support a portion of the barrier 101, e.g., can be located within the barrier 101 (here, between layers 1401 and 1402). Additionally, the annular portion 1410 may be inwardly tapered as illustrated in FIG. 14A. An outer portion of the molecule 1421 of the barrier 101 may be disposed on the support 1400 (e.g., the portion extending between the opening 1430 and the barrier periphery 1420), while an inner portion of the molecule may form a free-standing portion of the barrier 101 (e.g., the portion within the opening 1410 supported in part by the annular portion 1410). Using operations as described elsewhere herein, the barrier 101 may be prepared and the nanopore 110 may be inserted into the free-standing portion of the barrier 101. Although FIGS. 14A-B show the nanopore 110 in the barrier 101, it should be understood that the nanopore may be omitted and the barrier 101 may be used for any suitable purpose. More generally, while the barriers described herein are particularly suitable for use with nanopores (e.g., for nanopore sequencing as described with reference to Figures 4-7 and 17), it will be understood that the barriers do not necessarily have a nanopore inserted therein.

[0103] In a non-limiting example shown in Figure 14A, the barrier 101 may include a first layer 1401 including a first plurality of amphiphilic molecules 1421 and a second layer 1402 including a second plurality of amphiphilic molecules in contact with the first plurality of amphiphilic molecules. In the non-limiting example shown in Figure 14A, the copolymer is a diblock copolymer (AB) as described with reference to Figure 2B, where each molecule 1421 includes a hydrophobic "B" block 1431 (dark filled circles 1441 represent hydrophobic monomers) and a hydrophilic "A" block 1432 (light filled circles 1442 represent hydrophilic monomers) bonded directly or indirectly thereto. In other examples as described with reference to Figures 15 and 16, the copolymer may instead include a triblock copolymer (e.g., ABA or BAB, respectively). 14A , the hydrophilic blocks 1432 of the first plurality of molecules 1421 may form a first outer surface of the barrier 101, e.g., a surface of the barrier 101 that contacts the fluid 120 on the first side 111. The hydrophilic blocks 1432 of the second plurality of molecules 1421 may form a second outer surface of the barrier 101, e.g., a surface of the barrier 101 that contacts the fluid 120′ on the second side 112. The hydrophobic blocks 1431 of the first and second plurality of molecules 1421 may contact each other within the barrier.

[0104] 14A-14B show a suspended barrier comprising a diblock copolymer, it will be understood that suspended barriers comprising other types of polymers as provided herein are also contemplated. FIG. 15 shows a schematic of an alternative barrier that may be used in the examples described with reference to FIGS. 14A-14B. FIG. 15 shows a barrier 1501 suspended using a barrier support 1400 and an annular portion 1410 as described with reference to FIGS. 14A and 14B. In this example, the barrier 1501 comprises molecules of an ABA triblock copolymer as described with reference to FIG. 2A. Here, the triblock copolymer comprises hydrophobic "B" sections 1541 bound to hydrophilic "A" sections 1542. In the example shown in FIG. 15, each individual ABA molecule may be in one of two configurations. For example, the ABA molecule 1521 may have an "A" portion on each side of the barrier and a "B" portion in the center of the barrier, extending through the layer in a straight line. Alternatively, for example, the ABA molecules 1522 may extend to the middle of the barrier and then fold back on themselves such that both "A" sections are on the same side of the barrier and the "B" section is in the middle of the barrier. Thus, in this example, the barrier 1501 may be considered to be partially monolayer and partially bilayer. In other examples (not specifically shown) where the barrier 1501 substantially includes the molecules 1521 extending linearly through the barrier, the barrier 1501 may be substantially monolayer. In yet other examples (not specifically shown) where the barrier 1501 substantially includes the molecules 1522 extending approximately to the middle of the barrier and then fold back on itself, the barrier 1501 may be substantially bilayer. Although not specifically shown, a nanopore may optionally be inserted into any of such options for the barrier 1501 in a manner similar to that described elsewhere herein, for example, as shown in FIGS. 14A-14B.

[0105] FIG. 16 shows a schematic of an alternative barrier that may be used in the example described with reference to FIGS. 14A and 14B. FIG. 16 shows a barrier 1601 suspended using the barrier support 1400 and annular portion 1410 as described with reference to FIGS. 14A and 14B. In this example, the barrier 1601 includes molecules of a BAB triblock copolymer as described with reference to FIG. 2C. Here, the triblock copolymer includes hydrophilic "A" sections 1642 bonded to and between hydrophobic "B" sections 1641. In this example, the barrier 1601 may have a bilayer structure with the "B" sections 1641 oriented towards each other. The hydrophobic ends of the BAB molecules may generally be located approximately in the center of the barrier 1601, and the molecules then extend towards either outer surface of the barrier and then fold back on themselves. Thus, both "B" sections are located in the center of the barrier and the "A" sections are on one side or the other of the barrier. A nanopore, not specifically shown, may optionally be inserted into barrier 1601 in a manner similar to that described elsewhere herein, for example as shown in Figures 14A and 14B.

[0106] The diblock and triblock copolymers of the present invention may comprise any suitable combination of hydrophobic and hydrophilic blocks. In some examples, the hydrophilic A block may comprise a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, hydrophilic polypeptides, nitrogen-containing units, and poly(ethylene oxide) (PEO). Illustratively, the polyacrylamide may be selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamides, and phosphate-functionalized polyacrylamides. Non-limiting examples of zwitterionic monomers that can be polymerized to form zwitterionic polymers include:

[0107] [ka] Non-limiting examples of hydrophilic polypeptides include:

[0108] [ka] Non-limiting examples of charged polyacrylamides include:

[0109] [ka] where n is from about 2 to about 100. Non-limiting examples of nitrogen-containing units include:

[0110] [ka] Examples include:

[0111] In some examples, the hydrophobic B block may comprise a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Non-limiting examples of hydrogenated polydienes include saturated polybutadiene (PBu), saturated polyisoprene (PI), saturated poly(myrcene);

[0112] [ka] wherein n is from about 2 to about 100, x is from about 2 to about 100, y is from about 2 to about 100, and z is from about 2 to about 100; R1 is a functional group selected from the group consisting of carboxylic acid, carboxyl, methyl, hydroxyl, primary amine, secondary amine, tertiary amine, biotin, thiol, azide, propargyl, allyl, acrylate, zwitterionic, sulfate, sulfonate, alkyl, aryl, orthogonal functional groups, and hydrogen; and R2 is a reactive moiety selected from the group consisting of maleimide, allyl, propargyl, BCN, carboxylate, amine, thiol, DBCO, azide, N-hydroxysuccinimide, biotin, carboxyl, NHS activated esters, and other activated esters. In other non-limiting examples of hydrogenated polydienes, R1 is a reactive moiety selected from the group consisting of maleimide groups, allyl groups, propargyl groups, BCN groups, carboxylate groups, amine groups, thiol groups, DBCO groups, azide groups, N-hydroxysuccinimide groups, biotin groups, carboxyl groups, NHS activated esters, and other activated esters. Non-limiting examples of fluorinated polyethylenes are:

[0113] [ka] Non-limiting examples of hydrophobic polypeptides include (0 <x<1):

[0114] [ka] In the formula, n is about 2 to about 100.

[0115] In one non-limiting example, the AB diblock copolymer comprises PDMS-b-PEO, where "-b-" indicates that the polymer is a block copolymer. In another non-limiting example, the AB diblock copolymer comprises PBd-b-PEO. In another non-limiting example, the AB diblock copolymer comprises PIB-b-PEO. In another non-limiting example, the BAB triblock copolymer comprises PDMS-b-PEO-b-PDMS. In another non-limiting example, the BAB triblock copolymer comprises PBd-b-PEO-b-PBd. In another non-limiting example, the BAB triblock copolymer comprises PIB-b-PEO-b-PIB. In another non-limiting example, the ABA triblock copolymer comprises PEO-b-PBd-b-PEO. In another non-limiting example, the ABA triblock copolymer comprises PEO-b-PDMS-b-PEO. In another non-limiting example, the ABA triblock copolymer comprises PEO-b-PIB-b-PEO. It will be understood that any suitable hydrophilic block can be used with any suitable hydrophobic block. Additionally, in examples that include two hydrophilic blocks, the blocks can, but do not necessarily have to, comprise the same polymer as each other. Similarly, in examples that include two hydrophobic blocks, the blocks can, but do not necessarily have to, comprise the same polymer as each other.

[0116] The molecular weight, glass transition temperature, and chemical structure of each of the hydrophobic and hydrophilic blocks may be appropriately selected to provide a barrier with suitable stability for use and ability to insert a nanopore. For example, the molecular weight of each of the hydrophobic and hydrophilic blocks may affect the respective thickness of the blocks (and thus the layer of the barrier), which may affect the stability and ability to insert a nanopore, for example, by electroporation, pipette pump cycles, or surfactant-assisted pore insertion. Additionally or alternatively, the ratio of the molecular weights of the hydrophilic and hydrophobic blocks may affect the self-assembly of these blocks into the layer of the barrier. Additionally or alternatively, the glass transition temperature (T g) can affect the lateral flow properties of the barrier layer. Thus, in some instances, the hydrophobic and / or hydrophilic blocks may have a T below the operating temperature of the device, e.g., below room temperature, and in some instances, below about 0° C. g Additionally or alternatively, the chemical structures of the hydrophobic and hydrophilic blocks can affect the way the chains pack into the layers and the stability of those layers.

[0117] It will be understood that the diblock and triblock copolymers of the present invention can be made using any suitable combination of operations. Figures 3A-3C show schematic diagrams of exemplary schemes for preparing triblock copolymers for use in the nanopore compositions and devices of Figure 1. In some examples, the diblock and triblock copolymers of the present invention can be made using a "macroinitiator" approach as illustrated in Figure 3A, where one polymer block is first made and then used as an initiator (X in Figure 3A) to grow one or more additional blocks using monomers ([M] in Figure 3A). Illustratively, 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 polymerizing a plurality of hydrophobic monomers using the initiator to form a hydrophobic polymer attached to the hydrophilic polymer. Alternatively, the operations for making a diblock copolymer may include polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer, forming an initiator at the end of the hydrophobic polymer, and polymerizing a plurality of hydrophilic monomers using the initiator to form a hydrophilic polymer attached to the hydrophobic polymer. Similarly, the operations for making a triblock BAB copolymer may include polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer, forming an initiator at each end of the hydrophilic polymer, and polymerizing a plurality of hydrophobic monomers using the initiator to form a hydrophobic polymer attached to each end of the hydrophilic polymer. Similarly, the operations for making a triblock ABA copolymer may include polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer, forming an initiator at each end of the hydrophobic polymer, and polymerizing a plurality of hydrophilic monomers using the initiator to form a hydrophilic polymer attached to each end of the hydrophobic polymer. In such a "macroinitiator" approach, the initiator (X in FIG. 3A) may be appropriately selected based on the particular monomers used and the particular type of polymerization being performed.For example, in the case of atom transfer free radical polymerization (ATRP), the initiator may include bromine or chlorine. Alternatively, for example, in the case of reversible addition-fragmentation chain transfer (RAFT) polymerization, the initiator may include a chain transfer agent. After polymerization is completed, the end group (X in FIG. 3A) may be modified or removed (e.g., to provide end group Y in FIG. 3A).

[0118] In other examples, diblock and triblock copolymers of the present invention can be made using a "linking" approach as shown in FIG. 3B, where polymer blocks are made separately and then linked together using reactive moieties (X and Y in FIG. 3B). Illustratively, 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 linking the hydrophilic polymer to the hydrophobic polymer. Operations for making a triblock copolymer can include polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer having termini, polymerizing a plurality of hydrophobic monomers to form a first hydrophobic polymer and a second hydrophobic polymer, and linking the first hydrophobic polymer and the second hydrophobic polymer to the respective termini of the hydrophilic polymer. Alternatively, the operations for making a triblock copolymer may 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 an end, and coupling the first hydrophilic polymer and the second hydrophilic polymer to the respective ends of the hydrophobic polymer. In such a "coupling" approach, the end of the hydrophobic polymer may include a first reactive moiety (Y in FIG. 3B) and the end of the hydrophilic polymer may include a second reactive moiety (X in FIG. 3B) that reacts with the first reactive moiety to couple the hydrophilic polymer to the hydrophobic polymer. The reactive moieties (X and Y in FIG. 3B) may be appropriately selected based on the particular polymer to be coupled and the type of coupling to be performed. For example, a "click" chemistry moiety may be used. Illustratively, one of the first and second reactive moieties can include an azide and the other of the first and second reactive moieties can include an alkyne, or one of the first and second reactive moieties can include a thiol and the other of the first and second reactive moieties can include an alkene, or one of the first and second reactive moieties can include a thiol and the other of the first and second reactive moieties 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 comprise an amine, and the other of the first reactive moiety and the second reactive moiety may comprise N-hydroxysuccinimide (NHS). FIG. 3C shows a non-limiting example, where the hydrophobic polymer is PDMS with an amine (NH2) group at one of its termini, and the hydrophilic polymer is PEO with NHS at its termini, and the amine and NHS groups react with each other in the presence of triisopropylamine to provide an ABA triblock copolymer. Another non-limiting example of forming a triblock copolymer 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 under an inert atmosphere using a degassed solvent (e.g., chloroform), in the presence of a photoinitiator (e.g., irgacure 2959), and under UV exposure for 5-30 minutes.

[0119] [ka]

[0120] Another non-limiting example of forming a triblock copolymer is shown below. In this example, PIB-bisallyl is reacted with PEG-thiol via thiol-ene click chemistry. The reaction proceeds using a degassed solvent (e.g., chloroform) in the presence of a photoinitiator (e.g., irgacure 2959) under an inert atmosphere (dry argon or dry nitrogen) for 5-60 minutes under UV exposure. In some examples, the reaction proceeds at 1 mW / cm 2 ~100mW / cm 2 The UV exposure is carried out for 2 to 180 minutes with a UV output ranging from 100 nm to 200 nm. In some examples, the UV wavelength used is 365 nm.

[0121] [ka]

[0122] For nanopore sequencing applications, membrane fluidity may be beneficial. Without wishing to be bound by any theory, it is believed that the fluidity of block copolymer membranes is primarily imparted by the physical properties of the hydrophobic "B" block. More specifically, the "low T g " Hydrophobic polymers (e.g., T g The B block containing g " Polymers (e.g., T above room temperature g For example, in certain instances, the hydrophobic B block of the copolymer may have a T of less than about 20° C., less than about 0° C., or less than about −20° C. g has.

[0123] Low T g A hydrophobic B block having a T sufficiently low for use in nanopore sequencing may be used to help maintain membrane flexibility under conditions suitable for performing nanopore sequencing, for example, in a manner as described with reference to Figures 4, 5, 6, 7, or 17. In some instances, g The hydrophobic B block has a T in the range of about -75°C to about -25°C. g In another example, the PIB may comprise or consist essentially of a PIB that may be expected to have a T sufficiently low for use in nanopore sequencing. g The hydrophobic B block has a T in the range of about -135°C (or lower) to about -115°C. g In yet another example, the nanopore may comprise or consist essentially of PDMS, which may be expected to have a T sufficiently low for use in nanopore sequencing. g The hydrophobic B block having the formula: may comprise or consist essentially of PBd. Different forms of PBd may be used as the B block in the present barrier. For example, the cis-1,4 form of PBd has a T ranging from about -105°C to about -85°C. g Alternatively, for example, the cis-1,2 form of PBd can be expected to have a T in the range of about -25°C to about 0°C. gAlternatively, for example, the trans-1,4 form of PBd can be expected to have a T in the range of about -95°C to about -5°C. g In yet another example, the T may be expected to be sufficiently low for use in nanopore sequencing. g The hydrophobic B block has a T in the range of about -75°C to about -45°C. g In yet another example, the nanopore sequencing apparatus may comprise or consist essentially of polymyrcene (PMyr), which may be expected to have a T sufficiently low for use in nanopore sequencing. g The hydrophobic B block having the formula (I) may comprise or consist essentially of polyisoprene (PIP). Different forms of PIP may be used as the B block in the barrier of the present invention. For example, the cis-1,4 form of PIP has a T ranging from about -85°C to about -55°C. g Alternatively, for example, the trans-1,4 form of PIP can be expected to have a T in the range of about -75°C to about -45°C. g It can be expected that the

[0124] Hydrophobic B blocks with fully saturated carbon backbones (e.g., PIB) may also be expected to enhance the chemical stability of block copolymer membranes. Additionally or alternatively, branched structures in hydrophobic B blocks such as PIB may be expected to induce chain entanglement, which may be expected to enhance the stability of block copolymer membranes. This allows the use of smaller hydrophobic blocks, ameliorating the hydrophobic mismatch penalty for inserted nanopores. Additionally or alternatively, hydrophobic B blocks with relatively low polarity may be expected to be better electrical insulators and thus improve the electrical performance of devices for nanopore sequencing (e.g., those described with reference to Figures 4-7 or 17).

[0125] In some examples of AB copolymers shown below with PBd as the B block and PEO as the A block, R is a functional group selected from the group consisting of carboxylic acid, carboxyl, methyl, hydroxyl, primary amine, secondary amine, tertiary amine, biotin, thiol, azide, propargyl, allyl, acrylate, zwitterionic, sulfate, sulfonate, alkyl, aryl, orthogonal functional groups, and hydrogen. m=about 2 to about 100 and n=about 2 to about 100.

[0126] [ka]

[0127] In some non-limiting examples, R=OH, n=about 8 to about 50, and m=about 1 to about 20. In some non-limiting examples, R=OH, n=about 10 to about 15, and m=about 5 to about 15.

[0128] In some examples of ABA copolymers shown below, including one or more PIB blocks as B blocks and PEO as A blocks, R1 and R2 are independently a moiety selected from the group consisting of carboxylic acid, carboxyl, methyl, hydroxyl, primary amine, secondary amine, tertiary amine, biotin, thiol, azide, propargyl, allyl, acrylate, zwitterionic, sulfate, sulfonate, alkyl, aryl, orthofunctional, and hydrogen. V is an optional group corresponding to a bis-functional 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 positions, m=about 2 to about 100, and n=about 2 to about 100. V may optionally be flanked by functional groups selected from the group consisting of carboxylic acid, carboxyl, methyl, hydroxyl, primary amine, secondary amine, tertiary amine, biotin, thiol, azide, propargyl, allyl, acrylate, zwitterionic, sulfate, sulfonate, alkyl, aryl, orthogonal functional groups, and hydrogen. When V is absent, only one PIB block is present and n is from about 2 to about 100. L1 and L2 are linkers that may independently include at least one moiety selected from the group consisting of amide, thioether (sulfide), succinic acid, maleic acid, methylene, ether, and the product of a click reaction.

[0129] [ka]

[0130] Some non-limiting examples of the above structures are n=about 2 to about 50, and m=about 1 to about 50, R1=R2=COOH, V=tert-butylbenzene, and L1=L2=ethylsulfide. Other non-limiting examples are n=about 5 to about 20, m=about 2 to about 15, R1=R2=COOH, V=tert-butylbenzene, and L1=L2=ethylsulfide. Other non-limiting examples are n=about 13 to about 19, m=about 2 to about 5, R1=R2=COOH, V=tert-butylbenzene, and L1=L2=ethylsulfide. Other non-limiting examples are n=about 7 to about 13, m=about 7 to about 13, R1=R2=COOH, V=tert-butylbenzene, and L1=L2=ethylsulfide. In particular, in one non-limiting example (the structure of which is shown below), n=16, m=3, R1=R2=COOH, V=tert-butylbenzene, and L1=L2=ethylsulfide. In another non-limiting example (the structure of which is shown below), n=10, m=10, and R1=R2=COOH, V=tert-butylbenzene, and L1=L2=ethylsulfide. In another non-limiting example (the structure of which is shown below), n=16, m=8, R1=R2=CH3, V=tert-butylbenzene, and L1=L2=ethylsulfide.

[0131] [ka]

[0132] In some examples, a multifunctional precursor is provided, and further in the above example, V can be used as a precursor to synthesize the corresponding bifunctional initiator. For example, the multifunctional 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 bifunctional initiators allows cationic polymerization on both sides of the initiator to produce a bifunctional PIB, such as allyl-PIB-allyl, which can then be attached to a hydrophilic A block to produce an ABA block copolymer containing PIB as the B block. It should be understood that although the difunctional initiator may be located between the first PIB polymer and the second PIB polymer, the first PIB polymer and the difunctional initiator (V) together may be considered to form, for example, the B block of an ABA triblock copolymer.

[0133] In another non-limiting example, the ABA triblock copolymer is

[0134] [ka] wherein m=about 2 to about 100, n=about 2 to about 100, and p=about 2 to about 100; and R1 and R2 are independently 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 some non-limiting examples, m=about 2 to about 30, n=about 25 to about 45, p=about 2 to about 30, and R1 and R2 are independently 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 some non-limiting examples, m=about 2 to about 15, n=about 30 to about 40, p=about 2 to about 15, and R1 and R2 are independently 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 some non-limiting examples, m=about 7 to about 11, n=about 35 to about 40, p=about 7 to about 11, and R1 and R2 are independently a functional group selected from the group consisting of a carboxyl group, a methyl group, a hydroxyl group, a primary amine, a secondary amine, a tertiary amine, biotin, a thiol, an azide, a propargyl group, an allyl group, an acrylate group, a zwitterionic group, a sulfate, a sulfonate, an alkyl group, an aryl group, an orthogonal functional group, and hydrogen.In some non-limiting examples, m=about 2 to about 5, n=about 30 to about 37, p=about 2 to about 5, and R1 and R2 are independently 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.

[0135] In particular, in one non-limiting example, m=3, n=34, p=3, and R1=R2=COOH, as shown below. In another non-limiting example, m=9, n=37, p=9, and R1=R2=COOH, as shown below.

[0136] [ka]

[0137] In some examples of AB copolymers shown below, including a PIB block as the B block and a PEO as the A block, R is a moiety selected from the group consisting of carboxylic acid, carboxyl, methyl, hydroxyl, primary amine, secondary amine, tertiary amine, biotin, thiol, azide, propargyl, allyl, acrylate, zwitterionic, sulfate, sulfonate, alkyl, aryl, orthogonal functional groups, and hydrogen; m=about 2 to about 100; n=about 2 to about 100; and L is a linker selected from the group consisting of amide, thioether (sulfide), succinic acid, maleic acid, methylene, ether, or the product of a click reaction.

[0138] [ka]

[0139] In particular, in one non-limiting example shown below, 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 carboxyl, 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 carboxyl, and L is ethyl sulfide.

[0140] [ka]

[0141] It will be appreciated that a nanopore device as described with reference to FIG. 1 may be fabricated using any suitable barrier, including, but not limited to, those described with reference to FIGS. 2A-2C. Additionally, the barrier may be fabricated using any suitable copolymer, such as, but not limited to, those described with reference to FIGS. 3A-3C. For example, the device 100 described with reference to FIG. 1 may be fabricated using operations including forming a barrier in a fluidic well and inserting a nanopore into the barrier. The barrier may be suspended using a barrier support 1400, as described with reference to FIGS. 14A-14B, 15, and 16. Forming the barrier may include "painting" as known in the art. Known techniques for painting a barrier suspended by a barrier support include brush painting (manual), mechanical painting (e.g., using a stir bar), and bubble painting (e.g., using a flow through the device). Known techniques for inserting a nanopore into a suspended barrier include electroporation, pipette pump cycles, and surfactant-assisted pore insertion. Tools are commercially available for forming suspended barriers using synthetic polymers and inserting nanopores into the suspended barriers, such as the Orbit 16 TC platform available from Nanion Technologies Inc. (California, USA).

[0142] In some examples, the barrier may 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 barrier has an open pore current of 95 pA or more, or 100 pA or more at 100 mV. In some examples, the barrier 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 barrier 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 barrier has a signal to noise ratio of 40 or more, 50 or more, 60 or more, or 70 or more. In some examples, the barrier has a membrane coating yield of 90% or more, or 95% or more.

[0143] In some examples, the barrier has a pore insertion voltage of about 300 mV to about 1100 mV. In some examples, the barrier has a single pore percentage after insertion of 85% or more, 90% or more, or 95% or more. In some examples, the barrier has a single pore remaining rate of 90% or more, or 95% or more. In some examples, the barrier has a single pore current standard deviation of 2 pA or less, 1 pA or less, or 0.5 pA or less.

[0144] In some examples, a waveform consisting of a train of positive voltage micropulses spaced by 100 ms negative voltage periods at -100 mV is applied to the barrier. The positive voltage pulse train has a total of 20 pulses with a duration of 10 μs. The intervals between them have a set duration value of 30 ms and the voltage held at +50 mV. During the first cycle, the waveform may be applied continuously for 5 minutes, with the pulse magnitude maintained at +700 mV. In further applied cycles (again applied for 5 minutes each), the pulse strength is increased by 100 mV from +700 mV to +1200 mV for a total of 6 cycles. In some examples, the membrane survival under such a waveform is 60% or more, 80% or more, 90% or more, or 95% or more. In some examples, the voltage at 50% membrane survival is 1000 mV or more, or 1200 mV or more. In some instances, the voltage at 50% membrane and single pore remaining is 900 mV or greater, or 1000 mV or greater.

[0145] It will be further understood that the present barriers may be used in any suitable device or application. For example, FIG. 4 shows a schematic cross-sectional view of an exemplary use of the composition and device of FIG. 1. The device 100 shown in FIG. 4 may be configured to include a fluid well 100′, a barrier 101 (i.e., the barrier 101 may optionally be suspended using a barrier support and may include any AB, ABA, or BAB copolymer provided herein), which may have a configuration as described with reference to FIGS. 2A-2C, 14A-14B, 15, and / or 16, a first fluid 120 and a second fluid 120′, and a nanopore 110 in a manner as described with reference to FIG. 1. In the non-limiting example shown in FIG. 4, the second fluid 120′ may optionally include a plurality of each of the nucleotides 121, 122, 123, 124, e.g., G, T, A, and C, respectively. Each of the nucleotides 121, 122, 123, 124 in the second fluid 120' may be optionally bound to a respective label 131, 132, 133, 134 bound to the nucleotide via an extension (particularly an unlabeled extension). Optionally, the device 100 may further comprise a polymerase 105. As shown in FIG. 4, the polymerase 105 may be in a second composition of the second fluid 120'. Optionally, the polymerase 105 may be bound to the nanopore 110 or the barrier 101, for example, via a suitable extension (not specifically shown). The device 100 may optionally further comprise a first polynucleotide 140 and a second polynucleotide 150, as shown in FIG. 4. The polymerase 105 may be for sequentially adding multiple nucleotides to the first polynucleotide 140 using the sequence of the second polynucleotide 150. For example, at a particular point in time shown in Figure 4, polymerase 105 incorporates nucleotide 122 (T) into first polynucleotide 140, which hybridizes to second polynucleotide 150 to form a duplex. At other points in time (not specifically shown), polymerase 105 may use the sequence of second polynucleotide 150 to sequentially incorporate others of nucleotides 121, 122, 123, 124 into first polynucleotide 140.

[0146] The circuit 180 shown in Figure 4 can be configured to detect a change in the electrical properties of the opening in response to a polymerase sequentially adding multiple nucleotides to the first polynucleotide 140 using the sequence of the second polynucleotide 150. In the non-limiting example shown in Figure 4, the nanopore 110 can be attached to a permanent tether 410, which can include a head region 411, a tail region 412, an extension 413, a reporter region 414 (e.g., an abasic nucleotide), and a portion 415. The head region 411 of the tether 410 is attached to the nanopore 110 via any suitable chemical bond, protein-protein interaction, or any other suitable attachment that is typically irreversible. Head region 411 may be attached to any suitable portion of nanopore 110 that positions reporter region 414 within opening 413 and positions portion 415 in sufficient proximity to polymerase 105 to interact with labels 131, 132, 133, 134, respectively, of nucleotides 121, 122, 123, 124 acted upon by polymerase 105. Portion 415 may interact with labels 131, 132, 133, 134, respectively, to displace reporter region 414 within opening 113, thereby altering the rate at which salt 160 moves through opening 113, thereby detectably altering the conductivity of opening 113 as detected by circuitry 180. For further details regarding the use of permanent tethers coupled to nanopores to sequence polynucleotides, see U.S. Pat. No. 9,708,655, the entire contents of which are incorporated herein by reference.

[0147] Figure 5 shows a schematic cross-sectional view of another exemplary use of the composition and device of Figure 1. As shown in Figure 5, the device 100 may include a fluid well 100', a barrier 101 (i.e., the barrier 101 may optionally be suspended using a barrier support and may include any AB, ABA, or BAB copolymer provided herein) that may have a configuration as described with reference to Figures 2A-2C, 14A-14B, 15, and / or 16, a first fluid 120 and a second fluid 120', a nanopore 110, and a first polynucleotide 140 and a second polynucleotide 150, all of which may be similarly configured as described with reference to Figure 4. However, in the non-limiting example shown in Figure 5, the nucleotides 121, 122, 123, 124 do not necessarily need to be bound to their respective labels. The polymerase 105 may be coupled to the nanopore 110 and may be attached to a permanent tether 510, which may include a head region 511, a tail region 512, an extender 513, and a reporter region 514 (e.g., an abasic nucleotide). The head region 511 of the tether 510 is coupled to the polymerase 105 via any suitable chemical bond, protein-protein interaction, or any other suitable attachment that is typically irreversible. The head region 511 may be attached to any suitable portion of the polymerase 105 that positions the reporter region 514 within the opening 113. When the polymerase 105 interacts with a nucleotide 121, 122, 123, 124, such interaction may cause the polymerase 105 to undergo a conformational change. Such a conformational change can move reporter region 514 within opening 113, thereby altering the rate at which salt 160 moves through opening 113, thereby detectably altering the conductivity of opening 113 in a manner that is detected by circuitry 180. For further details regarding the use of permanent tethers attached to polymerases to sequence polynucleotides, see U.S. Patent No. 9,708,655, the entire contents of which are incorporated herein by reference.

[0148] Figure 6 shows a schematic cross-sectional view of another exemplary use of the composition and device of Figure 1. As shown in Figure 6, device 100 may include a fluid well 100', a barrier 101 (i.e., barrier 101 may optionally be suspended using a barrier support and may include any AB, ABA, or BAB copolymer provided herein) that may have a configuration as described with reference to Figures 2A-2C, 14A-14B, 15, and / or 16, a first fluid 120 and a second fluid 120', and a nanopore 110, all of which may be similarly configured as described with reference to Figure 4. However, in the non-limiting example shown in Figure 6, polynucleotide 150 moves through nanopore 110 under an applied force, e.g., a bias voltage that circuit 180 applies between electrodes 102 and 103. As bases in polynucleotide 150 pass through nanopore 110, such bases may alter the rate at which salt 160 translocates through opening 113, thereby detectably altering the conductivity of opening 113 in a manner that is detected by circuitry 180. For further details regarding the use of nanopores to sequence polynucleotides translocated therethrough, see U.S. Patent No. 5,795,782, the entire contents of which are incorporated herein by reference.

[0149] FIG. 7 shows a schematic cross-sectional view of another exemplary use of the composition and device of FIG. 1. As shown in FIG. 7, device 100 may include a fluid well 100′, a barrier 101 (i.e., barrier 101 may optionally be suspended using a barrier support and may include any AB, ABA, or BAB copolymer provided herein) that may have a configuration as described with reference to FIGS. 2A-2C, 14A-14B, 15, and / or 16, a first fluid 120 and a second fluid 120′, and a nanopore 110, all of which may be similarly configured as described with reference to FIG. 4. In the non-limiting example shown in FIG. 7, an altering polymer 750 moves through nanopore 110 under an applied force, e.g., a bias voltage that circuit 180 applies between electrodes 102 and 103. As used herein, “altering polymer” is intended to mean an elongated strand of labels having a sequence that corresponds to a sequence of nucleotides in a polynucleotide. In the example shown in FIG. 7, surrogate polymer 750 includes labels 751 attached to each other via linker 752. XPANDOMERS™ is a particular type of surrogate 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™, an engineered polymerase uses the sequence of a target polynucleotide to polymerize xNTPs that contain nucleobases attached to labels via linkers. The polymerized nucleotides are then processed to generate extended strands of labels, which are separated from each other by linkers attached between the labels and have sequences complementary to the sequence of the target polynucleotide.For example, for a description of XPANDOMERS™, linkers (tethers), labels, engineered polymerases, and methods for SBX™, see U.S. Pat. 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.

[0150] FIG. 17 shows a schematic cross-sectional view of another exemplary use of the composition and device of FIG. 1. As shown in FIG. 17, device 100 may include a fluid well 100′, a barrier 101 (i.e., barrier 101 may be optionally suspended using a barrier support and may include any AB, ABA, or BAB copolymer provided herein) that may have a configuration as described with reference to FIGS. 2A-2C, 14A-14B, 15, and / or 16, a first fluid 120 and a second fluid 120′, and a nanopore 110, all of which may be similarly configured as described with reference to FIG. 4. In the non-limiting example shown in FIG. 17, a duplex between polynucleotide 140 and polynucleotide 150 is located within nanopore 110 under an applied force (e.g., a bias voltage that circuit 180 applies between electrodes 102 and 103). The combination of bases in the double-stranded portion (here, base pair GC 121, 124 at the end of the double strand) with bases in the single-stranded portion of polynucleotide 150 (here, bases A and T 123, 122) can change the rate at which salt 160 moves through opening 113, and thus detectably change the conductivity of opening 113 as detected by circuit 180. For further details regarding the use of nanopores to sequence polynucleotides 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.

[0151] It is understood that compositions and devices as described with reference to Figures 1-7, 14A-14B, 15, 16, and 17 may be prepared in any suitable manner. Figure 8 shows a flow of operations for forming a device as shown in Figure 1. The method 800 shown in Figure 8 includes forming a barrier between a first fluid and a second fluid, the barrier suspended by a barrier support defining an opening, the barrier suspended across the opening and including one or more layers including molecules of a block copolymer (operation 810). The barrier may be formed using any suitable combination of operations provided herein or otherwise known in the art. For example, forming the barrier may include "painting" as known in the art. Known techniques for painting a barrier suspended by a barrier support include brush painting (manual), machine painting (e.g., using a stir bar), and bubble painting (e.g., using a flow through the device).

[0152] Each molecule of the block copolymer may include one or more hydrophilic blocks having approximately a length A and one or more hydrophobic blocks having approximately a length B. The one or more hydrophilic blocks may form the outer surface of the barrier, and the hydrophobic blocks may be located within the barrier. For example, the barrier may include any AB, ABA, or BAB copolymer provided herein. In some examples, the one or more hydrophobic blocks may include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). For reasons as described elsewhere herein, such polymers may be expected to produce suspended membranes of particularly useful quality for use in nanopore sequencing operations, such as those described with reference to Figures 4-7 and 17.

[0153] Method 800 also optionally includes inserting a nanopore into the barrier (operation 820). The nanopore may provide contact between the first and second fluids. The nanopore may be inserted into the barrier using operations as described elsewhere herein or otherwise known in the art. Known techniques for inserting a nanopore into a suspended barrier include electroporation, pipette pump cycles, and surfactant-assisted pore insertion. Tools are commercially available for forming suspended barriers using synthetic polymers and inserting nanopores into suspended barriers, such as the Orbit 16 TC platform available from Nanion Technologies Inc. (California, USA). It will be appreciated that operation 820 does not necessarily have to be performed after operation 810 if it is desired to use a barrier without a nanopore.

[0154] Exemplarily, the block copolymer is an AB diblock copolymer as described with reference to Figures 2B and 14A-14B. Thus, the barrier may have a thickness of approximately 2A+2B. In one non-limiting example of such a diblock copolymer, the hydrophobic block may be polybutadiene (PBd). Alternatively, the block copolymer may be an ABA triblock copolymer having two hydrophilic blocks and one hydrophobic block. Thus, the barrier may 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. In yet another alternative, the block copolymer is a BAB triblock copolymer having two hydrophobic blocks and one hydrophilic block. Thus, the barrier may have a thickness of approximately A+2B. EXAMPLES

[0155] The following examples are intended to be purely illustrative and not limiting of the invention unless specifically recited in the claims.

[0156] Example 1. The performance of PDMS-b-PEO-b-PDMS and PBd-b-PEO was evaluated in terms of ease of membrane preparation, controlled single- versus multiple-nanopore insertion, and stability against osmotic pressure.

[0157] All materials were tested on an Orbit-16 instrument from NanION, a tool that allows mechanical coating by rotating a Teflon stir bar on top of the chip cavity, as well as electrical testing of the membrane / pore constructs (membrane capacitance measurements, nanopore I / V curves).

[0158] Membrane coating and nanopore insertion The phospholipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), PEO, which is widely used to form lipid bilayers 500 -b-PBd 700 ("P5", P41716a-BdEO, commercially available from Polymer Source, Inc., Quebec, Canada), and PDMS 500 -b-PEO 420 -b-PDMS 500 ("P2", P9091-DMSEODMS, commercially available from Polymer Source, Inc.) were each diluted in octane (5 mg / mL) prior to testing by suspended membrane formation (also called membrane painting) using supports containing circular apertures as described with reference to Figures 14A-14B, 15, and 16. These three materials have similar hydrophilicity ratios, as well as a relatively low T of the hydrophobic block to facilitate pore insertion. g , and sufficiently low M w More specifically, PDMS was selected for comparison because it has a T g The T of PBd is in the range of -130℃ to -120℃. g is about -95 °C, but can rise to -12 °C depending on the isomer. Thus, in some cases, a low T g can be considered a negative temperature.

[0159] [ka]

[0160] In terms of coating quality, both the AB and BAB polymers required less material than DPhPC to form a stable film and produced less stir bar rotation at the top of the cavity.

[0161] Furthermore, the capacitance values ​​and noise levels of films made using three different compounds were measured. For example, Figure 9 shows the capacitance and noise levels of films made using DPhPC, PEO 420 -b-PBd 700 (P5) and PDMS 500 -b-PEO 500 -b-PDMS 500 9 shows plots and a table setting forth the capacitance (plot 910) and noise (plot 920) measured for films formed from (P2). As can be seen from plot 910, DPhPC exhibited the highest capacitance value (about 2.5 pF), followed by PEO. 500 -b-PBd 700 and PDMS 500 -b-PEO 420 -b-PDMS 500 shows a lower capacitance: about 2±0.7 pF and 1.5, respectively. As can be seen from plot 920, the membrane noise is similar for the three materials.

[0162] Figure 10 shows DPhPC, PEO 500 -b-PBd 700 (P5), and PDMS 500 -b-PEO 420 -b-PDMS 500(P2) Plot of normalized number of membranes remaining as a function of measured voltage for membranes. The initial number of membranes (16) was used to normalize the number of membranes remaining at different voltages. Average membrane number ± STD (n=3). The normalized number of membranes remaining at a given voltage corresponds to the membrane resistance to breakdown voltage. In this test, the voltage applied to the membrane is gradually increased to identify the voltage at which the membrane begins to break down. As can be seen from FIG. 10, membranes made from both AB and BAB polymer materials break down at much higher voltages compared to DPhPC membranes, highlighting their greater stability.

[0163] Another difference between the membranes formed using DPhPC and the AB and BAB polymer materials is how difficult it was to insert a single nanopore into the membranes. For nanopore sequencing as described with reference to Figures 4-7, it is useful to insert a single nanopore into each membrane. However, single nanopore insertion proved to be relatively difficult to achieve with DPhPC, which resulted in a mixture of membranes with single nanopores and membranes with multiple nanopores. On the other hand, PEO 500 -b-PBd 700 and PDMS 500 -b-PEO 420 -b-PDMS 500 was found to offer significantly improved control over single nanopore insertion.

[0164] Regarding nanopore properties, it was observed that MspA nanopores had slightly lower current levels when trapped in AB or BAB polymer membranes than in DPhPC membranes, which is likely driven by the higher resistance of the polymer membranes. 500 -b-PBd 700 and PDMS 500 -b-PEO 420 -b-PDMS 500 The MspA nanopore in the membrane was observed to have approximately 25% higher resistance (lower conductance) than the MspA nanopore in the DPhPC membrane. The MspA nanopore noise level was approximately 25% higher than that in the DPhPC and PDMS membranes. 500-b-PEO 420 -b-PDMS 500 is equivalent between 500 -b-PBd 700 It was observed to be slightly higher when inserted into the membrane.

[0165] To evaluate the stability of the AB polymer membrane, MspA nanopore / PEO in 1 M KCl + 50 mM HEPES buffer under AC 500 -b-PBd 700 The stability of the membrane construct was evaluated. Figure 11 shows the stability of the MspA nanopore / PEO in 1 M KCl + 50 mM HEPES buffer. 500 -b-PBd 700 A plot of membrane construct stability is shown. As shown in FIG. 11, this nanopore / membrane assembly can remain stable for over 16 hours. In comparison, MspA inserted into a DPhPC membrane under similar conditions was observed to remain for approximately 1 hour. The increase in current recorded over time was attributed to evaporation of the buffer solution since the system is an open cell.

[0166] Stability of membranes and membrane / pore constructs under osmotic pressure In some configurations, a salt imbalance setup may improve performance during nanopore sequencing, as described with reference to Figures 4 and 5. For example, a lower salt concentration in fluid 120' (also referred to as the cis side) may help maintain polymerase activity, and a higher salt concentration in fluid 120 (also referred to as the trans side) may help increase current levels. In a non-limiting example of a balanced salt condition, fluids 120' and 120 both contain 150 mM KCl and 50 mM HEPES. In a non-limiting example of an unbalanced salt condition, fluid 120' (cis) contains 50 mM KCl, 50 mM HEPES, and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP). Fluid 120 (trans) contains 300 mM KCl and 50 mM HEPES. Such an imbalance in salt concentration may generate an osmotic pressure 191, in the manner described with reference to Figure 1. PEO 500 -b-PBd 700 (P5) and PDMS 500 -b-PEO420 -b-PDMS 500 (P2) Both membranes were observed to tolerate 500mM / 1000mM salt conditions. 500 -b-PBd 700 (P5) was also observed to remain in the 250 mM / 1000 mM unbalanced salt condition, which indicates that the diblock and triblock membranes of the present invention can be used under osmotic pressure.

[0167] Example 2. The performance of different copolymers was evaluated with respect to ease of membrane preparation, controlled single vs. multiple nanopore insertion, and stability against osmotic pressure.

[0168] All materials were tested on an Orbit-16 instrument from NanION, a tool that allows mechanical coating by rotating a Teflon stir bar on top of the chip cavity, as well as electrical testing of the membrane / pore constructs (membrane capacitance measurements, nanopore I / V curves).

[0169] Membrane coating and nanopore insertion The copolymers, each listed in Table 1 below, were dissolved in an octane:butanol (95:5 by volume) solvent mixture at a concentration of 5 mg / mL prior to testing by suspended film formation (also referred to as film painting) using a support containing a circular aperture such as those described with reference to Figures 14A, 14B, 15, and 16.

[0170] [Table 1]

[0171] Characterization studies were used to extract metrics deemed relevant for nanopore sensing applications of such membranes. These metrics fall into categories such as stability (e.g., elasticity of membrane / membrane-pore system against stress tests including accelerated testing, 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 / leaky nature of membrane).

[0172] The first characterization studies performed focused on the success rate in membrane formation (membrane coating yield), membrane resistance to breakdown voltage, biological pore insertion (MspA pores), and the resulting current and noise of pores inside the block copolymer membranes. These served to evaluate the performance of PIB-PEO-based membranes against each other and against membranes formed with other polymers.

[0173] Regarding coating quality, all PIB-b-PEO block copolymers (AB1, AB2, AB3, AB4) and PEO-b-PIB-b-PEO block copolymers (ABA1, ABA2, ABA3) could be coated to form suspension films. Particularly satisfactory performance was achieved by PEO-b-PIB-b-PEO block copolymers with ABA structure. For example, FIG. 12 shows a plot describing the breakdown voltage measured for films formed using P5, ABA1, AB1, AB2, AB3, AB4, and ABA2. For the film formed using P5, it can be seen in FIG. 12 that at voltages of about 300 mV or less, the normalized number of the film that remained substantially intact ranged from about 1.0 at 0 V to about 0.95 at 300 mV, and at voltages of about 350 mV or more, the normalized number of the film decreased from about 0.9 at 350 mV to about 0.22 at 500 mV. For films formed with ABA1, it can be seen in Figure 12 that at voltages of about 300 mV or less, the normalized number for films that remained substantially intact ranged from about 1.0 at 0 V to about 0.95 at 500 mV, and at voltages of about 350 mV or more, the normalized number for films decreased from about 0.9 at 350 mV to about 0.5 at 300 mV. For films formed with ABA2, it can be seen in Figure 12 that at voltages of about 300 mV or less, the normalized number for films that remained substantially intact ranged from about 1.0 at 0 V to about 0.95 at 500 mV, and at voltages of about 350 mV or more, the normalized number for films decreased from about 0.9 at 350 mV to about 0.5 at 300 mV.

[0174] For films formed with AB1, it can be seen in Figure 12 that at voltages of about 300 mV or less, the normalized number for films 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 more, the normalized number for films decreased from about 0.16 at 350 mV to about 0.04 at 500 mV. For films formed with AB2, it can be seen in Figure 12 that at voltages of about 300 mV or less, the normalized number for films 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 more, the normalized number for films decreased from about 0.45 at 350 mV to about 0.01 at 500 mV. For films formed with AB3, it can be seen in Figure 12 that at voltages of about 300 mV or less, the normalized number for films 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 more, the normalized number for films decreased from about 0.85 at 350 mV to about 0.035 at 500 mV. For films formed with AB4, it can be seen in Figure 12 that at voltages of about 300 mV or less, the normalized number for films 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 more, the normalized number for films decreased from about 0.9 at 350 mV to about 0.65 at 500 mV.

[0175] As can be seen from the plot shown in FIG. 12, the ABA type block copolymers ABA1 and ABA2, and the AB4 type block copolymer exhibited particularly high resistance to the breakdown voltage stress test.

[0176] Another difference between the membranes is how difficult it was to insert a single nanopore into the membranes. For nanopore sequencing as described with reference to Figures 4-7 and 17, it is useful to insert a single nanopore into each membrane.

[0177] FIG. 13 shows plots of MspA nanopore / membrane construct stability in 1M KCl+50 mM HEPES buffer. For membranes formed using P5 with an MspA nanopore inserted, it can be seen in FIG. 13 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 membranes formed using ABA1 with an MspA nanopore inserted, it can be seen in FIG. 13 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 membranes formed using ABA2 with an MspA nanopore inserted, it can be seen in FIG. 13 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 with the MspA nanopore inserted, it can be seen in FIG. 13 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 with the MspA nanopore inserted, it can be seen in FIG. 13 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 with the MspA nanopore inserted, it can be seen in FIG. 13 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 with the MspA nanopore inserted, it can be seen in FIG. 13 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.

[0178] ABA2 and ABA3 membranes were identified as having particularly good performance. A notable improvement shown in ABA2 is enhanced membrane elasticity. A notable improvement shown in ABA3 is enhanced insertion and retention of a single MspA nanopore into the membrane with lower variability. Various properties of the membranes are shown in Table 2 below.

[0179] [Table 2]

[0180] Film quality was measured by film survival at a current of 450 mV across the barrier. Both ABA2 and ABA3 had 100% survival, and ABA4 had approximately 95% survival.

[0181] The current through the membrane when the MspA nanopore was inserted was also measured. The open pore current was measured at a voltage across the barrier of 100 mV. ABA2 and ABA3 had similar currents of 103 pA and 104 pA, respectively, and ABA4 had a current of 104 pA. After the MspA nanopore was inserted into the membrane, the root mean square (RMS) average of the current noise through the barrier was also measured. ABA2 and ABA3 had similar RMS current noise averages of 1.46 pA and 1.62 pA, respectively, and ABA4 had an RNS current noise of 2.13 pA. The signal-to-noise ratio (SNR) of the current through the barrier was also measured. ABA2 and ABA3 had SNRs of 71 and 64, respectively, and ABA4 had an SNR of 49.

[0182] Various yield percentages were also measured. ABA2, ABA3, and ABA4 all had membrane coating yields greater than 95%. The voltage required to insert the MspA nanopore into the membrane was also measured. ABA2 required a voltage of about 800 mV to about 1000 mV, while ABA3 required a voltage of about 350 mV to 450 mV, and ABA4 required a voltage of about 500 mV to about 850 mV. After performing the nanopore insertion step, the percentage of membranes containing MspA nanopores was about 90% for ABA2, greater than 95% for ABA3, and greater than 95% for ABA4. The percentage of single-pore membranes that survived washing was also measured. Specifically, the membranes were washed three times with 250 μL of fluid, and then the survival rate was determined. Both ABA2 and ABA3 had survival rates greater than 95%, as did ABA4.

[0183] The tightness of the open pore current diffusion of MspA nanopores in the membrane was also measured. Specifically, currents were measured at 400 mM KCl and 50 mV of current was applied to the membrane. ABA2 and ABA3 had open pore currents of 35.02 pA and 36.04 pA, respectively, and ABA4 had an open pore current of 36.18 pA. The standard deviation of the current was measured similarly. The standard deviations for ABA2 and ABA3 were 0.8 pA and 0.4 pA, respectively, and for ABA4 was 1.93 pA. The elasticity of the barrier was also measured. Membrane survival was measured after subjecting the barrier to a waveform consisting of a train of positive voltage micropulses spaced by 100 ms negative voltage periods at -100 mV. The positive voltage pulse train had a total of 20 pulses of 10 μs duration. The intervals between them had a set duration value of 30 ms and the 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 (again applied for 5 minutes each), the pulse strength was increased from +700 mV to +1200 mV in 100 mV increments for a total of 6 cycles. Both ABA2 and ABA3 had a survival rate of about 95% after application of the described waveforms, while ABA4 had a survival rate of about 65%. In the same experiment / test, the voltage at which 50% of the membrane remained after exposure to the waveform was determined. For ABA2, ABA3, 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 membranes after insertion of a single MspA pore. For ABA2 and ABA4, the voltage was determined to be approximately 1000 mV, while for ABA3, the voltage was determined to be approximately 900 mV.

[0184] Based on different metrics selected to evaluate suitability for use in polymeric membranes for nanopore sequencing applications, these results demonstrate that the performance of membranes containing PIB as the hydrophobic B block are particularly suitable for use in such applications. For example, the different metrics indicate that ABA and AB copolymers using PIB as the hydrophobic B block, especially those containing -COOH as the end group, can form membranes with properties useful for nanopore sequencing applications, including relatively high membrane formation yields, relatively high pore insertion and retention yields, relatively high stability under sequencing conditions, and relatively good electrical properties for high read quality. We believe that such properties may correlate with ease of flow cell fabrication, overall instrument, instrument / chip reliability, and high read accuracy, which may be important for the commercial manufacture and use of nanopore sequencing devices, such as those described with reference to Figures 4-7 and 17.

[0185] Particular aspects of the present subject matter are expressed in the following clauses. The invention is not limited to such clauses unless expressly recited in the claims. Clause 1. A barrier between a first fluid and a second fluid, comprising: a first layer comprising a first plurality of molecules of a diblock copolymer, a first layer, each molecule of a diblock copolymer comprising a hydrophobic block bound to a hydrophilic block; a second layer comprising a second plurality of molecules of the diblock copolymer; a hydrophilic block of a first plurality of molecules forming a first exterior surface of the barrier; a hydrophilic block of a second plurality of molecules forms a second exterior surface of the barrier; A barrier, wherein the hydrophobic blocks of the first plurality of molecules and the second plurality of molecules contact one another within the barrier. Clause 2. The barrier of clause 1, wherein the hydrophilic block is approximately a length A, the hydrophobic block is approximately a length B, the first layer and the second layer each have a thickness of approximately A+B, and the barrier has a thickness of approximately 2A+2B. Clause 3. The barrier of clause 2, wherein the length A is from about 2 repeat units to about 100 repeat units. Clause 4. The barrier of clause 2 or clause 3, wherein the length A is from about 5 repeat units to about 15 repeat units. Clause 5. The barrier of any one of clauses 2 to 4, wherein the length B is from about 2 repeat units to about 100 repeat units. Clause 6. The barrier of any one of clauses 2 to 5, wherein the length B is from about 2 repeat units to about 50 repeat units. Clause 7. The barrier of any one of clauses 2 to 6, wherein the length B is from about 10 repeat units to about 15 repeat units. Clause 8. The barrier of any one of clauses 1 to 7, wherein the hydrophilic block comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO). Clause 9. The barrier of clause 8, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 10. The barrier of any one of clauses 1-9, wherein the hydrophobic block comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Clause 11. A barrier according to any one of clauses 1 to 10, wherein each of the molecules further comprises a linker connecting the hydrophobic block to the hydrophilic block. Clause 12. The barrier of clause 11, wherein the linker comprises at least one moiety selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Clause 13. A device comprising: a fluid well for holding a first fluid and a second fluid; A barrier according to any one of clauses 1 to 12, disposed between a first fluid and a second fluid in a fluid well; a nanopore disposed within the barrier, the nanopore providing an opening that fluidly couples the first fluid to the second fluid. Clause 14. The device of clause 13, wherein the first fluid has a first composition and the second fluid has a second composition different from the first composition. Clause 15. The device of clause 14, wherein the first fluid has a first concentration of salt and the second fluid has a second concentration of salt different from the first concentration. Clause 16. A method for producing a device according to any one of clauses 13 to 15, comprising the steps of: forming a barrier within the fluid well; and inserting the nanopore into the barrier. Clause 17. The method of clause 16, wherein forming a barrier comprises painting. Clause 18. The method of clause 17, wherein the coating comprises a technique selected from the group consisting of brush coating, machine coating, and bubble coating. Clause 19. The method of any one of clauses 16 to 18, wherein inserting the nanopore comprises a technique selected from the group consisting of electroporation, pipette pump cycles, and surfactant-assisted pore insertion. Clause 20. A method for making a diblock copolymer, comprising: polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer; forming an initiator at the end of a hydrophilic polymer; and polymerizing a plurality of hydrophobic monomers using an initiator to form a hydrophobic polymer attached to a hydrophilic polymer. Clause 21. A method for making a diblock copolymer, comprising: polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer; forming an initiator at the end of a hydrophobic polymer; and polymerizing a plurality of hydrophilic monomers using an initiator to form a hydrophilic polymer attached to the hydrophobic polymer. Clause 22. The method of clause 20 or clause 21, wherein the initiator comprises bromine, iodine, or chlorine. Clause 23. The method of clause 20 or clause 21, wherein the initiator comprises a chain transfer agent. Clause 24. The method of clause 23, wherein the chain transfer agent comprises S-1-dodecyl-S'-(α,α'-dimethyl-α''-acetic acid) trithiocarbonate (DDMAT). Clause 25. The method of any one of clauses 20 to 24, wherein the hydrophilic polymer comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, a zwitterionic polymer, a polypeptide, and poly(ethylene oxide) (PEO). Clause 26. The method of clause 25, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 27. The method of any one of clauses 20-26, wherein the hydrophobic polymer comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Clause 28. The method of any one of clauses 20 to 27, wherein the hydrophobic polymer is attached to the hydrophilic polymer via a linker. Clause 29. The method of clause 28, wherein the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Clause 30. A method for making a diblock copolymer, comprising: polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer; polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer; and attaching a hydrophilic polymer to a hydrophobic polymer. Clause 31. The method of clause 30, wherein an end of the hydrophilic polymer comprises a first reactive moiety and an end of the hydrophobic polymer comprises a second reactive moiety that reacts with the first reactive moiety to bond the hydrophilic polymer to the hydrophobic polymer. Clause 32. The method of clause 31, wherein one of the first reactive moiety and the second reactive moiety comprises an azide and the other of the first reactive moiety and the second reactive moiety comprises an alkyne. Clause 33. The method of clause 31, wherein one of the first reactive moiety and the second reactive moiety comprises a thiol and the other of the first reactive moiety and the second reactive moiety comprises an alkene. Clause 34. The method of clause 31, wherein one of the first reactive moiety and the second reactive moiety comprises a thiol and the other of the first reactive moiety and the second reactive moiety comprises an alkyne. Clause 35. The method of clause 31, wherein one of the first reactive moiety and the second reactive moiety comprises an amine and the other of the first reactive moiety and the second reactive moiety comprises N-hydroxysuccinimide. Clause 36. The method of any one of clauses 30 to 35, wherein the hydrophilic polymer comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO). Clause 37. The method of clause 36, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 38. The method of any one of clauses 30 to 37, wherein the hydrophobic polymer comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Clause 39. The method of any one of clauses 30 to 38, wherein the hydrophobic polymer is attached to the hydrophilic polymer via a linker. Clause 40. The method of clause 39, wherein the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Clause 41. A barrier between a first fluid and a second fluid, comprising: a first layer comprising a first plurality of molecules of a triblock copolymer, a first layer, each molecule of a triblock copolymer including a first hydrophobic block and a second hydrophobic block, and a hydrophilic block disposed between the first hydrophobic block and the second hydrophobic block; a second layer comprising a second plurality of molecules of the triblock copolymer; a hydrophilic block of a first plurality of molecules forming a first exterior surface of the barrier; a hydrophilic block of a second plurality of molecules forms a second exterior surface of the barrier; A barrier, wherein the hydrophobic blocks of the first plurality of molecules and the second plurality of molecules contact one another within the barrier. Clause 42. A barrier as described in clause 41, wherein the hydrophilic blocks are approximately of length A, each hydrophobic block is approximately of length B, the first layer and the second layer each have a thickness of approximately A / 2+B, and the barrier has a thickness of approximately A+2B. Clause 43. The barrier of clause 42, wherein the length A is from about 2 repeat units to about 100 repeat units. Clause 44. A barrier according to clause 42 or clause 43, wherein the length A is from about 2 repeat units to about 50 repeat units. Clause 45. A barrier according to any one of clauses 42 to 44, wherein the length A is from about 5 repeat units to about 20 repeat units. Clause 46. A barrier according to any one of clauses 42 to 45, wherein the length A is from about 13 repeat units to about 19 repeat units. Clause 47. A barrier according to any one of clauses 42 to 45, wherein the length A is from about 7 repeat units to about 13 repeat units. Clause 48. A barrier according to clause 42 or clause 43, wherein the length A is from about 2 repeat units to about 30 repeat units. Clause 49. A barrier according to any one of clauses 42-43 or clause 48, wherein the length A is from about 2 repeat units to about 15 repeat units. Clause 50. A barrier according to any one of clauses 42-43 or clauses 48-49, wherein the length A is from about 7 repeat units to about 11 repeat units. Clause 51. A barrier according to any one of clauses 42-43 or clauses 48-49, wherein the length A is from about 2 repeat units to about 5 repeat units. Clause 52. A barrier according to any one of clauses 42 to 51, wherein the length B is from about 2 repeat units to about 100 repeat units. Clause 53. A barrier according to any one of clauses 42 to 52, wherein the length B is from about 2 repeat units to about 50 repeat units. Clause 54. A barrier according to any one of clauses 42 to 53, wherein the length B is from about 2 repeat units to about 15 repeat units. Clause 55. A barrier according to any one of clauses 42 to 54, wherein the length B is from about 2 repeat units to about 5 repeat units. Clause 56. A barrier according to any one of clauses 42 to 54, wherein the length B is from about 7 repeat units to about 13 repeat units. Clause 57. A barrier according to any one of clauses 42 to 52, wherein the length B is from about 25 repeat units to about 45 repeat units. Clause 58. A barrier according to any one of clauses 42 to 52 or clause 57, wherein the length B is from about 30 repeat units to about 40 repeat units. Clause 59. A barrier according to any one of clauses 42 to 52 or clauses 57 to 58, wherein the length B is from about 35 repeat units to about 40 repeat units. Clause 60. A barrier according to any one of clauses 42-52 or clauses 57-58, wherein the length B is from about 30 repeat units to about 37 repeat units. Clause 61. A barrier according to any one of clauses 41 to 60, wherein the hydrophilic block comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO). Clause 62. The barrier of clause 61, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 63. A barrier according to any one of clauses 41 to 62, wherein each of the hydrophobic blocks comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Clause 64. A barrier according to any one of clauses 41 to 63, wherein each molecule of the first plurality of molecules and the second plurality of molecules further comprises a linker connecting each hydrophobic block to a hydrophilic block. Clause 65. The barrier according to clause 64, wherein the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Clause 66. A device comprising: a fluid well for holding a first fluid and a second fluid; A barrier according to any one of clauses 41-65, disposed between a first fluid and a second fluid in a fluid well; a nanopore disposed within the barrier, the nanopore providing an opening that fluidly couples the first fluid to the second fluid. Clause 67. The device of clause 66, wherein the first fluid has a first composition and the second fluid has a second composition different from the first composition. Clause 68. The device of clause 67, wherein the first fluid has a first concentration of salt and the second fluid has a second concentration of salt different from the first concentration. Clause 69. A method for producing a device according to any one of clauses 66 to 68, comprising the steps of: forming a barrier within the fluid well; and inserting the nanopore into the barrier. Clause 70. The method of clause 69, wherein the barrier comprises a coating. Clause 71. The method of clause 70, wherein the coating comprises a technique selected from the group consisting of brush coating, machine coating, and bubble coating. Clause 72. The method of any one of clauses 69 to 71, wherein inserting the nanopore comprises a technique selected from the group consisting of electroporation, pipette pump cycles, and surfactant-assisted pore selection. Clause 73. A method for making a triblock copolymer, comprising: polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer; forming an initiator at each end of a hydrophilic polymer; and polymerizing a plurality of hydrophobic monomers using an initiator to form hydrophobic polymers attached to each end of a hydrophilic polymer. Clause 74. The method of clause 73, wherein the initiator comprises bromine, iodine, or chlorine, respectively. Clause 75. The method of clause 73 or clause 74, wherein the initiator comprises a chain transfer agent, respectively. Clause 76. The method of clause 75, wherein the chain transfer agent comprises S-1-dodecyl-S'-(α,α'-dimethyl-α''-acetic acid) trithiocarbonate (DDMAT). Clause 77. The method of any one of clauses 73 to 76, wherein the hydrophilic polymer comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO). Clause 78. The method of clause 77, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 79. The method of any one of clauses 73 to 78, wherein each of the hydrophobic polymers comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Clause 80. The method of any one of clauses 73 to 79, wherein each of the hydrophobic polymers is attached to the hydrophilic polymer via a linker. Clause 81. The method of clause 80, wherein the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Clause 82. A method for making a triblock copolymer, comprising: polymerizing a plurality of hydrophilic monomers to form a hydrophilic polymer having termini; polymerizing a plurality of hydrophobic monomers to form a first hydrophobic polymer and a second hydrophobic polymer; and attaching a first hydrophobic polymer and a second hydrophobic polymer to respective ends of a hydrophilic polymer. Clause 83. The method of clause 82, wherein each end of the first hydrophobic polymer and the second hydrophobic polymer comprises a first reactive moiety, and each end of the hydrophilic polymer comprises a second reactive moiety that reacts with the first reactive moiety to bond the first hydrophobic polymer and the second hydrophobic polymer to the hydrophilic polymer. Clause 84. The method of clause 83, wherein one of the first reactive moiety and the second reactive moiety comprises an azide and the other of the first reactive moiety and the second reactive moiety comprises an alkyne. Clause 85. The method of clause 83, wherein one of the first reactive moiety and the second reactive moiety comprises a thiol and the other of the first reactive moiety and the second reactive moiety comprises an alkene. Clause 86. The method of clause 83, wherein one of the first reactive moiety and the second reactive moiety comprises a thiol and the other of the first reactive moiety and the second reactive moiety comprises an alkyne. Clause 87. The method of clause 83, wherein one of the first reactive moiety and the second reactive moiety comprises an amine and the other of the first reactive moiety and the second reactive moiety comprises N-hydroxysuccinimide. Clause 88. The method of any one of clauses 82 to 87, wherein the hydrophilic polymer comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO). Clause 89. The method of clause 88, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 90. The method of any one of clauses 82-89, wherein the first hydrophobic polymer and the second hydrophobic polymer each comprise a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene). Clause 91. The method of any one of clauses 82 to 90, wherein the first hydrophobic polymer and the second hydrophobic polymer are each attached to the hydrophilic polymer via a linker. Clause 92. The method of clause 91, wherein the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Clause 93. A barrier between a first fluid and a second fluid, comprising: a layer comprising a plurality of molecules of a triblock copolymer; each molecule of the triblock copolymer comprises a first hydrophilic block, a second hydrophilic block, and a hydrophobic block disposed between the first hydrophilic block and the second hydrophilic block; a first hydrophilic block of the first plurality of molecules forms a first exterior surface of the barrier; a second hydrophilic block of the second plurality of molecules forms a second exterior surface of the barrier; A barrier in which hydrophobic blocks of multiple molecules are in contact with each other within the barrier. Clause 94. A barrier as described in clause 93, wherein each hydrophilic block is approximately of length A, each hydrophobic block is approximately of length B, the layer has a thickness of approximately 2A+B, and the barrier has a thickness of approximately 2A+B. Clause 95. The barrier according to clause 94, wherein the length A is from about 2 repeat units to about 100 repeat units. Clause 96. A barrier according to clause 94 or clause 95, wherein the length A is from about 2 repeat units to about 50 repeat units. Clause 97. A barrier according to any one of clauses 94 to 96, wherein the length A is from about 5 repeat units to about 20 repeat units. Clause 98. A barrier according to any one of clauses 94 to 97, wherein the length A is from about 13 repeat units to about 19 repeat units. Clause 99. The barrier according to any one of clauses 94 to 97, wherein the length A is from about 7 repeat units to about 13 repeat units. Clause 100. The barrier of paragraph 94 or 95, wherein the length A is from about 2 repeat units to about 30 repeat units. Clause 101. The barrier according to any one of clauses 94-95 or clause 100, wherein the length A is from about 2 repeat units to about 15 repeat units. Clause 102. A barrier according to any one of clauses 94-95 or clauses 100-101, wherein the length A is from about 7 repeat units to about 11 repeat units. Clause 103. A barrier according to any one of clauses 94-95 or clauses 100-101, wherein the length A is from about 2 repeat units to about 5 repeat units. Clause 104. A barrier according to any one of clauses 94 to 103, wherein the length B is from about 2 repeat units to about 100 repeat units. Clause 105. A barrier according to any one of clauses 94 to 104, wherein the length B is from about 2 repeat units to about 50 repeat units. Clause 106. A barrier according to any one of clauses 94 to 105, wherein the length B is from about 2 repeat units to about 15 repeat units. Clause 107. A barrier according to any one of clauses 94 to 106, wherein the length B is from about 2 repeat units to about 5 repeat units. Clause 108. A barrier according to any one of clauses 94 to 106, wherein the length B is from about 7 repeat units to about 13 repeat units. Clause 109. A barrier according to any one of clauses 94 to 104, wherein the length B is from about 25 repeat units to about 45 repeat units. Clause 110. A barrier according to any one of clauses 94 to 104 or clause 109, wherein the length B is from about 30 repeat units to about 40 repeat units. Clause 111. A barrier according to any one of clauses 94-104 or clauses 109-110, wherein the length B is from about 35 repeat units to about 40 repeat units. Clause 112. A barrier according to any one of clauses 94-104 or clauses 109-110, wherein the length B is from about 30 repeat units to about 37 repeat units. Clause 113. A barrier according to any one of clauses 93 to 112, wherein each hydrophilic block comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO). Clause 114. The barrier of clause 113, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 115. A barrier according to any one of clauses 93 to 114, wherein the hydrophobic block comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Clause 116. A barrier according to any one of clauses 93 to 115, wherein each molecule of the plurality of molecules further comprises a linker connecting the hydrophobic block to each of the hydrophilic blocks. Clause 117. The barrier according to clause 116, wherein the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Article 118. A device, a fluid well for holding a first fluid and a second fluid; A barrier according to any one of clauses 93-117 disposed between a first fluid and a second fluid in a fluid well; a nanopore disposed within the barrier, the nanopore providing an opening that fluidly couples the first fluid to the second fluid. Clause 119. The device of clause 118, wherein the first fluid has a first composition and the second fluid has a second composition different from the first composition. Clause 120. The device of clause 119, wherein the first fluid has a first concentration of salt and the second fluid has a second concentration of salt different from the first concentration. Clause 121. A method for making a device according to clause 118, comprising the steps of: forming a barrier within the fluid well; and inserting the nanopore into the barrier. Clause 122. The method of clause 121, wherein forming a barrier comprises painting. Clause 123. The method of clause 122, wherein the coating comprises a technique selected from the group consisting of brush coating, machine coating, and bubble coating. Clause 124. The method of any one of clauses 121 to 123, wherein inserting the nanopore comprises a technique selected from the group consisting of electroporation, pipette pump cycles, and surfactant-assisted pore selection. Clause 125. A method for making a triblock copolymer, comprising: polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer; forming an initiator at each end of a hydrophobic polymer; and polymerizing a plurality of hydrophobic monomers using an initiator to form hydrophobic polymers attached to each end of a hydrophilic polymer. Clause 126. The method of clause 125, wherein the initiator comprises bromine, iodine, or chlorine, respectively. Clause 127. The method of clause 125 or clause 126, wherein the initiator comprises a chain transfer agent, respectively. Clause 128. The method of clause 127, wherein the chain transfer agent comprises S-1-dodecyl-S'-(α,α'-dimethyl-α''-acetic acid) trithiocarbonate (DDMAT). Clause 129. The method of any one of clauses 125 to 128, wherein each of the hydrophilic polymers comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, a zwitterionic polymer, a polypeptide, and poly(ethylene oxide) (PEO). Clause 130. The method of clause 129, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 131. The method of any one of clauses 125-130, wherein the hydrophobic polymer comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Clause 132. The method according to any one of clauses 125 to 131, wherein the hydrophobic polymer is attached to the hydrophilic polymer via a linker. Clause 133. The method according to clause 132, wherein the linker is selected from the group consisting of amide, thioether (sulfide), succinic acid group, maleic acid group, methylene, ether, and products of a click reaction. Clause 134. A method for making a triblock copolymer, comprising: polymerizing a plurality of hydrophobic monomers to form a hydrophobic polymer having termini; polymerizing a plurality of hydrophilic monomers to form a first hydrophilic polymer and a second hydrophilic polymer; and attaching a first hydrophilic polymer and a second hydrophilic polymer to respective ends of the hydrophobic polymer. Clause 135. The method of clause 134, wherein each end of the first hydrophilic polymer and the second hydrophilic polymer comprises a first reactive moiety and each end of the hydrophobic polymer comprises a second reactive moiety that reacts with the first reactive moiety to bond the first hydrophilic polymer and the second hydrophilic polymer to the hydrophobic polymer. Clause 136. The method of clause 135, wherein one of the first reactive moiety and the second reactive moiety comprises an azide and the other of the first reactive moiety and the second reactive moiety comprises an alkyne. Clause 137. The method of clause 135, wherein one of the first reactive moiety and the second reactive moiety comprises a thiol and the other of the first reactive moiety and the second reactive moiety comprises an alkene. Clause 138. The method of clause 135, wherein one of the first reactive moiety and the second reactive moiety comprises a thiol and the other of the first reactive moiety and the second reactive moiety comprises an alkyne. Clause 139. The method of clause 135, wherein one of the first reactive moiety and the second reactive moiety comprises an amine and the other of the first reactive moiety and the second reactive moiety comprises N-hydroxysuccinimide. Clause 140. The method of any one of clauses 134 to 139, wherein the first hydrophilic polymer and the second hydrophilic polymer each comprise a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, a zwitterionic polymer, a polypeptide, and poly(ethylene oxide) (PEO). Clause 141. The method of clause 140, wherein the polyacrylamide is selected from the group consisting of poly(N-isopropylacrylamide) (PNIPAM), charged polyacrylamide, and phosphate-functionalized polyacrylamide. Clause 142. The method of any one of clauses 134 to 141, wherein the hydrophobic polymer comprises a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene). Clause 143. The method of any one of clauses 134 to 142, wherein the first hydrophilic polymer and the second hydrophilic polymer are attached to the hydrophobic polymer via a linker. Clause 144. The method of clause 143, wherein the linker is selected from the group consisting of amides, thioethers (sulfides), succinic acid groups, maleic acid groups, methylenes, ethers, and products of click reactions.

[0186] Further comments While various illustrative examples have been described above, it will be apparent to one skilled in the art that various changes and modifications can be made herein without departing from the present invention. It is intended that the appended claims cover all such changes and modifications that fall within the true spirit and scope of the present invention.

[0187] 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 together in any suitable combination with any of the features of the other aspects described herein, in order to achieve the benefits described herein.

Claims

1. A barrier for use in determining the arrangement of nanopores, wherein the barrier is located between a first fluid and a second fluid, the barrier is suspended by a barrier support defining an opening, and comprises one or more layers of a. and nanopores of b. as follows: a. One or more layers suspended across the opening and containing molecules of a block copolymer, Each molecule of the block copolymer comprises one or more hydrophilic blocks having approximately length A and hydrophobic blocks having approximately length B. The one or more hydrophilic blocks form the outer surface of the barrier, and the hydrophobic blocks are located within the barrier. The hydrophobic block comprises poly(isobutylene) (PIB), The block copolymer is as follows: i and ii: i. It is a diblock copolymer; and ii. A triblock copolymer having two hydrophilic blocks and one hydrophobic block; one or more layers selected from, and b. A nanopore disposed within the barrier and providing contact between the first fluid and the second fluid, wherein the nanopore is a polypeptide nanopore.

2. The barrier according to claim 1, wherein the block copolymer is a diblock copolymer, and the barrier has a thickness of approximately 2A + 2B.

3. The barrier is One or more layers comprising molecules of a triblock copolymer, wherein the triblock copolymer is It includes a first hydrophilic block and a second hydrophilic block, and a hydrophobic block disposed between the first hydrophilic block and the second hydrophilic block, The first hydrophilic block and the second hydrophilic block form the outer surface of the barrier, and the hydrophobic block is located within the barrier, comprising one or more layers. The barrier according to claim 1, comprising:

4. The barrier according to claim 3, wherein the first hydrophilic block and the second hydrophilic block have a length of approximately A, the hydrophobic block has a length of approximately B, at least a portion of one or more layers has a thickness of approximately 2A + B, and the barrier has a thickness of approximately 2A + B.

5. i. The length A is approximately 2 repeat units to approximately 100 repeat units, and / or ii. The barrier according to claim 4, wherein the length B is approximately 2 repeat units to approximately 100 repeat units.

6. The barrier according to claim 3, wherein the first hydrophilic block and the second hydrophilic block each contain a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO).

7. At least one of the first hydrophilic block and the second hydrophilic block includes a portion selected from the group consisting of carboxylic acids, carboxyl groups, methyl groups, hydroxyl groups, primary amines, secondary amines, tertiary amines, biotin, thiols, azides, propargyl groups, allyl groups, acrylate groups, zwitterionic groups, sulfates, sulfonates, alkyl groups, aryl groups, orthogonal functional groups, and hydrogen. Preferably, the barrier according to claim 4 or 5, wherein the portion of the first hydrophilic block contains a methyl group or a carboxyl group, and the portion of the second hydrophilic block contains a methyl group or a carboxyl group.

8. The barrier comprises a first layer comprising a first plurality of molecules of the diblock copolymer, and the barrier further comprises a second layer comprising a second plurality of molecules of the diblock copolymer. The hydrophilic blocks of the first plurality of molecules form the first outer surface of the barrier. The hydrophilic blocks of the second plurality of molecules form the second outer surface of the barrier. The barrier according to claim 1, wherein the hydrophobic blocks of the first plurality of molecules and the second plurality of molecules are in contact with each other within the barrier.

9. The barrier according to claim 8, wherein the hydrophilic block has a length of approximately A, the hydrophobic block has a length of approximately B, the first layer and the second layer each have a thickness of approximately A + B, and the barrier has a thickness of approximately 2A + 2B.

10. The length A is approximately 2 repeat units to approximately 100 repeat units, and / or The barrier according to claim 9, wherein the length B is approximately 2 repeat units to approximately 100 repeat units.

11. The barrier according to any one of claims 8 to 10, wherein the hydrophilic block comprises a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymer, polypeptide, and poly(ethylene oxide) (PEO).

12. The barrier according to any one of claims 8 to 10, wherein the linker that bonds the hydrophobic block to the hydrophilic block comprises at least one portion selected from the group consisting of amides, thioethers (sulfides), succinic acid groups, maleic acid groups, methylene, ethers, and products of click reactions.

13. The barrier according to any one of claims 8 to 10, wherein the first hydrophilic block and the second hydrophilic block each contain a portion independently selected from the group consisting of carboxylic acids, carboxyl groups, methyl groups, hydroxyl groups, primary amines, secondary amines, tertiary amines, biotin, thiols, azides, propargyl groups, allyl groups, acrylate groups, zwitterionic groups, sulfates, sulfonates, alkyl groups, aryl groups, orthogonal functional groups, and hydrogen.

14. A method for forming the barrier described in claim 1.

15. The barrier is formed using paint. The method according to claim 14, wherein the coating may be selected from the group consisting of brush coating, machine coating, and bubble coating.

16. The method according to any one of claims 14 or 15, wherein the one or more hydrophilic blocks include a polymer selected from the group consisting of N-vinylpyrrolidone, polyacrylamide, zwitterionic polymers, polypeptides, and poly(ethylene oxide) (PEO).

17. The method further includes inserting nanopores into the barrier, The method according to claim 14 or 15, wherein the polypeptide nanopore may be inserted into barrier electroporation, a pipette pump cycle, or a surfactant-assisted pore insertion.

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