Barriers comprising cross-linked

Abstract

Provided herein are barriers comprising cross-linked amphiphilic molecules and methods of making the same. In some examples, the barrier between the first fluid and the second fluid includes at least one layer comprising a plurality of amphiphilic molecules. Amphiphilic molecules of the plurality of amphiphilic molecules are crosslinked with each other.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 325,741, filed on March 31, 2022, entitled “BARRIERS INCLUDING CROSS-LINKED AMPHIPHILIC MOLECULES, AND METHODS OF MAKING THE SAME,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to barriers comprising amphiphilic molecules. Background Art

[0004] A great deal of academic and corporate time and energy has been invested in sequencing polynucleotides using nanopores. For example, the residence time of a complex of DNA with the Klenow fragment (KF) of DNA polymerase I at the top of a nanopore in an applied electric field has been measured. Or, for example, current or flux measurement sensors have been used in experiments involving DNA captured in an α-hemolysin nanopore. Or, for example, when captured in an electric field at the top of an α-hemolysin nanopore, KF-DNA complexes have been distinguished based on their properties. In another example, polynucleotide sequencing is performed using a single polymerase complex and nucleotide analogs in solution, the single polymerase complex comprising a polymerase and a template nucleic acid attached to the proximal side of the nanopore. The nucleotide analogs include a charge-blocked label attached to a polyphosphate portion of the nucleotide analog so that when the nucleotide analog is incorporated into the polynucleotide being synthesized, the charge-blocked label is cleaved. The charge-blocked label is detected by the nanopore to determine the presence and identity of the incorporated nucleotides, thereby determining the sequence of the template polynucleotide. In other examples, the construct includes a transmembrane protein nanopore subunit and a nucleic acid processing enzyme.

[0005] However, such previously known devices, systems and methods may not necessarily be sufficiently robust, reproducible or sensitive, and may not have sufficiently high throughput for practical implementation, e.g., required for commercial applications such as genome sequencing in the clinic and other environments requiring cost-effective and highly accurate operation. Therefore, what is needed is an improved device, system and method for sequencing polynucleotides, which may include the use of a membrane having nanopores disposed therein. Summary of the invention

[0006] Provided herein are barriers comprising cross-linked amphiphilic molecules and methods of making the same.

[0007] Some examples herein provide a barrier between a first fluid and a second fluid. The barrier may include at least one layer, the at least one layer including a plurality of amphiphilic molecules. The amphiphilic molecules in the plurality of amphiphilic molecules are cross-linked with each other.

[0008] In some examples, at least one layer includes: a first layer including a first plurality of amphiphilic molecules; and a second layer including a second plurality of amphiphilic molecules in contact with the first plurality of amphiphilic molecules. The amphiphilic molecules of the first layer may be cross-linked to each other, and the amphiphilic molecules of the second layer may be cross-linked to each other.

[0009] In some examples, the amphiphilic molecules include at least one hydrophobic block coupled to at least one hydrophilic block at an interface. In some examples, the amphiphilic molecules are cross-linked to each other at the hydrophilic blocks. In some examples, the amphiphilic molecules are cross-linked to each other at the hydrophobic blocks. In some examples, the amphiphilic molecules are cross-linked to each other at the interface.

[0010] In some examples, the amphiphilic molecules include molecules of a diblock copolymer including a hydrophobic block coupled to a hydrophilic block.

[0011] In some examples, the amphiphilic molecule includes a molecule of a triblock copolymer. In some examples, each molecule of the triblock copolymer includes a first hydrophobic block and a second hydrophobic block and a hydrophilic block coupled to the first hydrophobic block and the second hydrophobic block and located between the first hydrophobic block and the second hydrophobic block. In some examples, each molecule of the triblock copolymer includes a first hydrophilic block and a second hydrophilic block and a hydrophobic block coupled to the first hydrophilic block and the second hydrophilic block and located between the first hydrophilic block and the second hydrophilic block.

[0012] In some examples, the amphiphilic molecules are cross-linked by the product of the polymerization reaction. In some examples, the product of the polymerization reaction includes reacted itaconic acid moieties, reacted N-carboxyanhydride moieties, reacted disulfonylpyridyl moieties, reacted N-hydroxysuccinimide (NHS) esters, reacted acrylate moieties, reacted methacrylate moieties, reacted acrylamide moieties, reacted methacrylamide moieties, reacted styrene moieties, reacted maleic acid moieties, reacted carboxylic acid moieties, reacted thiol moieties, reacted allyl moieties, reacted vinyl moieties, reacted propargyl moieties, or reacted maleimide moieties.

[0013] In some examples, the amphiphilic molecules are cross-linked via the product of a coupling reaction. In some examples, the coupling reaction comprises a thiol-ene click reaction, a thiol-alkyne click reaction, a strain-promoted alkyne-azide cycloaddition, an amide coupling, a thiol / aza-Michael reaction, a [2+2] cycloaddition, a thio-Michael click reaction, a condensation reaction, a [2+2] photocycloaddition, a protein-ligand interaction, a host-guest chemistry, a disulfide formation, an imine formation, or an enamine formation.

[0014] In some examples, the barrier further comprises a nanopore within the barrier. In some examples, the nanopore comprises α-hemolysin or MspA.

[0015] In some examples, the barrier is suspended from a barrier support that defines an aperture, with one or more layers suspended across the aperture.

[0016] Some examples herein provide a barrier between a first fluid and a second fluid. The barrier may include at least one layer, the at least one layer including a plurality of amphiphilic molecules. The amphiphilic molecules include reactive moieties to crosslink with each other.

[0017] In some examples, the at least one layer includes a first layer including a first plurality of amphiphilic molecules and a second layer including a second plurality of amphiphilic molecules in contact with the first plurality of amphiphilic molecules.

[0018] In some examples, the reactive moiety is selected from the group consisting of an itaconic acid moiety, an N-carboxyanhydride moiety, a disulfonylpyridyl moiety, an N-hydroxysuccinimide (NHS) ester, an acrylate moiety, a methacrylate moiety, an acrylamide moiety, a methacrylamide moiety, a styrene moiety, a maleic acid moiety, a carboxylic acid moiety, a thiol moiety, an allyl moiety, a vinyl moiety, a propargyl moiety, and a maleimide moiety.

[0019] In some examples, the reactive moieties include a mixture of moieties that react with each other via thiol-ene click reactions, thiol-alkyne click reactions, strain-promoted alkyne-azide cycloadditions, amide couplings, thiol / aza-Michael reactions, [2+2] cycloadditions, thio-Michael click reactions, condensation reactions, [2+2] photocycloadditions, protein-ligand interactions, host-guest chemistry, disulfide formation, imine formation, or enamine formation.

[0020] In some examples, the amphiphilic molecule includes at least one hydrophobic block coupled to at least one hydrophilic block at an interface. In some examples, the reactive portion is located at the hydrophilic block of the corresponding amphiphilic molecule. In some examples, the reactive portion is located at the hydrophobic block of the corresponding amphiphilic molecule. In some examples, the reactive portion is located at the interface of the corresponding amphiphilic molecule.

[0021] In some examples, the amphiphilic molecules include molecules of a diblock copolymer including a hydrophobic block coupled to a hydrophilic block.

[0022] In some examples, the amphiphilic molecule includes a molecule of a triblock copolymer. In some examples, each molecule of the triblock copolymer includes a first hydrophobic block and a second hydrophobic block and a hydrophilic block coupled to the first hydrophobic block and the second hydrophobic block and located between the first hydrophobic block and the second hydrophobic block. In some examples, each molecule of the triblock copolymer includes a first hydrophilic block and a second hydrophilic block and a hydrophobic block coupled to the first hydrophilic block and the second hydrophilic block and located between the first hydrophilic block and the second hydrophilic block.

[0023] In some examples, the barrier further comprises a nanopore within the barrier. In some examples, the nanopore comprises α-hemolysin or MspA.

[0024] In some examples, the barrier is suspended from a barrier support that defines an aperture, with one or more layers suspended across the aperture.

[0025] Some examples herein provide methods of forming a barrier between a first fluid and a second fluid. The method may include forming at least one layer, the at least one layer including a plurality of amphiphilic molecules, wherein the amphiphilic molecules include reactive moieties. The method may include cross-linking the plurality of amphiphilic molecules to each other using a cross-linking reaction of the reactive moieties.

[0026] In some examples, forming at least one layer includes forming a first layer comprising a first plurality of amphiphilic molecules, and forming a second layer comprising a second plurality of amphiphilic molecules.

[0027] In some examples, the cross-linking reaction includes a polymerization reaction. In some examples, the reactive part is selected from the group consisting of itaconic acid part, N-carboxylic anhydride part, disulfonyl pyridyl part, N-hydroxysuccinimide (NHS) ester, acrylate part, methacrylate part, acrylamide part, methacrylamide part, styrene part, maleic acid part, carboxylic acid part, thiol part, allyl part, vinyl part, propargyl part and maleimide part. In some examples, the polymerization reaction includes ring-opening polymerization or step-growth polymerization. In some examples, the method also includes using an initiator to initiate the polymerization reaction. In some examples, the initiator includes a photoinitiator, a redox system or a photon. In some examples, the photoinitiator is selected from the group consisting of: 2,2-dimethoxy-2-phenylacetophenone, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate. In some examples, the redox system includes potassium persulfate or ammonium persulfate and N,N,N',N'-tetramethylethylenediamine.

[0028] In some examples, the cross-linking reaction includes a coupling reaction. In some examples, the coupling reaction includes a thiol-ene click reaction, a thiol-alkyne click reaction, a strain-promoted alkyne-azide cycloaddition, an amide coupling, a thiol / aza-Michael reaction, a [2+2] cycloaddition, a protein-ligand interaction, a host-guest chemistry, a disulfide formation, an imine formation, or an enamine formation. In some examples, an initiator is used to initiate the coupling reaction. In some examples, the initiator includes a free radical initiator, a redox system, a reducing agent, or a photon. In some examples, the free radical initiator includes 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone or 2,2'-azobis(2-methylpropionamidine) dihydrochloride. In some examples, the redox system includes potassium persulfate or ammonium persulfate and N,N,N',N'-tetramethylethylenediamine. In some examples, the reducing agent includes tris(2-carboxyethyl)phosphine, dithiothreitol, sodium ascorbate, or phosphine. In some examples, the reactive moiety includes a disulfide pyridyl moiety, an aliphatic amide moiety, a propargyl moiety, an azido moiety, a DBCO moiety, a BCN moiety, an amine moiety, an activated carboxyl moiety, a dimethylmaleimide moiety, or a maleimide moiety.

[0029] In some examples, the reactive moiety is located at the hydrophilic block of the amphiphilic molecule. In some examples, the reactive moiety is located at the interface between the hydrophilic block and the hydrophobic block of the amphiphilic molecule. In some examples, the reactive moiety is located at the hydrophilic block of the amphiphilic molecule.

[0030] In some examples, the amphiphilic molecule has an AB structure. In some examples, the amphiphilic molecule has an ABA structure. In some examples, the amphiphilic molecule has a BAB structure.

[0031] In some examples, the amphiphilic molecule includes poly(dimethylsiloxane) (PDMS). In some examples, the amphiphilic molecule includes poly(ethylene oxide) (PEO).

[0032] It should be understood that any corresponding features / examples of each of the aspects of the present disclosure as described herein may be implemented together in any appropriate combination, and any features / examples from any one or more of these aspects may be implemented together with any of the features of the other (multiple) aspects as described herein in any appropriate combination to achieve the benefits as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Cross-sectional views of exemplary nanoporous compositions and devices including barriers are schematically shown.

[0034] FIG. 2A to FIG. 2B Schematically shows Figure 1 Plan and cross-sectional views of further details of the nanopore composition and device.

[0035] FIG. 3A to FIG. 3D Exemplary procedures for forming a barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0036] Figure 4 Schematically illustrates an executable reference Figure 3D An alternative to the described operation.

[0037] FIG. 5A to FIG. 5B Exemplary procedures for forming an alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0038] FIG. 6A to FIG. 6B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0039] FIG. 7A to FIG. 7B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0040] FIG. 8A to FIG. 8B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0041] FIG. 9A to FIG. 9B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0042] FIG. 10A to FIG. 10BExemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0043] FIG. 11A to FIG. 11B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0044] FIG. 12A to FIG. 12B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated.

[0045] Fig. 12C Schematically shows a reference FIG. 3A to FIG. 3D or FIG. 12A to FIG. 12B Exemplary diblock copolymer molecules in the described procedures.

[0046] Fig.13 Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are shown.

[0047] Fig.14A Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are shown.

[0048] Fig. 14B Schematically shows a reference FIG. 11A to FIG. 11B or Fig.14A Exemplary triblock copolymer molecules from the described procedures.

[0049] FIG. 15A to FIG. 15C Further details of membranes using block copolymers are schematically shown, which may be included in Figure 1 Nanopore compositions and devices and for reference FIG. 3A to FIG. 14B The corresponding operation described.

[0050] Fig.16 An exemplary process flow in a method for forming a barrier comprising cross-linked amphiphilic molecules is shown.

[0051] Fig.17 The voltage breakdown waveform used to evaluate the stability of the polymer film is shown.

[0052] Fig.18A is a graph of measured film stability of films cross-linked using photoinitiators under different conditions.

[0053] Fig.18B is a graph of measured membrane stability of membranes cross-linked using a redox system under different conditions.

[0054] Fig.18C Schematically shown as reference FIG. 18A to FIG. 18B Exemplary reaction products in the cross-linked film.

[0055] Fig.19A is a graph of measured membrane stability of membranes cross-linked using a redox system.

[0056] Fig.19B Schematically shown as reference Fig.19A Exemplary reaction products in the cross-linked film.

[0057] Fig. 20 An exemplary reaction product in a film cross-linked as described in Example 3 is schematically shown.

[0058] Fig.21A is a graph of measured film stability of films crosslinked using a first photoinitiator under different conditions.

[0059] Fig.21B is a graph of measured film stability of films crosslinked using a second photoinitiator.

[0060] Fig. 21C is a graph of measured membrane stability of membranes cross-linked using a redox system.

[0061] Fig.21D Schematically shown as reference FIG. 21A to FIG. 21C Exemplary reaction products in the cross-linked film.

[0062] Fig. 22 An exemplary reaction product in a membrane cross-linked as described in Example 5 is schematically shown.

[0063] Fig.23A is a graph of membrane stability for membranes cross-linked with a reducing agent.

[0064] Fig. 23B Schematically shown as reference Fig.23A Exemplary reaction products in the cross-linked film.

[0065] Fig.24 An exemplary reaction product in a membrane cross-linked as described in Example 7 is schematically shown.

[0066] Fig.25A is a graph of measured membrane stability of membranes cross-linked using a reducing agent.

[0067] Fig.25B Schematically shown as reference Fig.25A Exemplary reaction products in the cross-linked film.

[0068] Fig.26 An exemplary reaction product in a membrane cross-linked as described in Example 9 is schematically shown.

[0069] Fig. 27An exemplary reaction product in a film cross-linked as described in Example 10 is schematically shown.

[0070] Figure 28 to Figure 31 Depicted are exemplary chemical reactions between different reactive moieties in different positions of the block copolymer.

[0071] Fig.32 Schematically shows Figure 1 Cross-sectional views of exemplary uses of compositions and devices.

[0072] Fig.33 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device.

[0073] Fig.34 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device.

[0074] Fig.35 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device.

[0075] Fig.36 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device. DETAILED DESCRIPTION

[0076] Provided herein are barriers comprising cross-linked amphiphilic molecules and methods of making the same.

[0077] For example, nanopore sequencing can utilize a nanopore inserted into a barrier such as a polymer membrane and including an orifice through which ions and / or other molecules can flow from one side of the membrane to the other side. The circuit can be used to detect nucleotide sequences. For example, during synthesis sequencing (SBS), on a first side of the barrier, a polymerase adds nucleotides to a growing polynucleotide in the order of the sequence of a template polynucleotide to which the growing polynucleotide hybridizes. The sensitivity of the circuit can be improved by using a fluid with different compositions on the respective sides of the membrane, for example, to provide suitable ion transport for detection on one side of the barrier while appropriately promoting the activity of the polymerase on the other side of the membrane. Therefore, barrier stability is beneficial.

[0078] As provided herein, the barrier including amphiphilic molecules can be stabilized by cross-linking amphiphilic molecules.Illustratively, amphiphilic molecules can be or include polymer chains, which are at their respective hydrophilic (A) ends, at their respective hydrophobic (B) ends or at hydrophilic-hydrophobic (AB) interfaces or at such positions (for example, at hydrophilic ends and / or at hydrophobic ends and / or at hydrophilic-hydrophobic interfaces) including functional groups. Functional groups can react in the manner of cross-linking amphiphilic molecules, thereby enhancing the stability of the film. In the example where nanopores are inserted into the film, it is expected that cross-linking will not adversely affect the nanopore functionality. For example, nanopores can maintain their relaxation ability and their mobility in the film. Therefore, the cross-linking of the present invention may not completely harden the film. Therefore, it can be expected that the film is sufficiently strong and stable, so as to be used for a long time under the force applied during the use (illustratively, genome sequencing) of the device including such films. Additionally, as described in more detail below, a variety of different cross-linking chemistries, such as polymerization or covalent coupling reactions, can be appropriately used.

[0079] First, some of the terms used herein will be briefly explained. Then, some exemplary methods for forming barriers comprising cross-linked amphiphilic molecules and intermediate structures formed using such methods will be described.

[0080] the term

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. The use of the term "including" and other forms such as "include", "includes", and "included" is not restrictive. The use of the term "having" and other forms such as "have", "has", and "had" is not restrictive. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "including" and "including" will be interpreted as having an open meaning. That is, the above terms should be interpreted synonymously with the phrase "at least having" or "at least including". For example, when used in the context of a process, the term "including" means that the process includes at least the steps listed, but may also include additional steps. When used in the context of a compound, composition, or system, the term "including" means that the compound, composition, or system includes at least the listed features or components, but may also include additional features or components.

[0082] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0083] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to describe and account for smaller fluctuations, such as due to variations in processing. For example, they may refer to less than or equal to ±10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0084] As used herein, the term "nucleotide" is intended to mean a molecule comprising a sugar and at least one phosphate group, and in some examples, a nucleobase. Nucleotides lacking a nucleobase may be referred to as "abasic". Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP ), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0085] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analogs, which are nucleotides that contain a type of 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, 6- Azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thiol adenine or guanine, 8-sulfanyl adenine or guanine, 8-hydroxy adenine or guanine, 5-halogen substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. As known in the art, some nucleotide analogs cannot be incorporated into polynucleotides, for example nucleotide analogs such as 5'-phosphosulfate adenosine. Nucleotide can include any suitable number of phosphates, for example three, four, five, six or more than six phosphates. Nucleotide analogs also include locked nucleic acid (LNA), peptide nucleic acid (PNA), and 5-hydroxybutyne-2'-deoxyuridine ("Super T").

[0086] As used herein, the term "polynucleotide" refers to a molecule comprising a sequence of nucleotides bound to each other. 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 can be included. Double-stranded DNA (dsDNA) includes genomic DNA, as well as PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. A polynucleotide may include non-naturally occurring DNA, such as enantiomeric DNA, LNA, or PNA. The precise sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, a primer, an expressed sequence tag (EST) or a serial analysis of gene expression (SAGE) tag), genomic DNA, a genomic DNA fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a synthetic polynucleotide, a branched polynucleotide, a plasmid, a vector, an isolated DNA of any sequence, an isolated RNA of any sequence, a nucleic acid probe, a primer, or an amplified copy of any of the foregoing.

[0087] As used herein, "polymerase" is intended to mean an enzyme with an active site for assembling polynucleotides by polymerizing nucleotides into polynucleotides. Polymerases can bind to the single-stranded target polynucleotides initiated, and nucleotides can be sequentially added to the growing primer to form a "complementary copy" polynucleotide having a sequence complementary to the sequence of the target polynucleotide. DNA polymerases can be bound to the target polynucleotides, and then move down along the target polynucleotides, adding nucleotides sequentially to the free hydroxyl groups at the 3' end of the growing polynucleotide chain. DNA polymerases can synthesize complementary DNA molecules from DNA templates. RNA polymerases can synthesize RNA molecules (transcription) from DNA templates. Other RNA polymerases, such as reverse transcriptases, can synthesize cDNA molecules from RNA templates. There are also other RNA polymerases that can synthesize RNA molecules from RNA templates, such as RdRP. Polymerases can use short RNA or DNA chains (primers) to start chain growth. Some polymerases can cause them to add bases to the chain site upstream of the chain. Such polymerases can be referred to as chain shifts, meaning that they have the activity of removing complementary chains from the template chain read by the polymerase.

[0088] 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 TM (Exo-)DNA polymerase, Deep VentR TM DNA polymerase, DyNAzyme TM EXT DNA、DyNAzyme TM II Hot Start DNA Polymerase, Phusion TM High-fidelity DNA polymerase, Therminator TM DNA polymerase, Therminator TM II DNA polymerase, DNA polymerase, (Exo-)DNA polymerase, RepliPHI TM Phi29 DNA polymerase, rBst DNA polymerase, rBst DNA polymerase (large), fragment (IsoTherm TM DNA polymerase), MasterAmp TM AmpliTherm TM DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase β, ThermoPhi DNA polymerase and Isopol TM SD+ polymerase. In a specific non-limiting example, the polymerase is selected from Bst, Bsu and Phi29. Some polymerases have activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been modified by mutation or otherwise to reduce or eliminate 3' and / or 5' exonuclease activity.

[0089] Example RNA polymerases include RdRp (RNA-dependent RNA polymerase), which catalyzes the synthesis of an RNA strand complementary to a given RNA template. Examples of RdRp include poliovirus 3Dpol, vesicular stomatitis virus L, and hepatitis C virus NS5B protein. Example RNA reverse transcriptases. A non-limiting example list includes RNA reverse transcriptases derived from avian myeloma virus (AMV), murine moloney leukemia virus (MMLV) and / or human immunodeficiency virus (HIV), telomerase reverse transcriptases such as (hTERT), SuperScript TM III. SuperScriptTM IV reverse transcriptase, II reverse transcriptase of the reverse transcriptase.

[0090] As used herein, the term "primer" is defined as a polynucleotide to which nucleotides can be added by free 3'OH groups. Primers may include a 3' block that inhibits polymerization until the block is removed. Primers may include modifications at the 5' end to allow a coupling reaction or to couple the primer to another portion. Primers may include one or more parts, such as 8-oxo-G, which may be cleaved under suitable conditions (such as UV light, chemistry, enzymes, etc.). Primer length may be the length of any suitable number of bases, and may include any suitable combination of natural and non-natural nucleotides. The target polynucleotide may include an "amplification adapter" or more simply an "adapter", which hybridizes with a primer (having a sequence complementary to the primer), and may be amplified so as to generate a complementary replication polynucleotide by adding nucleotides to the free 3'OH group of the primer.

[0091] As used herein, the term "plurality" is intended to represent a group of two or more different members. Multiple numbers can be in the range of small, medium, large to extremely large sizes. The size of a small multiple number can be in the range of, for example, several members to tens of members. A medium-sized multiple number can be in the range of, for example, tens of members to about 100 members or hundreds of members. A large multiple number can be in the range of, for example, about hundreds of members to about 1000 members, to thousands of members, and up to tens of thousands of members. Extremely large multiple numbers can be in the range of, for example, tens of thousands of members to about hundreds of thousands, one million, several million, tens of millions, and up to or exceeding hundreds of millions of members. Therefore, a plurality of numbers can be in the range of two to far more than 100 million members and all sizes as measured by the number of members, between the above exemplary ranges, and exceeding the above exemplary ranges. Therefore, the definition of the term is intended to include all integer values ​​greater than two.

[0092] As used herein, the term "double-stranded" when used with reference to a polynucleotide is intended to mean that all or substantially all of the nucleotides in the polynucleotide are hydrogen bonded to corresponding nucleotides in a complementary polynucleotide. A double-stranded polynucleotide may also be referred to as a "duplex."

[0093] As used herein, the term "single-stranded" when used with reference to a polynucleotide means that substantially none of the nucleotides in the polynucleotide are hydrogen bonded to corresponding nucleotides in a complementary polynucleotide.

[0094] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the purpose of an analysis or action, and may also be referred to using terms such as "library polynucleotide", "template polynucleotide" or "library template". The analysis or action includes subjecting the polynucleotide to amplification, sequencing, and / or other procedures. The target polynucleotide may include a nucleotide sequence other than the target sequence to be analyzed. For example, the target polynucleotide may include one or more adapters, including an amplification adapter that acts as a primer binding site, and the amplification adapter flanks the target polynucleotide sequence to be analyzed. In a specific example, the target polynucleotide may have sequences that are different from each other, but may have a first adapter and a second adapter that are identical to each other. Two adapters that may be located on the flanks of a specific target polynucleotide sequence may have sequences that are identical to each other, or sequences that are complementary to each other, or the two adapters may have different sequences. Therefore, the species in a plurality of target polynucleotides may include a region of known sequence that is located on the flanks of an unknown sequence region that will be evaluated by, for example, sequencing (e.g., SBS). In some examples, the target polynucleotide carries an amplification adapter at a single end, and such adapters may be located at the 3' end or the 5' end of the target polynucleotide. The target polynucleotide may be used without any adaptors, in which case the primer binding sequence may be derived directly from a sequence found in the target polynucleotide.

[0095] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. Unless specifically indicated otherwise, the different terms are not intended to represent any specific differences in size, sequence, or other properties. For clarity of description, the terms may be used to distinguish one polynucleotide species from another polynucleotide species when describing a particular method or composition comprising several polynucleotide species.

[0096] As used herein, the term "substrate" refers to the material used as a support for the composition described herein. Exemplary substrate materials may include glass, silicon dioxide, plastic, quartz, metal, metal oxide, organosilicate (e.g., polyhedral organosiliconesquioxane (POSS)), polyacrylate, tantalum oxide, complementary metal oxide semiconductor (CMOS), or a combination thereof. The example of POSS may be described in Kehagias et al., Microelectronic Engineering (Microelectronic Engineering) 86 (2009), POSS in pages 776-778, which is incorporated by reference in its entirety. In some examples, the substrate used in the present application includes a silicon dioxide-based substrate, such as glass, fused silica, or other silicon dioxide-containing materials. In some examples, a substrate based on silicon dioxide may include silicon, silicon dioxide, silicon nitride, or silane. In some examples, the substrate used in the present application includes a plastic material or component, such as polyethylene, polystyrene, poly (vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly (methyl methacrylate). Exemplary plastic materials include poly(methyl methacrylate), polystyrene, and cycloolefin polymer substrates. In some examples, the substrate is or includes a silicon dioxide-based material or a plastic material or a combination thereof. In a specific example, the substrate has at least one surface including glass or a silicon-based polymer. In some examples, the substrate may include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface including a metal oxide. In one example, the surface includes tantalum oxide or tin oxide. Acrylamide, enone, or acrylate may also be used as a substrate material or component. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers, and copolymers. In some examples, the substrate and / or the substrate surface may be or include quartz. In some other examples, the substrate and / or the substrate surface may be or include a semiconductor, such as GaAs or ITO. The foregoing list is intended to illustrate but is not limited to the present application. The substrate may include a single material or a plurality of different materials. The substrate may be a composite material or a laminate. In some examples, the substrate includes an organic silicate material.

[0097] The substrate can be flat, round, 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.

[0098] The substrate can be non-patterned, textured, or patterned on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns may include posts, pads, holes, ridges, channels, or other three-dimensional concave or convex structures. The pattern may be regular or irregular across the substrate surface. For example, the pattern may be formed by nanoimprint lithography or by using, for example, metal pads that form features on a non-metallic surface.

[0099] In some examples, the substrate described herein forms at least a portion of a flow cell, is located in a flow cell, or is coupled to a flow cell. The flow cell may include a flow chamber divided into a plurality of lanes or a plurality of partitions. Exemplary flow cells and substrates for making flow cells that can be used in the methods and compositions described herein include, but are not limited to, those commercially available from Illumina, Inc., San Diego, CA.

[0100] As used herein, the term "electrode" is intended to refer to a solid structure that conducts electricity. The electrode may include any suitable conductive material, such as gold, palladium, silver, or platinum, or a combination thereof. In some examples, the electrode may be disposed on a substrate. In some examples, the electrode may define a substrate.

[0101] As used herein, the term "nanopore" is intended to mean a structure including an orifice that allows molecules to pass from a first side of the nanopore to a second side of the nanopore, wherein a portion of the orifice of the nanopore has a width of 100 nm or less, such as 10 nm or less, or 2 nm or less. The orifice extends through the first and second sides of the nanopore. Molecules that can pass through the orifice of the nanopore may include, for example, ions or water-soluble molecules, such as amino acids or nucleotides. The nanopore may be disposed in a membrane, or may be provided by a substrate. Optionally, a portion of the orifice may be narrower than one or both of the first and second sides of the nanopore, in which case the portion of the orifice may be referred to as a "constriction". Alternatively or in addition thereto, the orifice of the nanopore or the constriction of the nanopore (if present) or both may be greater than 0.1 nm, 0.5 nm, 1 nm, 10 nm or greater. The nanopore may include a plurality of constrictions, such as at least two, or three, or four, or five, or more than five constrictions, and the nanopore includes a biological nanopore, a solid-state nanopore, or a biological and solid-state hybrid nanopore.

[0102] Biological nanopores include, for example, polypeptide nanopores and polynucleotide nanopores. "Polypeptide nanopore" is intended to mean a nanopore made of one or more polypeptides. The one or more polypeptides may include monomers, homopolymers or heteropolymers. The structures of polypeptide nanopores include, for example, α-helical bundle nanopores and β-barrel nanopores and all other nanopores known in the art. Example polypeptide nanopores include Aeromonas lysin, α-hemolysin, Mycobacterium smegmatis porin A, Gramicidin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F-sex cilium, 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, thereby allowing hydrophilic molecules to enter the bacteria. MspA forms a tightly interconnected octamer and a transmembrane β-barrel, which is similar to a goblet and contains a constriction. For further details of α-hemolysin, see US6,015,714, the entire contents of which are incorporated herein by reference. For further details of SP1, see Wang et al., Chem. Commun., 49: 1741-1743 (2013), the entire contents of which are incorporated herein by reference. For further details of MspA, see Butler et al., "Single-molecule DNA detection with engineered MspA protein nanopore", Proc. Inc. Natl. Acad. Sci. 105: 20647-20652 (2008) and Derrington et al., "Nanopore DNA sequencing with MspA", Proc. Natl. Acad. Sci. USA, 107: 16060-16065 (2010), the entire contents of which are incorporated herein by reference. Other nanopores include, for example, an MspA homolog from Norcadia farcinica, and lysins. For further details on lysins, see PCT Publication No. WO2013 / 153359, the entire contents of which are incorporated herein by reference.

[0103] "Polynucleotide nanopore" is intended to mean a nanopore made from one or more nucleic acid polymers. A polynucleotide nanopore may comprise, for example, polynucleotide origami.

[0104] "Solid-state nanopore" is intended to mean a nanopore made of one or more materials of non-biological origin. Solid-state nanopores can be made of 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. A solid-state nanopore may include an orifice formed in a solid-state membrane (e.g., a membrane including any such material).

[0105] "Biological and solid-state hybrid nanopores" are intended to mean hybrid nanopores made from materials of biological and non-biological origin. Materials of biological origin are as 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.

[0106] As used herein, "barrier" is intended to mean a structure that generally inhibits the passage of molecules from one side of the barrier to the other side of the barrier. The molecules whose passage is inhibited may include, for example, ions and water-soluble molecules, such as nucleotides or amino acids. However, if a nanopore is disposed within the barrier, the orifice of the nanopore may allow molecules to pass from one side of the barrier to the other side of the barrier. As a specific example, if a nanopore is disposed within the barrier, the orifice of the nanopore may allow molecules to pass 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.

[0107] As used herein, biological origin refers to materials derived from or isolated from a biological environment, such as an organism or a cell, or synthetically manufactured forms of biologically usable structures.

[0108] As used herein, "solid state" refers to materials of non-biological origin.

[0109] As used herein, "synthetic" refers to membrane materials that are of non-biological origin (eg, polymeric materials, synthetic phospholipids, solid-state membranes, or combinations thereof).

[0110] As used herein, "polymer membrane" refers to a synthetic barrier composed primarily of polymers of non-biological origin. In some examples, the polymer membrane consists essentially of polymers of non-biological origin. Block copolymers are one example of a polymer of non-biological origin that can be included in the barriers of the present invention. Hydrophobic polymers with ionic end groups are another example of a polymer of non-biological origin that can be included in the barriers of the present invention. Because the barriers of the present invention involve polymers of non-biological origin, the terms "polymer membrane," "membrane," and "barrier" are used interchangeably herein when referring to the barriers of the present invention, even though the terms "barrier" and "membrane" can generally cover other types of materials.

[0111] As used herein, the term "block copolymer" is intended to refer to a polymer having at least a first portion or "block" comprising a first type of monomer and at least a second portion or "block" coupled directly or indirectly to the first portion and comprising a different second type of monomer. The first portion may comprise a polymer of a first type of monomer, or the second portion may comprise a polymer of a second type of monomer, or the first portion may comprise a polymer of a first type of monomer and the second portion may comprise a polymer of a second type of monomer. The first portion may optionally comprise end groups having a different hydrophilicity than the first type of monomer, or the second portion may optionally comprise end groups having a different hydrophilicity than the second type of monomer, or the first portion may optionally comprise end groups having a different hydrophilicity than the first type of monomer and the second portion may optionally comprise end groups having a different hydrophilicity than 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. Depending on the particular configuration, the end groups of any hydrophobic block may be located on the inner surface of the barrier or on the outer surface of a barrier formed using such hydrophobic block.

[0112] Block copolymers include, but are not limited to, diblock copolymers and triblock copolymers.

[0113] "Diblock copolymer" is intended to mean a block copolymer comprising or consisting essentially of a first and a second block coupled 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, wherein "A" refers to the hydrophilic block and "B" refers to the hydrophobic block.

[0114] "Triblock copolymer" is intended to refer to a block copolymer comprising or consisting essentially of a first, second, and third block coupled directly or indirectly to each other. The first block and the third block may comprise or consist essentially of the same type of monomer as each other, and the second block may comprise a different type of monomer. In some examples, the first block may be hydrophobic, the second block may be hydrophilic, and the third block may be hydrophobic and comprise the same type of monomer as the first block, in which case the triblock copolymer may be referred to as a "BAB" copolymer, where "A" refers to a hydrophilic block and "B" refers to a hydrophobic block. In other examples, the first block may be hydrophilic, the second block may be hydrophobic, and the third block may be hydrophilic and comprise the same type of monomer as the first block, in which case the triblock copolymer may be referred to as an "ABA" copolymer, where "A" refers to a hydrophilic block and "B" refers to a hydrophobic block.

[0115] The specific arrangement of the molecules of a polymer chain (e.g., a block copolymer) within a polymer film may depend, among other things, on the respective block lengths, the types of monomers used for the different blocks, the relative hydrophilicity and hydrophobicity of the blocks, the composition of the fluid in which the film is formed, and / or the density of the polymer chains within the film. During the formation of the film, these and other factors generate forces between the molecules of the polymer chain that laterally position and reorient the molecules in a manner that substantially minimizes the free energy of the film. Once the polymer chains have completed these rearrangements, the film may be considered to be substantially "stable," even though the molecules may maintain a certain degree of mobility within the film.

[0116] The "AB interface" of a block copolymer, such as a diblock or triblock copolymer, refers to the interface where the hydrophilic block is coupled to the hydrophobic block.

[0117] As used herein, the term "hydrophobicity" is intended to mean the tendency to repel 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 will tend to associate with each other in a manner that minimizes contact with polar (hydrophilic) molecules to reduce the free energy of the system as a whole.

[0118] As used herein, the term "hydrophilic" is intended to mean a tendency to bind water molecules. Polar (hydrophilic) molecules in a polar environment will tend to associate with each other in a manner that minimizes contact with non-polar (hydrophobic) molecules to reduce the free energy of the system as a whole.

[0119] As used herein, the term "amphiphilic" is intended to mean having both hydrophilic and hydrophobic properties. For example, a block copolymer comprising a hydrophobic block and a hydrophilic block may be considered "amphiphilic". Illustratively, AB copolymers, ABA copolymers, and BAB copolymers may all be considered amphiphilic. Additionally, molecules comprising a hydrophobic polymer coupled to an ionic end group may be considered amphiphilic.

[0120] As used herein, "solution" is intended to refer to a homogeneous mixture containing two or more substances. In such a mixture, a solute is a substance that is uniformly dissolved in another substance called a solvent. A solution may include a single solute, or may include multiple solutes. Additionally or alternatively, a solution may include a single solvent, or may include multiple solvents. "Aqueous solution" refers to a solution in which the solvent is or includes water.

[0121] A first liquid that forms a homogeneous mixture with a second liquid is referred to herein as being "miscible" or "soluble" with the second liquid.

[0122] As used herein, the term "electroporation" means applying a voltage across a membrane such that a nanopore is inserted into the membrane.

[0123] As used herein, terms such as "cross-linked" and "cross-linking" refer to the formation of bonds between molecules. The bonds may include covalent bonds or non-covalent bonds, such as ionic bonds, hydrogen bonds, or π-π stacking. Cross-linked molecules may include polymers, proteins, or polymers and proteins.

[0124] As used herein, the term "initiator" is intended to mean an entity that can initiate a polymerization reaction. Non-limiting examples of initiators include moieties, molecules, and / or photons that can initiate a polymerization reaction.

[0125] As used herein, terms such as "covalently coupled" or "covalently bonded" refer to the formation of chemical bonds characterized by the sharing of electron pairs between atoms. For example, covalently coupled molecules refer to molecules that form chemical bonds, as opposed to non-covalent bonds such as electrostatic interactions.

[0126] As used herein, "C" wherein "a" and "b" are integers a To C b " or "C a-b " refers to the number of carbon atoms in a particular group. That is, the group may contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, "C1 to C4 alkyl" or "C 1-4 Alkyl" or "C 1-4 The term "alkyl" group refers to all alkyl groups having 1 to 4 carbons, namely CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-.

[0127] As used herein, the term "halogen" or "halo" means fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being exemplary.

[0128] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double bonds and triple bonds). An alkyl group may have 1 to 20 carbon atoms (whenever it appears in this article, a numerical range such as "1 to 20" refers to each integer in the given range; for example, "1 to 20 carbon atoms" means that the alkyl group may be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also covers the term "alkyl" in which no numerical range is specified). The alkyl group may also be a medium-sized alkyl group having 1 to 9 carbon atoms. The alkyl group may also be a low-sized alkyl group having 1 to 4 carbon atoms. The alkyl group may be designated as "C 1-4 Alkyl" or similar designations. By way of example only, "C 1-4 Alkyl" or "C 1-4"Alkyl" means that there are from one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

[0129] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. An alkenyl group may have from 2 to 20 carbon atoms, but this definition also encompasses the term "alkenyl" where no numerical range is specified. An alkenyl group may also be a medium-sized alkenyl group having from 2 to 9 carbon atoms. An alkenyl group may also be a lower alkenyl group having from 2 to 4 carbon atoms. An alkenyl group may be designated as "C 2-4 "C-alkenyl" or similar designations. By way of example only, "C 2-4 “Alkenyl” means that there are two to four carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are by no means limited to, ethenyl, propenyl, butenyl, pentenyl and hexenyl, and the like.

[0130] Groups including alkenyl groups include optionally substituted alkenyl, cycloalkenyl and heterocycloalkenyl groups.

[0131] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. Alkynyl groups can have from 2 to 20 carbon atoms, but this definition also covers the term "alkynyl" where no numerical range is specified. Alkynyl groups can also be medium-sized alkynyl groups having from 2 to 9 carbon atoms. Alkynyl groups can also be low-sized alkynyl groups having from 2 to 4 carbon atoms. Alkynyl groups can be designated as "C 2-4 "alkynyl" or similar designations. By way of example only, "C 2-4 Alkynyl" or "C 2-4 "Alkynyl" means that there are two to four carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl and 2-butynyl. Typical alkynyl groups include, but are by no means limited to, ethynyl, propynyl, butynyl, pentynyl and hexynyl, etc.

[0132] Groups including alkynyl groups include optionally substituted alkynyl, cycloalkynyl and heterocycloalkynyl groups.

[0133] As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) that contains only carbon in the ring backbone. When aryl is a ring system, each ring in the ring system is aromatic. An aryl group can have 6 to 18 carbon atoms, but this definition also covers the term "aryl" where no numerical range is specified. In some examples, an aryl group has 6 to 10 carbon atoms. An aryl group can be designated as "C 6-10 Aryl", "C6 or C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

[0134] As used herein, "heterocycle" refers to a cyclic compound that includes carbon atoms together with another atom (heteroatom) such as nitrogen, oxygen or sulfur. The heterocycle can be aromatic (heteroaryl) or aliphatic. Aliphatic heterocycles can be fully saturated or can contain one or more or two or more double bonds, for example, the heterocycle can be a heterocycloalkyl. The heterocycle can include a single heterocycle or multiple heterocycles that are fused together.

[0135] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more condensed rings sharing two adjacent atoms) containing one or more heteroatoms (i.e., elements other than carbon, including but not limited to nitrogen, oxygen and sulfur) in the ring skeleton. When heteroaryl is a ring system, each ring in the ring system is aromatic. The heteroaryl group can have 5 to 18 ring members (i.e., the number of atoms (including carbon atoms and heteroatoms) constituting the ring skeleton), but this definition also encompasses the term "heteroaryl" in which no numerical range is specified. In some examples, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group can be named "5 to 7 heteroaryl", "5 to 10 heteroaryl" or similar names. Examples of heteroaryl rings include, but are not limited to, furanyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothiophenyl.

[0136] As used herein, "cycloalkyl" means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0137] As used herein, "cycloalkenyl" or "cycloalkene" refers to a carbocyclic ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic. One example is cyclohexenyl or cyclohexene. Another example is norbornene or norbornenyl.

[0138] As used herein, "heterocycloalkenyl" or "heterocycloalkene" means a carbocyclic ring or ring system having at least one heteroatom in the ring backbone, at least one double bond, wherein no ring in the ring system is aromatic. In some examples, the heterocycloalkenyl or heterocycloalkene ring or ring system is 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, or 10-membered.

[0139] As used herein, "cycloalkynyl" or "cycloalkyne" refers to a carbocyclic ring or ring system having at least one triple bond, wherein no ring in the ring system is aromatic. One example is cyclooctyne. Another example is bicyclononyne. Another example is dibenzocyclooctyne (DBCO).

[0140] As used herein, "heterocycloalkynyl" or "heterocycloalkyne" means a carbocyclic ring or ring system having at least one heteroatom in the ring backbone, having at least one triple bond, wherein no ring in the ring system is aromatic. In some examples, the heterocycloalkynyl or heterocycloalkyne ring or ring system is 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, or 10-membered.

[0141] As used herein, "heterocycloalkyl" means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring skeleton. Heterocycloalkyl can be joined together in a fused, bridged or spiro manner. Heterocycloalkyl can have any degree of saturation, provided that at least one heterocycle in the ring system is not aromatic. The heterocycloalkyl group can have 3 to 20 ring members (i.e., the number of atoms (including carbon atoms and heteroatoms) constituting the ring skeleton), but this definition also covers the term "heterocycloalkyl" in which no numerical range is specified. The heterocycloalkyl group can also be a medium-sized heterocycloalkyl with 3 to 10 ring members. The heterocycloalkyl group can also be a heterocycloalkyl with 3 to 6 ring members. The heterocycloalkyl group can be named "3 to 6-membered heterocycloalkyl" or similar names. In some six-membered monocyclic heterocycloalkyls, the heteroatoms are selected from one to a maximum of three in O, N or S, and in some five-membered monocyclic heterocycloalkyls, the heteroatoms are selected from one or two heteroatoms selected from O, N or S. Examples of heterocycloalkyl rings include, but are not limited to, azepanyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidinedione, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxinyl, 1,4-dioxinyl, 1,4-dioxinyl, 1,3-oxathianyl, 1,4-oxathianyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithienyl, 1,3-dithianyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinone, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiomorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl and tetrahydroquinoline.

[0142] As used herein, the substituted group is derived from an unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms with another atom or group. Unless otherwise specified, when a group is considered to be "substituted", it means that the group is substituted with one or more substituents independently selected from: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heterocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heterocyclyl-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (C1-C6) alkyl (optionally substituted with halogen, C1-C6 C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy substituted), 5- to 10-membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy substituted), 5- to 10-membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy substituted), halo, cyano, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy (C1-C6) alkyl (i.e. , ether), aryloxy, sulfhydryl (thiol), halo (C1-C6) alkyl (e.g., -CF3), halo (C1-C6) alkoxy (e.g., -OCF3), C1-C6 alkylthio, arylthio, amino, amino (C1-C6) alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, and oxo (=O). Wherever a group is described as "optionally substituted", the group may be substituted by the above substituents.

[0143] In the case where the compounds disclosed herein have at least one stereocenter, they can exist as a single enantiomer or diastereomer, or as a mixture of such isomers (including racemates). Separation of a single isomer or selective synthesis of a single isomer is achieved by applying various methods well known to those skilled in the art. In the case where the compounds disclosed herein are understood to exist in tautomeric form, all tautomeric forms are included in the scope of the depicted structure. Unless otherwise indicated, all such isomers and their mixtures are included in the scope of the compounds disclosed herein. In addition, the compounds disclosed herein may exist in one or more crystalline or amorphous forms. Unless otherwise indicated, all such forms are included in the scope of the compounds disclosed herein, including any polymorphic form. In addition, some compounds disclosed herein can form solvates with water (i.e., hydrates) or common organic solvents. Unless otherwise indicated, such solvates are included in the scope of the compounds disclosed herein.

[0144] As used herein, the term "adduct" is intended to mean the product of a chemical reaction between two or more molecules, wherein the product contains all of the atoms of the reacting molecules.

[0145] As used herein, the term "joint" is intended to mean one or more molecules through which an element is attached to another element. For example, a joint can attach a first reactive portion to a second reactive portion. The joint can be covalent, or can be non-covalent. Non-limiting examples of covalent linkers include alkyl chains, polyethers, amides, esters, aryl groups, polyaryls, etc. Non-limiting examples of non-covalent joints include host-guest complexes, cyclodextrin / norbornene, inclusion complexes of adamantane and β-CD, DNA hybridization interactions, streptavidin / biotin, etc.

[0146] As used herein, the term "barrier support" is intended to refer to a structure from which a barrier can be suspended. In examples where the barrier support is used to support a polymer film, the barrier support may be referred to as a "membrane support". The barrier support may define an orifice so that a first portion of the barrier is suspended across the orifice, 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 to define an orifice and suspend the barrier across the orifice. In some examples, the barrier support may include a substrate having an orifice defined therethrough, and the barrier may be suspended across the orifice. Additionally or alternatively, the barrier support may include one or more first features (such as one or more edges or flanges of a hole within a substrate) protruding relative to one or more second features (such as the bottom surface of a hole), wherein the height difference between (a) one or more first features and (b) one or more second features defines an orifice, and the barrier may be suspended across the orifice. The orifice may have any suitable shape, such as a circular, elliptical, polygonal, or irregular shape. The barrier support may comprise any suitable material or combination of materials. For example, the barrier support may be of biological origin, or may be solid. In some examples, the barrier support may include, or may consist essentially of, an organic material, for example, a curable resin such as SU-8; polytetrafluoroethylene (PTFE), poly(methyl methacrylate) (PMMA), polyparaxylene, etc. Additionally or alternatively, in various examples, the barrier support may include, or may consist essentially of, an inorganic material, for example, silicon nitride, silicon oxide, or molybdenum disulfide.

[0147] As used herein, the term "annulus" is intended to refer to a liquid attached to a barrier support, located within the barrier, and partially extending into an orifice defined by the barrier support. Thus, it should be understood that the annulus can follow the shape of the orifice of the barrier, for example, can have a circular, elliptical, polygonal or irregular shape.

[0148] Barriers comprising cross-linked amphiphilic molecules and methods for their preparation

[0149] Now refer to Figures 1 to 16 , Figure 28 to Figure 31 and Figure 38 describe barriers comprising cross-linked amphiphilic molecules and methods for making the same.

[0150] Figure 1A cross-sectional view of an exemplary nanopore composition and device 100 including a polymer membrane is schematically shown. Device 100 includes a fluid pore 100', which includes a barrier 101 (such as a polymer membrane) having a first (trans) side 111 and a second (cis) side 112, a first fluid 120 within the fluid pore 100' and in contact with the first side 111 of the membrane, and a second fluid 120' within the fluid pore and in contact with the second side 112 of the membrane. Barrier 101 can have any suitable structure that generally inhibits the passage of molecules from one side of the membrane to the other side of the membrane, for example, generally inhibits contact between fluid 120 and fluid 120'. Illustratively, barrier 101 can include a polymer membrane that can include a diblock or triblock copolymer and can have a structure such as the following reference FIG. 2A to FIG. 2B , FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 6A to FIG. 6B , FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B , FIG. 9A to FIG. 9B , FIG. 10A to FIG. 10B , FIG. 11A to FIG. 11B , FIG. 12A to FIG. 12C , Fig.13 , FIG. 14A to FIG. 14B , FIG. 15A to FIG. 15C and Figure 28 to Figure 31 Describe the structure in more detail.

[0151] The first fluid 120 may have a first composition comprising a first concentration of salt 160, which for simplicity may be represented as positive ions, but it should be understood that counter ions may also be present. The second fluid 120' may have a second composition comprising a second concentration of salt 160, which may be the same or different from the first concentration. Any suitable one or more salts 160 may be used for the first fluid 120 and the second fluid 120', for example ranging from common salts to ionic crystals, metal complexes, ionic liquids or even water-soluble organic ions. For example, the salt may include any suitable combination of cations (such as, but not limited to, H, Li, Na, K, NH4, Ag, Ca, Ba and / or Mg) and anions (such as, but not limited to, OH, Cl, Br, I, NO3, ClO4, F, SO4 and / or CO3 2- ...). In a non-limiting example, the salt includes potassium chloride (KCl). It should also be understood that the first fluid and the second fluid may optionally include any suitable combination of other solutes. Illustratively, the first fluid 120 and the second fluid 120' may include an aqueous buffer (such as N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), commercially available from Fisher BioReagents).

[0152] Still refer to Figure 1, the device 100 may also include a nanopore disposed within the membrane 101 and providing an orifice 113 that fluidically couples the first side 111 with the second side 112. Thus, the orifice 113 of the nanopore 110 may provide a passage for the fluid 120 and / or the fluid 120' (e.g., the salt 160) to flow through the membrane 101. The nanopore 110 may include a solid-state nanopore, a biological nanopore (e.g., such as Figure 1 MspA) or biological and solid-state hybrid nanopores as shown in . Non-limiting examples and properties of membranes and nanopores are described elsewhere herein and in US 9,708,655, the entire contents of which are incorporated herein by reference. Figure 1 In the manner shown, the device 100 can optionally include a first electrode 102 in contact with the first fluid 120, a second electrode 103 in contact with the second fluid 120', and a circuit 180 in operative communication with the first and second electrodes and configured to detect a change in an electrical property of the orifice. Such a change can be, for example, in response to any suitable stimulus. In fact, it should be understood that the methods, compositions and devices of the present invention can be used in any suitable application or context, including any suitable method or device for sequencing (e.g., polynucleotide sequencing).

[0153] In some examples, the polymer film 101 between the first fluid 120 and the second fluid 120' comprises a block copolymer. FIG. 2A to FIG. 2B Schematically shows Figure 1 Further details of the nanopore composition and device are shown in plan and cross-sectional views. Figure 2A As shown, the membrane 101 may include a first layer 201 including a first plurality of amphiphilic molecules 221 and a second layer 202 including a second plurality of amphiphilic molecules in contact with the first plurality of amphiphilic molecules. Figure 2A In the non-limiting example shown, the copolymer is a diblock copolymer (AB), such that each molecule 221 includes a hydrophobic "B" block 231 (wherein the darker filled circles 241 represent hydrophobic monomers) and a hydrophilic "A" block 232 (wherein the lighter filled circles 242 represent hydrophilic monomers) coupled directly or indirectly thereto. FIG. 5A to FIG. 5B , FIG. 8A to FIG. 8B and Fig.13 In other examples described, the copolymer may alternatively include an ABA triblock copolymer. FIG. 6A to FIG. 6B , FIG. 9A to FIG. 9B , FIG. 11A to FIG. 11B and FIG. 14A to FIG. 14B In yet other examples described, the copolymer may alternatively include a BAB triblock copolymer.

[0154] exist Figure 2AIn the example shown, the hydrophilic blocks 232 of the first plurality of molecules 221 are cross-linked to each other via bonds 281 at a first outer surface of the membrane 101 (e.g., a surface of the membrane 101 that contacts the fluid 120 on the first side 111). The hydrophilic blocks 232 of the second plurality of molecules 221 may optionally also be cross-linked to each other via bonds 281 at a second outer surface of the membrane 101 (e.g., a surface of the membrane 101 that contacts the fluid 120' on the second side 112). Thus, the bonds 281 may strengthen and stabilize the membrane, thereby improving performance and durability. The hydrophobic blocks 231 of the first and second plurality of molecules 221 may contact each other within the membrane. Although Figure 2A An example of a hydrophilic block being cross-linked by bonds 281 formed in corresponding planes at the ends of the hydrophilic block is shown, but such cross-linked bonds may also be formed in any other suitable plane within the membrane. FIG. 10A to FIG. 10B , FIG. 11A to FIG. 11B and FIG. 14A to FIG. 14B In the manner described, the hydrophobic blocks 231 can be cross-linked by bonds formed in one or more planes at the ends of the hydrophobic blocks. Alternatively, for example, in a manner such as that described in reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B , FIG. 9A to FIG. 9B and Fig.13 In the manner described, the hydrophilic-hydrophobic (AB) interfaces within the membrane can be cross-linked via bonds formed in the corresponding planes at these interfaces.

[0155] exist FIG. 2A to FIG. 2B In the example shown, the membrane 101 can be suspended using a barrier support (e.g., membrane support 200) that defines an aperture 230. For example, the membrane support 200 can include a substrate having an aperture 230 defined therethrough, such as a substantially circular aperture, or an aperture having another shape. Additionally or alternatively, the barrier support can include one or more features in which a nanopore device is formed, such as edges or flanges on either side of the aperture. Non-limiting examples of materials that can be included in the barrier support are further provided above. An annulus 210 comprising a hydrophobic (non-polar) solvent and which can also comprise polymer chains and / or other compounds can be attached to the membrane support 200 and can support a portion of the membrane 101, for example, can be located within the barrier 101 (here, between layer 201 and layer 202). Alternatively, the annulus 210 can be as Figure 2A The outer portion of the molecule 221 of the membrane 101 can be disposed on the support 200 (e.g., the portion extending between the orifice 230 and the membrane perimeter 220), while the inner portion of the molecule can form an independently supported portion of the membrane 101 (e.g., the portion within the orifice 210, a portion of which is supported by the ring 210). Note that although the overall assembly of molecules that are cross-linked to each other can itself be considered to form a larger molecule (e.g., partially or substantially spanning FIG. 2A to FIG. 2Bmolecule in the pore 230 of the membrane support 220), but such larger molecular components may also be referred to as molecules in this article to facilitate discussion of such components.

[0156] The membrane 101 can be stabilized and the nanopore 110 can be inserted into a freestanding portion of the membrane 101, for example using a method such as will now be referred to as FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 6A to FIG. 6B , FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B , FIG. 9A to FIG. 9B , FIG. 10A to FIG. 10B , FIG. 11A to FIG. 11B , FIG. 12A to FIG. 12C , Fig.13 , FIG. 14A to FIG. 14B , FIG. 15A to FIG. 15C , Fig.16 , Figure 28 to Figure 31 and the operation described in Figure 38. Although FIG. 2A to FIG. 2B Nanopore 110 is shown within barrier 101, but it will be appreciated that the nanopore may be omitted and barrier 101 may be used for any suitable purpose. More generally, it will be appreciated that while the barriers described herein are particularly suitable for use with nanopores (e.g., for nanopore sequencing, such as in reference Figure 32 to Figure 36 ), but the barrier of the present invention does not necessarily have to have nanopores inserted therein.

[0157] FIG. 3A to FIG. 3D Exemplary procedures for forming a barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Figure 3A A barrier 301 is shown, which may be similar to that of reference FIG. 2A to FIG. 2B The described method uses a membrane support 200 and an optional ring 210 for suspension. Figure 3A As shown, in some aspects, barrier 301 can be constructed similar to reference FIG. 2A to FIG. 2B The film 101, for example, may include a layer 201 including a first plurality of amphiphilic molecules 221 and a layer 202 including a second plurality of amphiphilic molecules 221. However, the amphiphilic molecules in the barrier 301 have not yet been crosslinked. Instead, the amphiphilic molecules of the layer 201 (and optionally also the layer 202) may include reactive moieties 311. The reactive moieties 311 may react with each other in a manner that may fully or partially crosslink the amphiphilic molecules 221 with each other. In a process such as Figure 3A In the example shown, the amphiphilic molecules include molecules of a diblock copolymer oriented such that the hydrophobic "B" segments of the AB diblock copolymer are oriented toward each other and disposed within the membrane, while the hydrophilic "A" segments form the outer surface of the membrane. Figure 3AIn the non-limiting example shown, the hydrophilic "A" segment 332 may include, for example, a reactive portion 311 coupled to a terminal hydrophilic monomer 342. Suitable methods for forming a membrane suspended from a barrier support are known in the art, such as "coating", e.g., brush coating (manually), mechanical coating (e.g., using a stir bar), and bubble coating (e.g., using flow through a device).

[0158] In some examples, such as Figure 3B As shown, barrier 301 can be contacted with a fluid having dissolved initiator 321. Initiator 321 can be selected so as to chemically react with reactive moieties 311, for example so as to form a product in which amphiphilic molecules 221 are cross-linked with each other, such as via polymerization. In other examples, the initiator can be omitted, and the reactive moieties can react directly with each other without the use of an initiator.

[0159] Figure 3C The product of the polymerization reaction between amphiphilic molecules 221 is shown, where bonds 281 are formed between reactive moieties 311 (whose fill changes from cross-hatching to white to indicate that such moieties have reacted and are no longer available for reaction). Figure 3CIt may be suggested that each reactive portion 311 is cross-linked with two other portions via respective bonds 281, but it should be understood that each reactive portion may form bonds with any suitable number of other such reactive portions (e.g., one, two, three, or more than three other such reactive portions). The relative proportions of such products may be controlled, for example, by the type of reactive portion used, the type of initiator used, the reaction time, and the reaction conditions, so as to control the amount of cross-linking provided by the reaction between the reactive portions 311 of the molecules 221. Cross-linking may also be controlled by a coupling strategy. For example, a thiol-ene or thiol-yne reaction based on the generation of free radicals and controllable by the type and concentration of the initiator may be used. Alternatively, a cross-linking triggered by a reducing agent may be used, and the concentration and type of the reducing agent may be used to control the reaction. Alternatively, an initiator-free strategy may be used that uses UV light to trigger cross-linking, and the reaction may be controlled by UV dose (irradiance, wavelength, and time); in such examples, the barrier may be enclosed in a structure that is at least partially transparent to UV light. Other strategies can use two amphiphilic polymers with different reactive parts, wherein the ratio between the amphiphilic polymers can be selected to achieve substantially complete crosslinking. Depending on the strategy, this substantially complete crosslinking can be achieved in an exemplary ratio of 1:1 or 2:1. If a lower degree of crosslinking is required, the ratio can be adjusted to achieve partial crosslinking. Additionally, in some examples, the amount of crosslinking can be controlled by mixing the amphiphilic molecules 221 in a suitable ratio with other amphiphilic molecules that do not include reactive parts 311 or include different reactive parts and / or have different architectures (e.g., AB can be mixed with ABA and / or BAB; ABA can be mixed with AB and / or BAB; and / or BAB can be mixed with AB and / or ABA).

[0160] In some examples, after cross-linking of the amphiphilic molecules 221, the nanopore 110 may be Figure 3D Insert into the barrier in the manner shown. Figure 4 Schematically illustrates an executable reference Figure 3D More specifically, in Figure 4 In the example shown, the nanopore 110 can be inserted into the suspended barrier 301 before the amphiphilic molecules in the cross-linked barrier. The amphiphilic molecules can then be inserted into the suspended barrier 301, such as in FIG. FIG. 3A to FIG. 3C The nanopore 110 is cross-linked in the manner described. Non-limiting examples of techniques for inserting the nanopore 110 into the membrane (whether before or after cross-linking) include electroporation, pipetting pump circulation, and detergent-assisted nanopore insertion. Tools for forming membranes using synthetic polymers and inserting nanopores in the membrane are commercially available, such as the Orbit 16TC platform from Nanion Technologies Inc. in California, USA.

[0161] although FIG. 3A to FIG. 3D The manipulation of cross-linking the hydrophilic blocks of a diblock copolymer is shown, but it is understood that such manipulation can be similarly used to cross-link other portions of a diblock copolymer or to cross-link other types of amphiphilic molecules, such as other types of polymers. FIG. 5A to FIG. 5B Exemplary procedures for forming an alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Figure 5A A suspended membrane 501 is shown comprising an ABA triblock copolymer molecule comprising a hydrophobic "B" segment 541 coupled to and located between hydrophilic "A" segments 542. The membrane 501 may be similar to that of reference FIG. 2A to FIG. 2B The described approach uses a membrane support 200 and an optional ring 210 for suspension. Each individual ABA molecule can be in one of two arrangements. For example, an ABA molecule 521 can extend through the layer in a linear manner, with the "A" segment on each side of the membrane and the "B" segment in the middle of the membrane. Or, for example, an ABA molecule 522 can extend to the middle of the membrane and then fold back on itself, so that both "A" segments are on the same side of the membrane, and the "B" segment is in the middle of the membrane. Therefore, in this example, the barrier 501 can be considered to be partially monolayer and partially bilayer. In other examples (not specifically shown) in which the barrier 501 substantially includes molecules 521 extending through the barrier in a linear manner, the barrier 501 can be substantially monolayer. In yet other examples (not specifically shown) in which the barrier 501 substantially includes molecules 522 extending to approximately the middle of the barrier and then folding back on itself, the barrier 501 can be substantially bilayer. The reactive portion 311 can be coupled to the hydrophilic segment 541, for example, to the terminal hydrophilic monomer of such segment. The reactive portion 311 can be similar to the reference FIG. 3B to FIG. 3C The described manner reacts with each other so as to form Figure 5B Bonds 281 are shown to cross-link molecules 521, 522. The nanopores may be inserted into the barrier at any suitable time, such as before or after cross-linking.

[0162] FIG. 6A to FIG. 6B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Fig. 6A A suspended membrane 601 is shown that includes molecules 621 of a BAB triblock copolymer that include hydrophilic "A" segments 642 coupled to and located between hydrophobic "B" segments 641. The membrane 601 can be similar to the reference FIG. 2A to FIG. 2BThe described approach uses a membrane support 200 and an optional ring 210 for suspension. In this example, the membrane 601 can have a two-layer architecture in which the "B" segments 641 are oriented toward each other. The hydrophobic ends of the BAB molecules can typically be located in the approximately middle of the membrane 601, and then the molecules extend toward either outer surface of the membrane and then fold back on themselves. Thus, the "B" segments are all located in the middle of the membrane, while the "A" segments are on one side or the other of the membrane. The reactive portion 311 can be coupled to the hydrophilic segment 642, for example, to one or more hydrophilic monomers of such a segment. In Figure 6B In the example shown, the reactive portion 311 may be similar to the reference FIG. 3B to FIG. 3C The described means react with each other to crosslink molecules 621 via bonds 281. The nanopores may be inserted into the barrier at any suitable time, for example before crosslinking or after crosslinking.

[0163] although FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B and FIG. 6A to FIG. 6B Reactive moieties are shown to be present at the ends of the hydrophilic A blocks on both sides of the hanging barrier, but it will be appreciated that such reactive moieties may be provided at any suitable location within the barrier and reacted to crosslink the amphiphilic molecules at such location. For example, the reactive moieties may be located at the AB interface. Illustratively, FIG. 7A to FIG. 7B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Fig. 7A The suspended barrier 701 shown comprises an AB diblock copolymer molecule 721, wherein the reactive portion 311 is located at the AB interface between the hydrophilic block 742 and the hydrophobic block 741. The barrier 701 may be similar to the reference FIG. 2A to FIG. 2B The membrane support 200 and the optional ring 210 are used for suspension in the manner described. FIG. 3B to FIG. 3C In the manner described, the reactive portion 311 can react so as to Figure 7B Bonds 281 are shown cross-linking amphiphilic molecules 721 .

[0164] Or, for example, FIG. 8A to FIG. 8B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Fig. 8A The suspended barrier 801 shown comprises an ABA triblock copolymer molecule 821, wherein the reactive portion 311 is located at the AB interface between the hydrophilic block 842 and the hydrophobic block 841. The barrier 801 can be similar to the reference FIG. 2A to FIG. 2B The described approach uses a membrane support 200 and an optional ring 210 for suspension. FIG. 5A to FIG. 5BAs described, each individual ABA molecule can be in one of two arrangements. For example, ABA molecule 821 can extend through the layer in a linear manner, with the "A" segment on each side of the membrane and the "B" segment in the middle of the membrane. Or, for example, ABA molecule 822 can extend to the middle of the membrane and then fold back on itself, so that both "A" segments are on the same side of the membrane and the "B" segment is in the middle of the membrane. Therefore, in this example, barrier 801 can be considered to be partially monolayer and partially bilayer. In other examples (not specifically shown) in which barrier 501 substantially includes molecules 821 extending through the barrier in a linear manner, barrier 501 can be substantially monolayer. In still other examples (not specifically shown) in which barrier 801 substantially includes molecules 822 extending to approximately the middle of the barrier and then folding back on itself, barrier 801 can be substantially bilayer. With reference to, for example, FIG. 3B to FIG. 3C In the manner described, the reactive portion 311 can react so as to Figure 8B Bonds 281 are shown cross-linking amphiphilic molecules 821 .

[0165] Or, for example, FIG. 9A to FIG. 8B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Fig. 9A The suspended barrier 901 shown comprises a BAB triblock copolymer molecule 921, wherein the reactive portion 311 is located at the AB interface between the hydrophilic block 942 and the hydrophobic block 941. The barrier 901 may be similar to the reference FIG. 2A to FIG. 2B The membrane support 200 and the optional ring 210 are used for suspension in the manner described. FIG. 3B to FIG. 3C In the manner described, the reactive portion 311 can react so as to Fig. 9B Bonds 281 are shown cross-linking amphiphilic molecules 921 .

[0166] In other examples, the reactive moiety may be located at the end of the hydrophobic B block. Illustratively, FIG. 10A to FIG. 10B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Fig. 10A The suspended barrier 1001 shown includes an AB diblock copolymer molecule 1021, wherein the reactive moiety 311 is located at the hydrophobic block 1041, for example, coupled to the terminal monomer 1043 of the hydrophobic block. The barrier 1001 can be similar to the reference FIG. 2A to FIG. 2B The membrane support 200 and the optional ring 210 are used for suspension in the manner described. FIG. 3B to FIG. 3C In the manner described, the reactive portion 311 can react so as to Fig. 10B The bonds 281 shown cross-link the amphiphilic molecules 1021. Alternatively, for example, FIG. 11A to FIG. 11BExemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Fig.11A The hanging barrier 1101 shown includes a BAB triblock copolymer molecule 1121, wherein the reactive moiety 311 is located at the hydrophobic block 1141, for example, coupled to the terminal monomer 1143 of the hydrophobic block. FIG. 3B to FIG. 3C In the manner described, the reactive portion 311 can react so as to Fig. 11B Bonds 281 are shown cross-linking amphiphilic molecules 1121.

[0167] Note that, in various examples provided herein, bonds 281 may be located in one or more specific planes within the barrier, depending on the specific arrangement and proximity of reactive moieties 311. For example, when bonds 281 crosslink the hydrophilic portion of an amphiphilic molecule, such as in reference FIG. 3A to FIG. 3D , FIG. 5A to FIG. 5B and FIG. 6A to FIG. 6B As described, and as will be referred to below FIG. 12A to FIG. 12C As further described, one set of keys 281 may be substantially located in a first plane providing a first outer surface of the barrier, and another set of keys 281 may be substantially located in a second plane providing a second outer surface of the barrier. For example, when the membrane is partially or substantially a double layer, the keys 281 of one of the membrane layers may be substantially located in a first plane providing the first outer surface of the membrane, and the keys 281 of another of the membrane layers may be substantially located in a second plane providing the second outer surface of the membrane; alternatively, when the membrane is substantially a single layer, one set of keys 281 of the membrane may be substantially located in a first plane providing the first outer surface of the membrane, and another set of keys 281 may be substantially located in a second plane providing the second outer surface of the membrane.

[0168] Alternatively, for example, when bond 281 cross-links the hydrophilic-hydrophobic interface of an amphiphilic molecule, for example, such as reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B and FIG. 9A to FIG. 9B As described, and as will be referred to below Fig.13 As further described, one set of keys 281 may be substantially located in a first plane within the layer, and another set of keys 281 may be substantially located in a second plane within the layer. For example, when the membrane is partially or substantially a double layer, the keys 281 of one of the membrane layers may be substantially located in a first plane within the first layer of the membrane, and the keys 281 of another of the membrane layers may be substantially located in a second plane within the second layer of the membrane; alternatively, when the membrane is substantially a single layer, one set of keys 281 of the membrane may be substantially located in a first plane within the membrane, and another set of keys 281 may be substantially located in a second plane within the membrane.

[0169] Alternatively, for example, when bond 281 cross-links the hydrophobic portion of an amphiphilic molecule, for example, such as reference FIG. 10A to FIG. 10B and FIG. 11A to FIG. 11B As described, and as will be referred to below FIG. 14A to FIG. 14B Further described, the key 281 of each of the membrane layers may be located in one or more planes between the two layers. Fig. 10B and Fig. 11B In the manner shown, bonds 281 may be formed between reactive moieties 311 within the plane of the respective layers and / or may be formed between reactive moieties 311 in planes different from each other.

[0170] A variety of reactive moieties are available for polymerization and cross-linking reactions, such as those described in FIG. 3A to FIG. 11B For example, the reactive moiety 311 can be selected from the group consisting of itaconic acid moiety, N-carboxylic anhydride moiety, disulfonyl pyridyl moiety, acrylate moiety, methacrylate moiety, acrylamide moiety, methacrylamide moiety, styrene moiety, maleic acid moiety, carboxylic acid moiety, thiol moiety, allyl moiety, vinyl moiety, propargyl moiety and maleimide moiety. In some examples, the polymerization reaction includes ring-opening polymerization or step-growth polymerization.

[0171] For both AB and BAB architectures, there are ways to have reactive moieties at the ends of the B blocks, and these moieties can be crosslinked / polymerized (so the crosslinks can extend laterally within the film). Examples of polymerizable moieties include, but are not limited to, acrylate or acrylamide derivatives; examples of crosslinkable moieties include, but are not limited to, thiols and alkenes / alkynes (to form sulfides), thiols and maleimides (to form sulfosuccinimide), azides and alkynes / BCN / DBCO, thiols and thiols (to form disulfides), dimethylmaleimide moieties, etc.

[0172] For the ABA architecture, the B block itself has no "free ends", however, some B blocks may be flanked by central reactive moieties. Illustratively, such B blocks may be synthesized as follows: a homobifunctional initiator containing a third central reactive moiety (such as those described above); the latter may not participate in the polymerization reaction (this may be achieved by ensuring orthogonality or by being protected). Such polymerization may generate a telechelic B block that can be terminated in a manner that generates reactive ends that can react with A blocks to generate the ABA architecture while retaining the aforementioned central reactive moiety for subsequent crosslinking / polymerization purposes in the film. Alternative ways to generate such B blocks include, but are not limited to: using a heterobifunctional initiator (one functional group is intended for A block coupling and the other functional group is the initiation moiety) to polymerize the B block, wherein the termination step uses a homobifunctional initiator containing a third central reactive moiety (such as those described above) and two reactive moieties that can react with the two growing B blocks.

[0173] For AB, BAB, and ABA architectures, reactive moieties may be provided at the AB interface and these moieties may be cross-linked / polymerized (so that the cross-links may extend laterally within the film). Examples of polymerizable moieties include, but are not limited to, itaconic acid derivatives of maleic acid; examples of cross-linkable moieties include, but are not limited to, thiols and alkenes / alkynes (to generate sulfides), thiols and thiols (to generate disulfides), dimethylmaleimide moieties, and the like.

[0174] For both AB and ABA architectures, reactive moieties may be provided at the ends of the A blocks, and these moieties may be crosslinked / polymerized (so the crosslinks may extend laterally across the exterior of the membrane). Examples of polymerizable moieties include, but are not limited to, acrylate or acrylamide derivatives; examples of crosslinkable moieties include, but are not limited to, thiols and alkenes / alkynes (to generate sulfides), thiols and thiols (to generate disulfides), azides and alkynes / BCN / DBCO, dimethylmaleimide moieties, and the like.

[0175] For the BAB architecture, the A block itself has no "free ends", however, there may be central reactive moieties flanking the A block. Illustratively, such A blocks may be synthesized as follows: a homobifunctional initiator including a third central reactive moiety (such as those described above); the latter may not participate in the polymerization reaction (this may be achieved by ensuring orthogonality or by being protected). Such polymerization will generate a telechelic A block, which can be terminated in a manner to generate reactive ends that can react with B blocks to generate the BAB architecture while retaining the aforementioned central reactive moieties for subsequent crosslinking / polymerization purposes in the membrane. There are alternative ways to generate such A blocks, including but not limited to: using a heterobifunctional initiator (one functional group is intended for B block coupling and the other functional group is the initiation moiety) to polymerize the A block, wherein the termination step uses a homobifunctional initiator containing a third central reactive moiety (such as those described above) and two reactive moieties that can react with the two growing A blocks.

[0176] Figure 28 to Figure 31 Depicted are exemplary chemical reactions between different reactive moieties in different positions of a block copolymer, such as described above. For simplicity, only AB block copolymers are depicted, but such reactions are also applicable to ABA or BAB block copolymers if relevant.

[0177] More specifically, Fig.28 Examples are shown where the A block and B block of a block copolymer are coupled together in a manner that creates / generates / leaves reactive moieties at the AB interface, and these moieties then react to make the block copolymer molecule in a manner such as reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B , FIG. 9A to FIG. 9B , FIG. 12A to FIG. 12B and / or Fig.13The described methods are interconnected with each other. Fig.28 In the example (A) shown, the A block and the B block of the block copolymer molecule are coupled together using itaconic acid moieties, and the itaconic acid moieties are polymerized using a free radical polymerization process to reinforce at least one layer of the film, for example, in a manner such as reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B or FIG. 9A to FIG. 9B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). Fig.28 In the example (B) shown, the A block and the B block of the block copolymer molecule are coupled together using an acrylamide moiety, and the acrylamide moiety is polymerized using a free radical polymerization process to strengthen at least one layer of the film, for example, in a manner such as reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B or FIG. 9A to FIG. 9B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). Fig.28 In the example (C) shown, the A block and the B block of the block copolymer molecule are coupled together using maleic acid moieties, and the maleic acid moieties are polymerized using a free radical polymerization process to strengthen at least one layer of the film, for example, in a manner such as reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B or FIG. 9A to FIG. 9B Optionally, a similar process may be used to enhance the second layer of the membrane (if present).

[0178] exist Fig.28 In the example (D) shown, the A block and the B block of the first block copolymer molecule are coupled together using a first part, which includes one or more primary amines (-NH2) in the example shown, here the first primary amine and the second primary amine; and the A block and the B block of the second block copolymer molecule are coupled together using a second part, which includes one or more NHS esters (-ONHS) in the example shown, here the first NHS ester and the second NHS ester. The first part (e.g., the amine part) is reacted with the second part (e.g., the NHS ester) using a condensation process to enhance at least one layer of the membrane, for example, such as reference FIG. 12A to FIG. 12B or Fig.13 The described approach uses a first reactive moiety and a second reactive moiety to obtain a reaction similar to that described in reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B or FIG. 9A to FIG. 9B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). In the example where each molecule includes two or more amines or two or more NHS esters, two or more such molecules can be cross-linked to each other. For example, when each molecule includes three or more amines or three or more NHS esters, three or more such molecules can be cross-linked to each other. Fig.28 The R groups shown in Example (A) and Example (D) may include any suitable moiety, such as an aliphatic or aromatic or other non-reactive spacer.

[0179] Fig.29 Examples are shown where the polymerizable moiety is at the end group of the A block or at the end group of the B block, and the moiety is then polymerized to make the molecules of the block copolymer in a manner such as reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B or FIG. 11A to FIG. 11B The described methods are interconnected with each other. Fig.29 In the example (A) shown, the acrylic moiety is located at the end of the A block or at the end of the B block, and the acrylic moiety is polymerized using a free radical polymerization process to strengthen at least one layer of the film, for example, in a manner such as reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B or FIG. 11A to FIG. 11B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). Fig.29 In the example (B) shown, the styrene moiety is located at the end of the A block or at the end of the B block, and the styrene moiety is polymerized using a free radical polymerization process to strengthen at least one layer of the film, for example, such as reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B or FIG. 11A to FIG. 11B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). Fig.29 In the example (C) shown, the N-carboxylic anhydride portion is located at the end of the A block or at the end of the B block, and the N-carboxylic anhydride portion is polymerized using a ring-opening polymerization process to strengthen at least one layer of the film, for example, in a manner such as reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B or FIG. 11A to FIG. 11B Optionally, a similar process may be used to enhance the second layer of the membrane (if present). Fig.29 The R group shown in example (C) may include any suitable moiety, such as an aliphatic or aromatic or other non-reactive spacer.

[0180] Fig.30 Additional examples are shown where the A block and B block of a block copolymer are coupled together at the AB interface using reactive moieties, and these moieties are then reacted to allow the molecules of the block copolymer to be structured as in reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B , FIG. 9A to FIG. 9B, FIG. 12A to FIG. 12B and / or Fig.13 The described methods are interconnected with each other. Fig.30 In the example (A) shown, the A block and the B block of the block copolymer molecule are coupled together using a moiety including a thiol group (-SH), and the moieties are coupled together using a disulfide formation process to strengthen at least one layer of the membrane, for example, in a manner such as reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B or FIG. 9A to FIG. 9B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). Fig.30 In example (A), the crosslinking is optionally reversible.

[0181] exist Fig.30 In the example (B) shown, the A block and the B block of the first block copolymer molecule are coupled together using a first part, which includes one or more thiol groups (-SH) in the example shown; and the A block and the B block of the second block copolymer molecule are coupled together using a second part, which includes one or more alkynes or alkenes in the example shown. For example, the first part (e.g., the thiol part) is reacted with the second part (e.g., the alkyne or alkene) using a thiol-ene / alkyne click chemistry process (which is irreversible) to enhance at least one layer of the film, for example, such as reference FIG. 12A to FIG. 12B or Fig.13 The described approach uses a first reactive moiety and a second reactive moiety to obtain a reaction similar to that described in reference FIG. 7A to FIG. 7B , FIG. 8A to FIG. 8B or FIG. 9A to FIG. 9B Optionally, a similar process can be used to strengthen the second layer of the film (if present). In the example where each molecule includes two or more thiols or two or more alkynes or alkenes, two or more such molecules can be cross-linked to each other. For example, when each molecule includes three or more thiols or three or more alkenes, three or more such molecules can be cross-linked to each other. Fig.30 The R groups shown in Example (A) and Example (B) may include any suitable moiety, such as an aliphatic or aromatic or other non-reactive spacer.

[0182] Fig.31 Additional examples are shown where the reactive moieties are at the end groups of the A blocks or at the end groups of the B blocks, and these moieties then react to make the molecules of the block copolymer in a manner such as reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B , FIG. 11A to FIG. 11B , FIG. 12A to FIG. 12B or FIG. 14A to FIG. 14B The described methods are interconnected with each other. Fig.31In the example (A) shown, the dimethylmaleimide is located at the end of the A block or at the end of the B block, and the dimethylmaleimide moiety reacts in a [2+2] cycloaddition process to strengthen at least one layer of the film, for example, in a manner such as reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B or FIG. 11A to FIG. 11B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). Fig.31 In example (A), the crosslinking is optionally reversible. Fig.31 In the example (B) shown, the disulfide pyridyl moiety is located at the end of the A block or at the end of the B block, and the disulfide pyridyl moiety is polymerized using a disulfide forming process (which can use a reducing agent or a free radical initiator) to strengthen at least one layer of the film, for example, such as reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B or FIG. 11A to FIG. 11B Optionally, a similar process can be used to enhance the second layer of the membrane (if present). Fig.31 In example (B), the cross-linking is optionally reversible.

[0183] exist Fig.31 In the example (C) shown, the first block copolymer molecule includes a first portion (e.g., a disulfide pyridyl group in the example shown), and the second block copolymer molecule includes a second portion (e.g., an alkene or an alkyne in the example shown). For example, the first portion (e.g., a disulfide pyridyl group) is reacted with one or more second portions (e.g., an alkyne or an alkene) using a thiol-ene / alkyne click chemistry process (which is irreversible) to enhance at least one layer of the film, for example, such as in reference FIG. 12A to FIG. 12B or FIG. 14A to FIG. 14B The described approach uses a first reactive moiety and a second reactive moiety to obtain a reaction similar to that described in reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B , FIG. 11A to FIG. 11B , FIG. 12A to FIG. 12B or FIG. 14A to FIG. 14B The structure described; Optionally, a similar process can be used to strengthen the second layer of the film (if present). In the example shown, the alkyne can react with up to two thiols. The first reaction between the alkyne and thiol moieties consumes the triple bond and generates a double bond, which in turn can react with another thiol.

[0184] exist Fig.31In the example (D) shown, the first block copolymer molecule includes a first portion (e.g., a disulfide pyridyl group in the example shown), and the second block copolymer molecule includes a second portion (e.g., a maleimide in the example shown). For example, the first portion (e.g., a disulfide pyridyl group) is reacted with the second portion (e.g., a maleimide) using a thiol-Michael click chemistry process (which is pH reversible) to enhance at least one layer of the membrane, for example, such as in reference FIG. 12A to FIG. 12B or FIG. 14A to FIG. 14B The described approach uses a first reactive moiety and a second reactive moiety to obtain a reaction similar to that described in reference FIG. 3A to FIG. 3D , Figure 4 , FIG. 5A to FIG. 5B , FIG. 10A to FIG. 10B , FIG. 11A to FIG. 11B , FIG. 12A to FIG. 12B or FIG. 14A to FIG. 14B The structure described; optionally, a similar process can be used to strengthen the second layer of the membrane (if present).

[0185] With reference such as Figure 3B The polymerization reaction may be initiated in an initiator. Non-limiting examples of suitable initiators include photoinitiators, redox systems or photons (such as ultraviolet (UV) light). Illustratively, the photoinitiator is UV activated and is selected from the group consisting of: 2,2-dimethoxy-2-phenylacetophenone, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and phenyl-2,4,6-trimethylbenzoylphosphinate lithium, whose structures are shown below:

[0186] 2,2-Dimethoxy-2-phenylacetophenone (DMAP):

[0187] 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (V50):

[0188] 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959): and

[0189] Lithium phenyl-2,4,6-trimethylbenzoylphosphinate: In the examples described herein using UV light, the barrier can be located within a structure that is at least partially transparent to the UV light in order to facilitate crosslinking and / or reversing crosslinking. For example, the barrier can be located within a flow cell, the cover of which can be at least partially transparent to the UV light used for crosslinking and / or reversing crosslinking, so that a sufficient amount of UV light reaches the barrier to adequately perform the reaction.

[0190] In some examples, the redox system includes potassium persulfate and N,N,N',N'-tetramethylethylenediamine, and their structures are shown below:

[0191] Potassium Persulfate (KPS): and

[0192] N,N,N',N'-Tetramethylethylenediamine (TEMED): Ammonium persulfate and TEMED can also be used as a redox system.

[0193] Although FIG. 3A to FIG. 11B Crosslinking using polymerized amphiphilic molecules may be shown, but it should be understood that other types of crosslinking reactions, such as coupling reactions, may also be appropriately used to crosslink amphiphilic molecules. For example, FIG. 12A to FIG. 12B Exemplary procedures for forming another alternative barrier comprising cross-linked amphiphilic molecules are schematically illustrated. Fig. 12A A hanging barrier 1200 is shown. Fig. 12A As shown, in some aspects, barrier 1200 can be constructed similar to reference Figure 1 and FIG. 2A to FIG. 2B The membrane 101, for example, may include a layer 1201 including a first plurality of amphiphilic molecules and a layer 1202 including a second plurality of amphiphilic molecules. Some of the amphiphilic molecules of the layer 1201 (and optionally also the layer 1202) may include a reactive portion 1211 (here, a molecule 1221, which is located in both the layer 1201 and the layer 1202), while other amphiphilic molecules of the layer 1201 (and optionally also the layer 1202) may include a reactive portion 1212 (here, a molecule 1221, which is located in both the layer 1201 and the layer 1202) different from the reactive portion 1211. In a method such as Fig. 12A In the example shown, amphiphilic molecules 1221, 1222 include molecules of an AB diblock copolymer, wherein the hydrophilic "A" segment 1232 of molecule 1221 may include a reactive portion 1211, and the A segment 1232 of molecule 1222 may include, for example, a reactive portion 1212 coupled to a terminal hydrophilic monomer 1242. In other examples, only one type of reactive portion is used. Suitable methods for forming a suspended film using a barrier support are known in the art, such as "coating", such as brush coating (manually), mechanical coating (e.g., using a stirring rod), and bubble coating (e.g., using flow through a device).

[0194] Reactive moieties 1211, 1212 can react with each other in a manner that allows the amphiphilic molecules to fully or partially cross-link with each other. Figure 3BIn the manner described, barrier 1200 may be contacted with a fluid having dissolved initiator 1221 which chemically reacts with reactive moieties 1211 and / or 1212, for example to form a product in which amphiphilic molecules 221 are cross-linked to one another, such as via coupling of moiety 1211 with moiety 1212.

[0195] Fig. 12B The product of the polymerization reaction between amphiphilic molecules is shown, where a bond 1281 is formed between reactive moiety 1211 and reactive moiety 1212 (whose fill changes from cross-hatching to white to indicate that such moieties have reacted and are no longer available for reaction). Fig. 12B It may be suggested that each reactive moiety 1211 is cross-linked with two moieties 1212 via respective bonds 1281 and each reactive moiety 1212 is cross-linked with two moieties 1211 via respective bonds 1281, but it is understood that each reactive moiety may form bonds with any suitable number of other such reactive moieties (e.g., one, two, three, or more than three other such reactive moieties), and that bonds 1281 may be of different types from one another, e.g., may include different moieties from one another. The relative proportions of such products may be controlled in a manner such as described elsewhere herein, e.g., by the type of reactive moiety used, the type of initiator used, and the reaction conditions, so as to control the amount of cross-linking provided using the reaction between reactive moieties 1211 and reactive moieties 1212. Additionally, in some examples, the amount of cross-linking can be controlled by mixing amphiphilic molecules including reactive moieties 1211, 1212, respectively, in appropriate proportions with other amphiphilic molecules that do not include reactive moieties 1211 and 1212 or that include different reactive moieties and / or have different architectures (e.g., AB can be mixed with ABA and / or BAB; ABA can be mixed with AB and / or BAB; and / or BAB can be mixed with AB and / or ABA). For example, the ratio between different types of amphiphilic molecules can be selected to determine the extent of cross-linking. Illustratively, in the case of using two different amphiphilic molecules, the ratio can be selected (e.g., a ratio of monofunctional molecules to bifunctional molecules of about 1:2) to provide substantially complete cross-linking between these molecules, while a lower ratio (e.g., a ratio of about 1:1) can leave some molecules unreacted, resulting in only partial cross-linking.

[0196] In some examples, after cross-linking of the amphiphilic molecules 1221, 1222, or before cross-linking of the amphiphilic molecules 1221, 1222, the nanopore 110 may be formed in a manner such as with reference to Figure 3D or Figure 4 Insert into the barrier in the manner described.

[0197] With reference FIG. 5A to FIG. 5B , FIG. 6A to FIG. 6B , FIG. 7A to FIG. 7B, FIG. 8A to FIG. 8B , FIG. 9A to FIG. 9B , FIG. 10A to FIG. 10B and FIG. 11A to FIG. 11B In a similar manner, the type of amphiphilic molecules used and the location of the reactive moieties 1211, 1212 within such molecules may be varied appropriately. For example, moieties 1211 and 1212 may be located opposite to each other. FIG. 12A to FIG. 12C At the AB interface of the molecule, or vice versa FIG. 12A to FIG. 12C Alternatively, for example, moieties 1211 and 1212 may instead be provided within an ABA triblock copolymer, such as at the A block, at the AB interface, or at the B block. Alternatively, for example, moieties 1211 and 1212 may instead be provided within a BAB triblock copolymer, such as at the A block, at the AB interface, or at the B block. Fig.13 FIG14 shows an exemplary operation for forming another alternative barrier comprising crosslinked amphiphilic molecules, wherein moieties 1211 and 1212 are provided at the AB interface of an ABA triblock copolymer. FIG15 shows an exemplary operation for forming another alternative barrier comprising crosslinked amphiphilic molecules, wherein moieties 1211 and 1212 are provided at the B block of a BAB triblock copolymer. The barrier is formed at Fig.13 and Fig.14A and may be appropriately formed into a document such as that described herein (e.g., reference FIG. 12A to FIG. 12C ) are cross-linked in the manner of the bond 1281 provided.

[0198] A variety of reaction schemes are available for the coupling reaction, such as reference FIG. 12A to FIG. 14BFor example, the coupling reaction may include a thiol-ene click reaction, a thiol-alkyne click reaction, a strain-promoted alkyne-azide cycloaddition (e.g., azide with DBCO or BCN), an amide coupling (primary amide with N-hydroxysuccinimide (NHS) or pentafluorophenyl (PFP) activated ester), a thiol / aza-Michael reaction (thiol / primary amine with maleimide, maleic acid, fumaric acid, acrylic acid or acrylamide), a [2+2] photocycloaddition (e.g., dimethylmaleimide, enone or coumarin), a protein-ligand interaction (e.g., biotin-avidin or biotin-streptavidin), a condensation (e.g., an amine with an NHS ester), or a host-guest chemistry (e.g., cyclodextrin-adamantane). Such reactions may be irreversible. Alternatively, a reversible reaction can be used, such as disulfide formation, imine formation, [2+2] cycloaddition, thiol-Michael click reaction or enamine formation (e.g., aldehyde / ketone). Non-limiting examples of reactive moieties 1211, 1212 may include disulfide pyridyl moieties, fatty acylamino moieties, propargyl moieties, azido moieties, DBCO moieties, BCN moieties, amine moieties, activated carboxyl moieties, dimethylmaleimide moieties, propargyl moieties, NHS esters or maleimide moieties. Optionally, the coupling reaction can be initiated using an initiator, such as a free radical initiator, a redox system, a reducing agent or a photon. Non-limiting examples of free radical initiators include 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and 2,2'-azobis(2-methylpropionamidine) dihydrochloride, the structures of which are provided above. Non-limiting examples of redox systems are potassium or ammonium persulfate and N,N,N',N'-tetramethylethylenediamine, the structures of which are provided above. Non-limiting examples of reducing agents include tris(2-carboxyethyl)phosphine, dithiothreitol, sodium ascorbate, and phosphine.

[0199] Fig. 12C Schematically shows a reference FIG. 3A to FIG. 3D or FIG. 12A to FIG. 12B An exemplary diblock copolymer molecule of the process described, and Fig. 14B Schematically shows a reference FIG. 11A to FIG. 11B or Fig.14A An exemplary triblock copolymer molecule in the described procedures. Fig. 12C and Fig. 14B In the figure, from left to right, the groups include acrylamide (for polymerization), methacrylamide (for polymerization), pentafluorobenzyl methacrylate (for polymerization), and thiol (for coupling). Figure 28 to Figure 31 Other non-limiting examples are described. Other non-limiting examples are described elsewhere herein.

[0200] FIG. 15A to FIG. 15CFurther details of membranes using block copolymers are schematically shown, which may be included in Figure 1 Nanopore compositions and devices and for reference FIG. 3A to FIG. 14B It should be understood that such membranes may be suitably adapted for use with any other composition or device and are not limited to use with nanopores. FIG. 15A to FIG. 15C The hydrophilic block of the membrane may include reactive moieties 311, 1211, or 1212, such as described elsewhere herein.

[0201] Reference now Fig. 15B , the membrane 1501 uses a diblock "AB" copolymer. The membrane 1501 includes a first layer 1507 that can contact the fluid 120 and a second layer 1508 that can contact the fluid 120', and the contact method is similar to that of the reference Figure 1 The first layer 1507 includes a first plurality of molecules 1502 of a diblock AB copolymer, and the second layer 1508 includes a second plurality of molecules 1502 of a diblock AB copolymer. Fig. 15B As shown, each molecule 1502 of the diblock copolymer includes a hydrophobic block (denoted as "B" and having a length of approximately "B"), which is coupled to a hydrophilic block (denoted as "A" and having a length of approximately "A"). The hydrophilic A blocks of the first plurality of molecules 1502 (molecules forming layer 1507) form a first outer surface of the membrane 1501 (e.g., in contact with fluid 120). The hydrophilic A blocks of the second plurality of molecules 1502 (molecules forming layer 1508) form a second outer surface of the membrane 1502 (e.g., in contact with fluid 120'). The respective ends of the hydrophobic B blocks of the first and second plurality of molecules are arranged within the membrane 1501 in a manner such as Fig. 15B As shown, substantially all molecules 1502 within layer 1507 may extend substantially linearly and in the same orientation as one another, and similarly, substantially all molecules 1502 within layer 1508 may extend substantially linearly and in the same orientation as one another (which is opposite to the orientation of the molecules within layer 1507). Thus, each of first layer 1507 and second layer 1508 may have a thickness of approximately A+B, and film 1501 may have a thickness of approximately 2A+2B. In some examples, length A is from about 2 repeating units (RU) to about 100RU, or from about 1 repeating unit (RU) to about 50RU, for example, from about 5RU to about 40RU, or from about 10RU to about 30RU, or from about 10RU to about 20RU, or from about 20RU to about 40RU. Additionally or alternatively, in some examples, the length B is from about 2RU to about 100RU, or from about 5RU to about 100RU, for example, from about 10RU to about 80RU, or from about 20RU to about 50RU, or from about 50RU to about 80RU. Optionally, with reference to Fig. 15BThe barrier 1501 may be such as reference FIG. 2A to FIG. 2B The described manner is suspended across the aperture.

[0202] Reference now Fig. 15C , the membrane 1511 uses a triblock "BAB" copolymer. The membrane 1511 includes a first layer 1517 that can contact the fluid 120 and a second layer 1518 that can contact the fluid 120', and the contact method is similar to that of the reference Figure 1 The first layer 1517 includes a first plurality of molecules 1512 of a triblock copolymer, and the second layer 1518 includes a second plurality of molecules 1512 of a triblock copolymer. Fig. 15C As shown, each molecule 1512 of the triblock copolymer includes a first hydrophobic block and a second hydrophobic block, each of which is represented by "B" and has a length of approximately "B", and a hydrophilic block disposed between the first hydrophobic block and the second hydrophobic block, which is represented by "A" and has a length of approximately "A". The hydrophilic A blocks of the first plurality of molecules 1512 (molecules forming layer 1517) form a first outer surface of the membrane 1511 (e.g., in contact with fluid 120). The hydrophilic A blocks of the second plurality of molecules 1512 (molecules forming layer 1518) form a second outer surface of the membrane 1511 (e.g., in contact with fluid 120'). The respective ends of the hydrophobic B blocks of the first and second plurality of molecules are arranged within the membrane 1511 in a manner such as Fig. 15C 1511. As shown in the figure, substantially all molecules 1512 in layer 1517 can extend in the same orientation as each other, and can be folded at the A block so that the A block can contact the fluid, and the B block is inside the film 1511. Similarly, substantially all molecules 1512 in layer 1518 can extend in the same orientation as each other (it is opposite to the orientation of the molecules in layer 1517), and can be folded at their A block so that the A block contacts the fluid, and the B block is inside the film 1511. Therefore, the first layer 1517 and the second layer 1518 can each have a thickness of about A / 2+B, and the film 1511 can have a thickness of about A+2B. In some examples, the length A is from about 2RU to about 100RU, such as from about 10RU to about 80RU, or from about 20RU to about 50RU, or from about 50RU to about 80RU. Additionally or alternatively, in some examples, the length B is from about 2RU to about 100RU, or from about 5RU to about 100RU, for example, from about 10RU to about 80RU, or from about 20RU to about 50RU, or from about 50RU to about 80RU. Optionally, with reference to Fig. 15B The barrier 1511 may be such as reference FIG. 2A to FIG. 2B The described manner is suspended across the aperture.

[0203] Reference now Fig.15A, membrane 1521 uses a triblock "ABA" copolymer. Membrane 1521 includes a layer 1529 that can contact both fluids 120 and 120'. Layer 1529 includes a plurality of molecules 1522 of a triblock ABA copolymer. Fig.15A As shown, each molecule 1522 of the triblock copolymer includes a first hydrophilic block and a second hydrophilic block, each of which is represented by "A" and has a length of about "A", and a hydrophobic block disposed between the first hydrophilic block and the second hydrophilic block, which is represented by "B" and has a length of about "B". The hydrophilic A blocks at the first ends of the molecules 1522 (molecules forming layer 1529) form a first outer surface of the membrane 1521 (e.g., in contact with the fluid 120). The hydrophilic A blocks at the second ends of the molecules 1522 form a second outer surface of the membrane 1521 (e.g., in contact with the fluid 120'). The hydrophobic B blocks of the molecules 1522 are arranged in a manner such as Fig. 15C As shown, most of the molecules 1522 in layer 1529 may extend substantially linearly and in the same orientation as each other. Optionally, as shown, Fig.15A As shown, some of the molecules 1522' may fold at their B blocks so that two hydrophilic A blocks of such molecules may contact the same fluid with each other. Fig.15A The examples shown can be considered to be partially monolayer and partially bilayer. In other examples (not specifically shown), layer 1529 can be a complete monolayer or can be a complete bilayer, for example, as also described elsewhere herein. Regardless of whether the film includes substantially linearly extending molecules 1522 and / or folded molecules 1522', such as Fig.15A As shown, layer 1529 may have a thickness of approximately 2A+B. In some examples, length A is from about 1RU to about 100RU, for example, from about 2RU to about 100RU, or from about 10RU to about 80RU, or from about 20RU to about 50RU, or from about 50RU to about 80RU. Additionally or alternatively, in some examples, length B is from about 2RU to about 100RU, or from about 5RU to about 100RU, for example, from about 10RU to about 80RU, or from about 20RU to about 50RU, or from about 50RU to about 80RU. It should be understood that any end group coupled to the hydrophilic or hydrophobic block contributes to the overall thickness of the barrier. Optionally, reference Fig.15A The barrier 1521 may be such as reference FIG. 2A to FIG. 2B The described manner is suspended across the aperture.

[0204] It should be understood that the layers of the various films provided herein can be configured to have any suitable size. Illustratively, to form films of similar size to each other:

[0205] ABA triblock copolymer ( Fig.15A) may have two hydrophilic blocks each of length A (M of each A block w = x) and a hydrophobic block (M w =y); when self-assembled, those ABA triblock copolymers will 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.

[0206] AB diblock copolymer ( Fig. 15B ) may have one hydrophilic block (M) of length A w = x) and a hydrophobic block (M w =y / 2); when self-assembled, those AB diblock copolymers will 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.

[0207] BAB triblock copolymer ( Fig. 15C ) may have one hydrophilic block (M) of length A w = x) and two hydrophobic blocks each of length B (M of each B block w =y / 2); when self-assembled, those BAB triblock copolymers will form a membrane 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. Additionally or alternatively, the polymers filled into the layers of the membrane can affect the hydrophilicity ratio of each membrane, where the hydrophilicity ratio can be defined as the molecular weight of the hydrophilic block to 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 ( Fig.15A ), hydrophilicity ratio = 2x / (2x+y);

[0208] AB diblock copolymer ( Fig. 15B ), hydrophilicity ratio = x / (x+y / 2); and

[0209] BAB triblock copolymer ( Fig. 15C ), hydrophilicity ratio = x / (x+y).

[0210] The diblock copolymers and triblock copolymers of the present invention may include any suitable combination of hydrophobic blocks and hydrophilic blocks. In some examples, the hydrophilic A block may include a polymer selected from the group consisting of: N-vinyl pyrrolidone, polyacrylamide, zwitterionic polymers, hydrophilic polypeptides, nitrogen-containing units, and poly(ethylene oxide) (PEO). Exemplarily, the polyacrylamide may be selected from the group consisting of: poly(N-isopropylacrylamide) (PNIPAM) and charged polyacrylamide and phosphoric acid functionalized polyacrylamide. Non-limiting examples of zwitterionic monomers that can be polymerized to form zwitterionic polymers include:

[0211]

[0212] Non-limiting examples of hydrophilic polypeptides include:

[0213]

[0214] Non-limiting examples of charged polyacrylamides are wherein n is from about 1 to about 100. Non-limiting examples of nitrogen-containing units include:

[0215] In some examples, the hydrophobic B block may include a polymer selected from the group consisting of poly(dimethylsiloxane) (PDMS), polybutadiene (PBd), polyisoprene, polymyrcene, polychloroprene, hydrogenated polydienes, fluorinated polyethylene, polypeptides, and poly(isobutylene) (PIB). Non-limiting examples of hydrogenated polydienes include saturated polybutadiene (PBu), saturated polyisoprene (PI), saturated poly(myrcene),

[0216]

[0217] 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, 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 group, zwitterionic group, sulfate, sulfonate, alkyl, aryl, orthogonal functional group and hydrogen, and R2 is a reactive moiety selected from the group consisting of maleimide group, allyl group, propargyl group, BCN group, carboxylate group, amine group, thiol group, DBCO group, azide group, N-hydroxysuccinimide group, biotin group, carboxyl group, NHS activated ester and other activated esters. In other non-limiting examples of hydrogenated polydiene, 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, azido groups, N-hydroxysuccinimide groups, biotin groups, carboxyl groups, NHS activated esters and other activated esters. Non-limiting examples of fluorinated polyethylene are Non-limiting examples of hydrophobic polypeptides include (0 <x<1):

[0218]

[0219] wherein n is from about 2 to about 100.

[0220] In one non-limiting example, the AB diblock copolymer includes PDMS-b-PEO, wherein "-b-" indicates that the polymer is a block copolymer. In another non-limiting example, the AB diblock copolymer includes PBd-b-PEO. In another non-limiting example, the AB diblock copolymer includes PIB-b-PEO. In another non-limiting example, the BAB triblock copolymer includes PDMS-b-PEO-b-PDMS. In another non-limiting example, the BAB triblock copolymer includes PBd-b-PEO-b-PBd. In another non-limiting example, the BAB triblock copolymer includes PIB-b-PEO-b-PIB. In another non-limiting example, the ABA triblock copolymer includes PEO-b-PBd-b-PEO. In another non-limiting example, the ABA triblock copolymer includes PEO-b-PDMS-b-PEO. In another non-limiting example, the ABA triblock copolymer includes PEO-b-PIB-b-PEO. It should be understood that any suitable hydrophilic block can be used with any suitable hydrophobic block. Additionally, in examples including two hydrophilic blocks, these blocks may, but need not, include the same polymer as each other. Similarly, in examples including two hydrophobic blocks, these blocks may, but need not, include the same polymer as each other.

[0221] The corresponding molecular weight, glass transition temperature and chemical structure of the hydrophobic block and the hydrophilic block can be appropriately selected to provide the membrane with appropriate stability in use and the ability to insert into the nanopore. For example, the corresponding molecular weight of the hydrophobic block and the hydrophilic block can affect the thickness of each block (and thus the layer of the membrane), and can affect the stability and the ability to insert into the nanopore, such as by electroporation, pipetting pump circulation or detergent-assisted nanopore insertion. Additionally or alternatively, the molecular weight ratio of the hydrophilic block and the hydrophobic block can affect the self-assembly of those blocks into the layer of the membrane. Additionally or alternatively, the corresponding glass transition temperature (T g ) can affect the lateral fluidity of the membrane layer; therefore, in some examples, the T of the hydrophobic block and / or the hydrophilic block g Less than the operating temperature of the device, such as less than room temperature, and in some examples less than about 0° C. is useful. Additionally or alternatively, the chemical structure of the hydrophobic and hydrophilic blocks can affect the way the chains pack into layers and the stability of those layers.

[0222] For nanopore sequencing applications, membrane fluidity may be considered beneficial. Without wishing to be bound by any theory, it is believed that the fluidity of the block copolymer membrane is largely imparted by the physical properties of the hydrophobic "B" block. More specifically, the "low T g "Hydrophobic polymers (e.g., having a T below about 0°C) g ) can be used to generate a B block having a high T g "Polymers (e.g., having a T above room temperature) g ) of the B block is more fluid. For example, in some examples, the hydrophobic B block of the copolymer has a T of less than about 20°C, less than about 0°C, or less than about -20°C. g .

[0223] With low T g The hydrophobic B block of can be used to help maintain membrane flexibility under conditions suitable for nanopore sequencing, for example, as in reference Figure 32 to Figure 36 In some examples, a sufficiently low T g The hydrophobic B block may include or may consist essentially of PIB, which PIB may be expected to have a T in the range of about -75°C to about -25°C. g In other examples, a sufficiently low T g The hydrophobic B block of may include or may consist essentially of PDMS, which PDMS may be expected to have a T in the range of about -135°C (or lower) to about -115°C. g In other examples, a sufficiently low T gThe hydrophobic B block of may include PBd or may consist essentially of PBd. Different forms of PBd may be used as the B block in the barrier of the present invention. For example, the cis-1,4 form of PBd may be expected to have a T in the range of 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. g Alternatively, 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 other examples, a sufficiently low T g The hydrophobic B block of may include or may consist essentially of polymyrcene (PMyr), which PMyr may be expected to have a T in the range of about -75°C to about -45°C. g In other examples, a sufficiently low T g The hydrophobic B block of may include or may 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, it is expected that the cis-1,4 form of PIP has a T in the range of 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 .

[0224] Hydrophobic B blocks with fully saturated carbon backbones (such as PIB) can also be expected to increase the chemical stability of block copolymer membranes. Additionally or alternatively, branched structures within the hydrophobic B blocks, such as using PIB, can be expected to induce chain entanglement, which can be expected to enhance the stability of block copolymer membranes. This can allow the use of smaller hydrophobic blocks, reducing the adverse effects of hydrophobic mismatches on inserted nanopores. Additionally or alternatively, hydrophobic B blocks with relatively low polarity can be expected to be better electrical insulators, thereby improving the electrical properties of devices for nanopore sequencing (e.g., such as reference Figure 32 to Figure 36 described above).

[0225] In some examples of AB copolymers including PBd as the B block and PEO as the A block shown below, R is a reactive group 311, 1211, or 1212; m = about 2 to about 100; and n = about 2 to about 100.

[0226]

[0227] In some non-limiting examples, R is a reactive group 311, 1211, or 1212; n=about 8 to about 50; and m=about 1 to about 20. In some non-limiting examples, R is a reactive group 311, 1211, or 1212; n=about 10 to about 15; and m=about 5 to about 15.

[0228] In some examples of ABA copolymers shown below that include one or more PIB blocks as B blocks and PEO as A blocks, at least one of R1 and R2 can be a reactive group 311, 1211, or 1212, and the other of R1 and R2 can be a reactive group 311, 1211, or 1212, or can be a group that is not reactive to the chemistry used to react 311, 1211, or 1212; V is an optional group corresponding to a bifunctional initiator from which isobutylene can be propagated, and can be tert-butylbenzene, a phenyl group connected to the hydrophobic block via the para, meta, or ortho position, naphthalene, another aromatic group, an alkane chain having about 2 to about 20 carbons, or another aliphatic group; m = about 2 to about 100; and n = about 2 to about 100. V may optionally be flanked by 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 group, zwitterionic group, sulfate, sulfonate, alkyl, aryl, any orthogonal functional group and hydrogen. When V is absent, there is only one PIB block and n = about 2 to about 100. L1 and L2 are independently linkers, which in some examples may be non-reactive, for example, may include at least one portion selected from the group consisting of: amide, thioether (sulfide), succinate, maleate, methylene, ether and the product of a "click" reaction. In other examples, L1 and / or L2 may be reactive and may correspond to reactive moieties 311, 1211 or 1212 and may be similar to reference FIG. 8A to FIG. 8B In such examples, R1 and / or R2 are not necessarily reactive.

[0229]

[0230] In some non-limiting examples of the above structure, n = about 2 to about 50, and m = about 1 to about 50, V = tert-butylbenzene, and L1 = L2 = ethyl sulfide. In other non-limiting examples, n = about 5 to about 20, m = about 2 to about 15, V = tert-butylbenzene, and L1 = L2 = ethyl sulfide. In other non-limiting examples, n = about 13 to about 19, m = about 2 to about 5, V = tert-butylbenzene, and L1 = L2 = ethyl sulfide. In other non-limiting examples, n = about 7 to about 13, m = about 7 to about 13, V = tert-butylbenzene, and L1 = L2 = ethyl sulfide. In particular, in one non-limiting example, n = 16, m = 3, V = tert-butylbenzene, and L1 = L2 = ethyl sulfide. In another non-limiting example, n = 10, m = 10, V = tert-butylbenzene, and L1 = L2 = ethyl sulfide. In another non-limiting example, n=16, m=8, V=tert-butylbenzene, and L1=L2=ethyl sulfide.

[0231] In some examples, the multifunctional precursor can be derived from and used as a precursor for synthesizing a bifunctional initiator, and in the example further described above, V corresponds to these bifunctional initiators. For example, the multifunctional precursor can be 5-tert-butylisophthalic acid (TBIPA), which can be synthesized as 1-(tert-butyl)-3,5-bis(2-methoxypropyl-2-yl)benzene (TBDMPB) using reactions known in the art. In another example, TBIPA can be synthesized as 1-tert-butyl-3,5-bis(2-chloropropyl-2-yl)benzene using reactions known in the art. The use of this bifunctional initiator allows cationic polymerization on both sides of the initiator to produce bifunctional PIB such as allyl-PIB-allyl, which can then be coupled with hydrophilic A blocks to produce an ABA block copolymer including PIB as a B block. Here, although the difunctional initiator may be located between the first and second PIB polymers, it is understood that the first and second PIB polymers and the difunctional initiator (V) together may be considered to form a B block, such as a B block of an ABA triblock copolymer.

[0232] In another non-limiting example, an ABA triblock copolymer comprises Wherein m=about 2 to about 100, n=about 2 to about 100, p=about 2 to about 100, at least one of R1 and R2 may be a reactive group 311, 1211, or 1212, and the other of R1 and R2 may be a reactive group 311, 1211, or 1212, or may be a group that is not reactive to the chemistry used to react 311, 1211, or 1212. In some non-limiting examples, m=about 2 to about 30, n=about 25 to about 45, and p=about 2 to about 30. In some non-limiting examples, m=about 2 to about 15, n=about 30 to about 40, and p=about 2 to about 15. In some non-limiting examples, m=about 7 to about 11, n=about 35 to about 40, and p=about 7 to about 11. In some non-limiting examples, m=about 2 to about 5, n=about 30 to about 37, and p=about 2 to about 5. In particular, in one non-limiting example, m = 3, n = 34, and p = 3. In another non-limiting example, m = 9, n = 37, and p = 9.

[0233] In some examples of AB copolymers shown below including PIB blocks as B blocks and PEO as A blocks, R is a reactive group 311, 1211, or 1212; m = about 2 to about 100; n = about 2 to about 100; and L is a linker. In some examples, L is non-reactive, for example, selected from the group consisting of: amide, thioether (sulfide), succinate, maleate, methylene, ether, or the product of a click reaction. In other examples, L can be reactive and can correspond to the reactive portion 311, 1211, or 1212 and can be similar to reference FIG. 8A to FIG. 8B In such examples, R is not necessarily reactive.

[0234]

[0235] In one non-limiting example, n=13, m=8, and L is ethyl sulfide. In another non-limiting example, n=13, m=3, and L is ethyl sulfide. In another non-limiting example, n=30, m=8, and L is ethyl sulfide. In another non-limiting example, n=30, m=3, and L is ethyl sulfide.

[0236] Thus, it will be appreciated that a variety of amphiphilic molecules and a variety of reactive moieties can be used to generate barriers that are stabilized using covalent bonds to molecules, such as those described in reference Figure 1 In the nanopore device. Fig.16An exemplary operational flow in method 1600 for forming a barrier comprising molecules covalently bonded to amphiphilic molecules is shown. Method 1600 may include forming at least one layer comprising a plurality of amphiphilic molecules, wherein the amphiphilic molecules include a reactive portion (operation 1610). For example, operation 1610 may include forming a first layer and a second layer comprising a first plurality of amphiphilic molecules and a second plurality of amphiphilic molecules, respectively. In other examples, operation 1610 may include forming a monolayer, or a layer that is partially monolayer and partially bilayer. That is, barrier 101 may include molecules of a block copolymer (e.g., AB, ABA, or BAB) having any suitable arrangement within the barrier, such as described elsewhere herein. The AB interface of a hydrophilic "A" block, a hydrophobic "B" block, or an amphiphilic molecule (e.g., a block copolymer) may be such as described in reference to FIG. 3A to FIG. 14B The described method is coupled to the reactive moiety (e.g., 311, 1211 or 1212).

[0237] Fig.16 The illustrated method 1600 may also include cross-linking the plurality of amphiphilic molecules to each other using a cross-linking reaction of the reactive moiety (operation 1620). In examples including a first layer and a second layer, the cross-linking reaction may be used to couple the amphiphilic molecules of the first layer to each other and / or to the amphiphilic molecules of the second layer, and / or may be used to cross-link the amphiphilic molecules of the second layer to each other and / or to the amphiphilic molecules of the first layer. In some examples of operation 1620, the reactive moiety 311 may be used to cross-link the plurality of amphiphilic molecules to each other using a cross-linking reaction of the reactive moiety, such as a reference FIG. 3A to FIG. 11B In other examples of operation 1620, reactive portions 1211 and 1212 may be used to polymerize amphiphilic molecules in a manner such as reference FIG. 12A to FIG. 14B The described manner couples the amphiphilic molecules to each other. Optionally, the nanopore may be inserted into the barrier at any suitable time, for example, before any of the reactions described herein, or after any of the reactions described herein.

[0238] It should also be understood that the barriers of the present invention may be used in any suitable device or application. Fig.32 Schematically shows Figure 1 Cross-sectional views of exemplary uses of compositions and devices. Fig.32 The device 100 shown may be configured such as with reference to Figure 1 The method includes a fluid pore 100', a barrier 101 which may have a configuration such as described elsewhere herein, a first fluid 120 and a second fluid 120', and a nanopore 110. Fig.32In the non-limiting example shown, the second fluid 120' may optionally include each of a plurality of nucleotides 121, 122, 123, 124, such as G, T, A, and C. Each of the nucleotides 121, 122, 123, 124 in the second fluid 120' may optionally be coupled to a corresponding label 131, 132, 133, 134, which is coupled to the nucleotide via an elongated body (an elongated body that is not specifically labeled). Optionally, the device 100 may also include a polymerase 105. Fig.32 As shown, polymerase 105 may be in the second composition of second fluid 120'. Alternatively, polymerase 105 may be coupled to nanopore 110 or barrier 101, for example via a suitable elongated body (not specifically shown). Device 100 may also optionally be provided in a manner such as Fig.32 The illustrated method includes a first polynucleotide 140 and a second polynucleotide 150. A polymerase 105 can be used to sequentially add multiple nucleotides to the first polynucleotide 140 using the sequence of the second polynucleotide 150. For example, in Fig.32 At the specific time shown, polymerase 105 incorporates nucleotide 122 (T) into first polynucleotide 140, which hybridizes with second polynucleotide 150 to form a duplex. At other times (not specifically shown), polymerase 105 can use the sequence of second polynucleotide 150 to sequentially incorporate other nucleotides of nucleotides 121, 122, 123, 124 into first polynucleotide 140.

[0239] Fig.32 The circuit 180 shown can be configured to detect a change in an electrical property of the pore in response to a polymerase sequentially adding multiple nucleotides to the first polynucleotide 140 using the sequence of the second polynucleotide 150. Fig.32In the non-limiting example shown, the nanopore 110 can be coupled to a permanent tether 3210, which can include a head region 3211, a tail region 3212, an elongated body 3213, a reporter region 3214 (e.g., an abasic nucleotide), and a portion 3215. The head region 3211 of the tether 3210 is coupled to the nanopore 110 via any suitable chemical bond, protein-protein interaction, or any other suitable attachment that is generally irreversible. The head region 3211 can be attached to any suitable portion of the nanopore 110, which places the reporter region 3214 within the orifice 3213 and places the portion 3215 in a position sufficiently close to the polymerase 105 to interact with the corresponding labels 131, 132, 133, 134 of the nucleotides 121, 122, 123, 124 acted upon by the polymerase 105. Portions 3215 can interact with labels 131, 132, 133, 134, respectively, in such a way that reporter region 3214 moves within pore 113 and thereby changes the rate at which salt 160 moves through pore 113, thereby being able to detectably change the conductivity of pore 113, in such a way that it is detected by circuit 180. For further details on sequencing polynucleotides using permanent tethers coupled to nanopores, see US 9,708,655, the entire contents of which are incorporated herein by reference.

[0240] Fig.33 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device. Fig.33 As shown, the device 100 may include a fluid pore 100', a barrier 101 which may have a configuration such as described elsewhere herein, 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 described with reference to Fig.32 The structure is similar to that described above. Fig.33In the non-limiting example shown, nucleotides 121, 122, 123, 124 do not have to be coupled to corresponding labels. Polymerase 105 can be coupled to nanopore 110 and can be coupled to permanent tether 3310, which can include head region 3311, tail region 3312, elongated body 3313 and reporter region 3314 (e.g., abasic nucleotides). The head region 3311 of tether 3310 is coupled to polymerase 105 via any suitable chemical bond, protein-protein interaction, or any other suitable attachment that is generally irreversible. Head region 3311 can be attached to any suitable portion of polymerase 105, which places reporter region 3314 in orifice 113. When polymerase 105 interacts with nucleotides 121, 122, 123, 124, such interactions may cause polymerase 105 to undergo conformational changes. Such conformational changes can cause the reporter region 3314 to move within the pore 113 and thereby change the rate at which the salt 160 moves through the pore 113, thereby being able to detectably change the conductivity of the pore 113 in a manner that allows detection by the circuit 180. For further details on sequencing polynucleotides using a permanent tether coupled to a polymerase, see US 9,708,655, the entire contents of which are incorporated herein by reference.

[0241] Fig.34 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device. Fig.34 As shown, the device 100 may include a fluid hole 100', a barrier 101 which may have a configuration such as described elsewhere herein, a first fluid 120 and a second fluid 120', and a nanopore 110, all of which may be described with reference to Fig.32 The structure is similar to that described above. Fig.34 In the non-limiting example shown, polynucleotide 150 is translocated through nanopore 110 under an applied force (e.g., a bias voltage applied between electrode 102 and electrode 103 by circuit 180). As bases in polynucleotide 150 pass through nanopore 110, such bases can change the rate at which salt 160 moves through pore 113, thereby being able to detectably change the conductivity of pore 113 in a manner that allows detection by circuit 180. For further details on using nanopores to sequence polynucleotides translocated therethrough, see US5,795,782, the entire contents of which are incorporated herein by reference.

[0242] Fig.35 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device. Fig.35As shown, the device 100 may include a fluid hole 100', a barrier 101 which may have a configuration such as described elsewhere herein, a first fluid 120 and a second fluid 120', and a nanopore 110, all of which may be described with reference to Fig.32 The structure is similar to that described above. Fig.35 In the non-limiting example shown, the surrogate polymer 3550 translocates through the nanopore 110 under an applied force (e.g., a bias voltage applied between the electrode 102 and the electrode 103 by the circuit 180). As used herein, "surrogate polymer" is intended to mean an elongated chain of a label having a sequence corresponding to a nucleotide sequence in a polynucleotide. Fig.35 In the example shown, the surrogate polymer 3550 includes labels 3551 coupled to each other via a linker 3552. TM XPANDOMERS are a specific type of alternative polymer developed by Roche Sequencing (Pleasanton, CA). TM Sequencing ByeXpansion TM (SBX TM , Roche Sequencing, Pleasanton CA). TM In the process, an engineered polymerase uses the sequence of a target polynucleotide to polymerize xNTPs, which include nucleobases coupled to labels via linkers. The polymerized nucleotides are then processed to produce elongated chains of labels that are separated from each other by linkers coupled between these labels and have a sequence complementary to the target polynucleotide. For example, for XPANDOMERS TM , linkers (tethers), labels, engineered polymerases, and for SBX TM For a description of the methods of, see the following patents, each of which is incorporated herein by reference in its entirety: US 7,939,249, US 8,324,360, US 8,349,565, US 8,586,301, US 8,592,182, US 9,670,526, US 9,771,614, US 9,920,386, US 10,301,345, US 10,457,979, US 10,676,782, US 10,745,685, US 10,774,105, and US 10,851,405.

[0243] Fig.36 Schematically shows Figure 1 A cross-sectional view of another exemplary use of the composition and device. Fig.36As shown, the device 100 may include a fluid hole 100', which may have a Figure 2A To Figure 2C, FIG. 14A to FIG. 14B , Figure 15 and / or Fig.16 The barrier 101 of the described construction (i.e., the barrier 101 may optionally be suspended using a barrier support and may include any AB, ABA, or BAB copolymer provided herein), the first fluid 120 and the second fluid 120', and the nanopore 110, all of which may be similar to those described in reference Figure 4 The structure is similar to that described above. Fig.36 In the non-limiting example shown, the duplex between polynucleotide 140 and polynucleotide 150 is located within nanopore 110 under an applied force (e.g., a bias voltage applied by circuit 180 between electrode 102 and electrode 103). The combination of bases in the double-stranded portion (here, base pairs GC 121, 124 at the ends of the duplex) and 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 pore 113, and can therefore detectably change the conductivity of pore 113 in a manner that is detected by circuit 180. For further details on using a nanopore to sequence a polynucleotide translocated therethrough, see U.S. Patent Publication No. 2023 / 0090867 to Mandell et al., the entire contents of which are incorporated herein by reference.

[0244] Working Example

[0245] The following examples are intended to be illustrative only and not limiting of the present invention.

[0246] The performance of different membranes was evaluated based on membrane stability. Membranes suspended by barriers with circular orifices were generated and then characterized using an automated patch clamp apparatus using Ag / AgCl electrodes. Fig.17 The voltage breakdown waveform used to evaluate the stability of the polymer film is shown. The film stability is quantified as the percentage of film remaining at the end of each step of the voltage ramp shown. Fig.17 As shown, the voltage ramp was stepped from 150 mV to 500 mV in steps of 50 mV. Each step lasted 10 seconds. The membranes were coated using the Orbit 16TC platform using standard buffer conditions (1 M KCl, 50 mM HEPES, pH=7.4).

[0247] Example 1

[0248] In Example 1, polymer 1, PDMS-b-PEO-acrylate polymer (AB polymer with reactive acrylate moieties at the ends of the A blocks) and polymer 2, succinate-b-PDMS-b-succinate polymer (ABA polymer with succinic acid as the A blocks) were mixed together in a 4:1 ratio and used to form a polymer such as reference Figure 3A The structures of these polymers are shown below:

[0249]

[0250] Polymer 1, PDMS-b-PEO-acrylate, wherein the A block comprises about 8 to 9 PEO repeating units (RU), and the B block comprises about 19 to 20 PDMS RUs, wherein the acrylate moiety is coupled to the terminal PEO via an ester bond; and

[0251]

[0252] Polymer 2, succinate-PDMS-succinate, wherein the A block includes about 1 RU of succinate and the B block includes about 41 RUs, wherein the succinate moiety is coupled to the terminal PDMS via an amide bond.

[0253] With reference such as Figure 3B The described method uses polymerization to crosslink the membrane under various conditions and uses reference Fig.17 The waveforms described measure their stability. Fig.18A is a graph of measured film stability of films crosslinked using a photoinitiator under different conditions. More specifically, during the formation of the film, a 4:1 mixture of polymer 1 and polymer 2 was mixed with 0.3 wt% of photoinitiator (PI) V50. A first subset of the films was exposed to UV light of about 1350 mW and about 365 nm wavelength for 10 minutes; a second subset of the films was exposed to UV light for 20 minutes; and a third subset of the films was not exposed to UV light to serve as a control. From Fig.18A It can be seen that the film exposed to UV light for 10 minutes had the greatest stability at increasing voltages, while the stability of the film exposed to UV light for 20 minutes was lower than that of the film exposed for 10 minutes, and in some cases lower than that of the film not exposed to UV. Films considered "unzappable" are those that remain stable for at least about 100 ms at a voltage of about 1 V (the highest voltage that can be generated using this system). From these results, it can be understood that the duration of UV exposure can be appropriately adjusted to produce the desired degree of crosslinking and stability.

[0254] Fig.18Bis a graph of measured membrane stability of membranes cross-linked using a redox system under different conditions. More specifically, membranes were formed using a 4:1 mixture of polymer 1 and polymer 2. For some membranes, the buffer solution on the second (cis) side 112 of the membrane was exchanged with similar buffer solutions containing 1 wt % of KPS and TEMED, respectively. A first subset of membranes was incubated with the KPS / TEMED mixture for 10 minutes; a second subset of membranes was incubated with the KPS / TEMED mixture for 20 minutes; and a third subset of membranes was not incubated with the KPS / TEMED mixture to serve as a control. From Fig.18B It can be seen that the membrane incubated with KPS / TEMED has the greatest stability at all voltages, while the stability of the membrane incubated for 10 minutes is lower than that of the membrane exposed for 20 minutes, and the membrane not incubated with KPS / TEMED has the lowest stability. From these results, it can be understood that the duration of exposure to the redox system can be appropriately adjusted to produce the desired crosslinking degree and stability.

[0255] Fig.18C Schematically shown as reference FIG. 18A to FIG. 18B Exemplary reaction products in the cross-linked film. Fig.18C As shown, the reaction product of the polymerization of the acrylate portion of polymer 1 includes polyacrylate formed in the plane at the ends of the A blocks of the block copolymer. Note that although a second layer of the AB copolymer is not shown, in some examples, similar reaction products may form in the plane at the ends of such A blocks in the second layer, such as in examples where both sides of the film are in contact with the initiator. In other examples, polyacrylate may be formed substantially only at the ends of the A blocks in the first layer, such as in examples where only the ends of that layer are in contact with the initiator.

[0256] Example 2

[0257] In Example 2, polymer 3, maleic acid-b-PDMS-b-maleic acid polymer (ABA polymer with reactive maleic acid moieties at the ends of the A blocks) was used to form a polymer such as reference Figure 5A The structure of polymer 3 is shown below:

[0258]

[0259] Polymer 3, wherein the A block comprises about 1 maleic acid RU, and the B block comprises about 41 PDMS RUs, wherein the maleic acid moiety is coupled to the A block via respective amide bonds.

[0260] With reference such as Figure 5B The membrane was cross-linked as described and the Fig.17 The waveforms described measure their stability. Fig.19Ais a graph of measured membrane stability of membranes crosslinked using a redox system. More specifically, after the membranes were formed, the buffer solution on the second (cis) side 112 of the membrane was exchanged with similar buffer solutions containing 1 wt % of KPS and TEMED, respectively. A first subset of the membranes was incubated with the KPS / TEMED mixture for 20 minutes; a second subset of the membranes was not incubated with the KPS / TEMED mixture to serve as a control. Fig.19A It can be seen that the stability of the membrane incubated with KPS / TEMED at higher voltages is higher than that of the membrane not incubated with KPS / TEMED. From these results, it can be understood that cross-linking enhances the stability of the membrane.

[0261] Fig.19B Schematically shown as reference Fig.19A Exemplary reaction products in the cross-linked film. Fig.19A As shown, the reaction product of the polymerization of the maleic acid portion of polymer 3 includes a poly (maleic acid derivative) formed in the plane at the end of the A block of the block copolymer, wherein in this particular example, the A block is formed by a single maleic acid portion, but in other specific implementations, the A block may be formed by a hydrophilic polymer having a terminal maleic acid portion. Note that although the maleic acid portion on the other side of the membrane is not shown, in some examples, similar reaction products may be formed in the plane on the other side of the membrane, such as in examples where both sides of the membrane are in contact with the initiator. In other examples, the reaction product may be formed substantially only at the end of a group of A blocks, such as in examples where only one side of the membrane is in contact with the initiator.

[0262] Example 3

[0263] In Example 3, PEO-b-maleic acid-PDMS-b-maleic acid-PEO polymer (ABA polymer with reactive maleic acid moieties at the AB interface) was used to form a polymer such as reference Fig. 8A The structure of polymer 4 is shown below:

[0264]

[0265] Polymer 4, wherein the A block comprises about 8 PEO RUs and the B block comprises about 41 PDMS RUs, wherein the maleic acid moiety is coupled to the A block and the B block via respective amide bonds.

[0266] With reference such as Figure 8B The membrane is cross-linked in the manner described. Fig. 20 An exemplary reaction product in a film cross-linked as described in Example 3 is schematically shown. Fig. 20As shown, the reaction product of the polymerization of the maleic acid portion of polymer 4 includes a poly(maleic acid derivative) formed in the plane at the AB interface of the block copolymer. Note that although the maleic acid portion on the other side of the film is not shown, in some examples, a similar reaction product may be formed in the plane on the other side of the film.

[0267] Example 4

[0268] In Example 4, polymer 5, propargyl-PEO-b-PDMS-b-PEO-propargyl polymer (ABA polymer having reactive propargyl moieties at the ends of the A block) and polymer 6, disulfide pyridyl-PEO-b-PDMS-b-PEO-disulfide pyridyl polymer (ABA polymer having disulfide pyridyl moieties at the ends of the A block) were mixed together in a ratio of 1:2 and used to form a polymer such as reference 4. FIG. 12A to FIG. 12B The structures of these polymers are shown below:

[0269]

[0270] Polymer 5, wherein the A block comprises about 8 PEO RUs and the B block comprises about 41 PDMS RUs, wherein the propargyl moiety is coupled to the terminal PEO via respective ether bonds; and

[0271]

[0272] Polymer 6, wherein the A block comprises about 8 PEO RUs and the B block comprises about 41 RUs, wherein the disulfide pyridyl moiety is coupled to the terminal PEO via respective amide bonds.

[0273] With reference such as FIG. 12A to FIG. 12B and Fig.13 The described method uses coupling reactions to crosslink the membrane under various conditions and uses reference Fig.17 The waveforms described measure their stability. Fig.21A is a graph of measured film stability of films crosslinked using a first photoinitiator under different conditions. More specifically, during film formation, a 1:2 mixture of polymer 5 and polymer 6 was mixed with 0.3 wt% of photoinitiator (PI) V50. A first subset of films was exposed to UV light of about 1350 mW and about 365 nm wavelength for 10 minutes; a second subset of films was exposed to UV light for 20 minutes; and a third subset of films was not exposed to UV light to serve as a control. From Fig.21AIt can be seen that the film exposed to UV light for 10 minutes has the greatest stability at higher voltages, while the stability of the film exposed to UV light for 10 minutes is lower than that of the film exposed for 10 minutes, and the film not exposed to UV has the lowest stability. From these results, it can be understood that the duration of UV exposure can be appropriately adjusted to produce the desired crosslinking degree and stability.

[0274] Fig. 21B is a graph of measured film stability of films crosslinked using a second photoinitiator. More specifically, during film formation, a 1:2 mixture of polymer 5 and polymer 6 was mixed with 0.5 wt % PI Irgacure 2959 and 5 wt % isopropyl alcohol (IPA). A first subset of the films was exposed to UV light at about 1350 mW and about 365 nm wavelength for 20 minutes; and a second subset of the films was not exposed to UV light to serve as a control. From Fig. 21B It can be seen that the film exposed to UV light for 20 minutes has the greatest stability at higher voltages, while the film not exposed to UV light has the lowest stability. From these results, it can be understood that the duration of UV exposure can be appropriately adjusted to produce the desired degree of crosslinking and stability.

[0275] Fig. 21C is a graph of measured membrane stability for membranes crosslinked using a redox system. More specifically, membranes were formed using a 1:2 mixture of polymer 5 and polymer 6. For some membranes, the buffer solution on the second (cis) side 112 of the membrane was exchanged with similar buffer solutions containing 1 wt % of KPS and TEMED, respectively. A first subset of membranes was incubated with the KPS / TEMED mixture for 20 minutes; and a second subset of membranes was not incubated with the KPS / TEMED mixture to serve as a control. From Fig. 21C It can be seen that the membrane incubated with KPS / TEMED has the greatest stability at higher voltages, and the membrane not incubated with KPS / TEMED has the lowest stability. From these results, it can be understood that the duration of redox system exposure can be appropriately adjusted to produce the desired degree of crosslinking and stability.

[0276] Fig.21D Schematically shown as reference FIG. 21A to FIG. 21C Exemplary reaction products in the cross-linked film. Fig.21DAs shown, the reaction product of the thiol-alkyne click coupling between the propargyl moiety of polymer 5 and the disulfide pyridyl moiety of polymer 6 includes a sulfide bond formed in the plane at the end of the A block of the block copolymer. Note that the other end of the ABA copolymer is not shown, and in some examples, similar reaction products can be formed in the plane at the end of the other A block, such as in examples where both sides of the membrane are in contact with the initiator. In other examples, the reaction product can be formed substantially only at the end of the A block on the first side of the membrane, such as in examples where only that side of the membrane is in contact with the initiator.

[0277] Example 5

[0278] In Example 5, polymer 5 (propargyl-PEO-b-PDMS-b-PEO-propargyl ABA polymer of Example 4) and polymer 7, acylamino-PEO-b-PDMS-b-PEO-acylamino polymer (ABA polymer having acylamino moieties at the ends of the A blocks) were mixed together in a 1:1 ratio and used to form a polymer such as reference 7. Fig. 12A and Fig.13 The structure of polymer 7 is shown below:

[0279]

[0280] Polymer 7, wherein the A block comprises about 8 PEO RUs, and the B block comprises about 41 RUs, wherein the fatty acylamino moiety is coupled to the terminal PEO via respective amide bonds.

[0281] With reference such as FIG. 12A to FIG. 12B The described approach uses a coupling reaction to cross-link the membrane. Fig. 22 An exemplary reaction product in a film cross-linked as described in Example 5 is schematically shown. Fig. 22 As shown, the reaction products of the ring opening and dithiol formation coupling between the propargyl moiety of polymer 5 and the acylamino moiety of polymer 7 include sulfide bonds formed in the plane at the end of the A block of the block copolymer. Note that the other end of the ABA copolymer is not shown, and in some examples, similar reaction products may be formed in the plane at the end of the other A block, such as in examples where both sides of the membrane are in contact with the initiator. In other examples, the reaction products may be formed substantially only at the end of the A block on the first side of the membrane, such as in examples where only that side of the membrane is in contact with the initiator.

[0282] Example 6

[0283] In Example 6, polymer 6 (the disulfide pyridyl-PEO-b-PDMS-b-PEO-disulfide pyridyl polymer of Example 4) is used to form a polymer such as reference Figure 3AThe membrane described. FIG. 12A to FIG. 12B and Fig.13 The described method uses a coupling reaction to crosslink the membrane and uses reference Fig.17 The waveforms described measure their stability. Fig.23A is a graph of membrane stability for membranes cross-linked with a reducing agent. More specifically, after membrane formation, the buffer solution on the second (cis) side 112 of the first set of membranes was exchanged with a similar buffer solution containing 1 mM sodium ascorbate as a reducing agent. The reducing agent cleaves the pyridyl groups from polymer 6, generating free thiols at the ends of the A block of the copolymer. The reducing agent and pyridyl groups are washed off several times with an aqueous buffer including 1 M KCl and 50 mM HEPES. The free thiols then react spontaneously to form disulfide bridges between pairs of copolymer molecules. A second subset of membranes was not incubated with the reducing agent to serve as a control. From Fig.23A It can be seen that the stability of the membrane incubated with the reducing agent for 20 minutes at higher voltages is higher than that of the membrane not incubated with the reducing agent. From these results, it can be understood that cross-linking enhances the stability of the membrane.

[0284] Fig. 23B Schematically shown as reference Fig.23A Exemplary reaction products in the cross-linked film. Fig.23A As shown, the reaction product of the coupling of the deprotected thiol moiety of polymer 6 includes a mixture of thiol groups and disulfide bridges formed in the plane at the end of the A block of the block copolymer. Note that although the thiol and disulfide bridges on the other side of the membrane are not shown, in some examples, similar reaction products can be formed in the plane on the other side of the membrane, such as in examples where both sides of the membrane are in contact with a reducing agent. In other examples, the reaction product can be formed essentially only at the end of a group of A blocks, such as in examples where only one side of the membrane is in contact with a reducing agent. The reaction can optionally be reversible. For example, a reducing agent can be used to cleave disulfide bridges to obtain free thiols and reverse crosslinking. Such reversibility may be useful, for example, in some applications, the membrane is crosslinked for stability and transported, and then the crosslinking is reversed, so that the membrane is more fluid during use (such as sequencing).

[0285] Example 7

[0286] In Example 7, polymer 7 (lipoylamino-PEO-b-PDMS-b-PEO-lipoylamino polymer of Example 5) is used to form a polymer such as reference Figure 3A The membrane described. FIG. 12A to FIG. 12B and Fig.13The described approach uses a coupling reaction to crosslink the membrane. More specifically, after the membrane is formed, the buffer solution on the second (cis) side 112 of the membrane is exchanged with a similar buffer solution containing a reducing agent. The reducing agent cleaves the disulfide within the acylamino groups of polymer 7, generating free thiols at the ends of the A blocks of the copolymer. The reducing agent is washed off. The free thiols are then oxidatively dimerized, resulting in the formation of disulfide bridges between pairs of copolymer molecules.

[0287] Fig.24 An exemplary reaction product in a film cross-linked as described in Example 7 is schematically shown. Fig.24 As shown, the reaction product of the coupling of the deprotected thiol moieties of polymer 7 includes a mixture of different disulfide bridges formed in the plane at the ends of the A blocks of the block copolymer. Note that although the disulfide bridges on the other side of the membrane are not shown, in some examples, similar reaction products may be formed in the plane on the other side of the membrane, such as in examples where both sides of the membrane are contacted with the reducing agent. In other examples, the reaction product may be formed substantially only at the ends of one set of A blocks, such as in examples where only one side of the membrane is contacted with the reducing agent.

[0288] Example 8

[0289] In Example 8, polymer 6 (the disulfide pyridyl-PEO-b-PDMS-b-PEO-disulfide pyridyl polymer ABA polymer of Example 4) and polymer 8, maleimide-PEO-b-PDMS-b-PEO-maleimide polymer (ABA polymer having maleimide moieties at the ends of the A block) were mixed together in a 1:1 ratio and used to form a polymer such as reference 4. Fig. 12A and Fig.13 The structure of polymer 8 is shown below:

[0290]

[0291] Polymer 8, wherein the A block comprises about 8 PEO RUs and the B block comprises about 41 RUs, wherein the maleimide moiety is coupled to the terminal PEO via respective amide bonds.

[0292] With reference such as FIG. 12A to FIG. 12B and Fig.13 The described method uses a coupling reaction to crosslink the membrane and uses reference Fig.17 The waveforms described measure their stability. Fig.25Ais a graph of measured membrane stability of membranes cross-linked using a reducing agent. More specifically, after the membranes were formed, the buffer solution on the second (cis) side 112 of the first set of membranes was exchanged with a similar buffer solution containing 1 mM sodium ascorbate as a reducing agent. The reducing agent cleaves the pyridyl groups from polymer 6, generating free thiols at the ends of the A block of the copolymer. The reducing agent and pyridyl groups are washed off using an aqueous buffer in the manner described in Example 6. The free thiols are then cross-linked with the maleimide moieties of polymer 8. A second subset of membranes was not incubated with the reducing agent to serve as a control. From Fig.25A It can be seen that the membrane incubated with the reducing agent for 3 hours is more stable at higher voltages than the membrane not incubated with the reducing agent.

[0293] In this example, a modified waveform is used, increasing the pulse duration from 900mV / 1000us every five minutes. More specifically, the complete waveform used is as follows:

[0294] Wave A lasts 5 minutes

[0295] -60mV / 180ms

[0296] 10mV / 20ms

[0297] 40mV / 480ms, 8 pulses every 30ms 900mV / 10us

[0298] Wave B lasts 5 minutes

[0299] -60mV / 180ms

[0300] 10mV / 20ms

[0301] 40mV / 480ms, 8 pulses every 30ms 900mV / 25us

[0302] Waveform C lasts 5 minutes

[0303] -60mV / 180ms

[0304] 10mV / 20ms

[0305] 40mV / 480ms, 8 pulses every 30ms 900mV / 50us

[0306] Wave D lasts 5 minutes

[0307] -60mV / 180ms

[0308] 10mV / 20ms

[0309] 40mV / 480ms, 8 pulses every 30ms 900mV / 100us

[0310] Wave E lasts 5 minutes

[0311] -60mV / 180ms

[0312] 10mV / 20ms

[0313] 40mV / 480ms, 8 pulses every 30ms 900mV / 1000us

[0314] The motivation for this improved waveform was to improve the resolution of the QC method. More specifically, the main waveform 150mV to 500mV showed that the membrane without cross-linking was very stable at 500mV, so it was decided to use a more harsh waveform to evaluate the membrane with and without cross-linking. From these results, it can be understood that cross-linking enhances the stability of the membrane.

[0315] Fig.25B Schematically shown as reference Fig.25A Exemplary reaction products in the cross-linked film. Fig.25B As shown, the reaction product of the coupling between the free thiol moieties of polymer 6 and the maleimide moieties of polymer 8 includes sulfosuccinimide formed in the plane at the end of the A block of the block copolymer. Note that the other end of the ABA copolymer is not shown, and in some examples, similar reaction products may be formed in the plane at the end of the other A block, such as in examples where both sides of the membrane are in contact with the reducing agent. In other examples, the reaction product may be formed substantially only at the end of the A block on the first side of the membrane, such as in examples where only that side of the membrane is in contact with the reducing agent.

[0316] Example 9

[0317] In Example 9, polymer 7 (lipoamido-PEO-b-PDMS-b-PEO-lipoamido polymer of Example 5) and polymer 8 (maleimide-PEO-b-PDMS-b-PEO-maleimide polymer of Example 8) were mixed together in a 1:1 ratio and used to form a polymer such as reference Fig. 12A and Fig.13 The membrane.

[0318] With reference such as FIG. 12A to FIG. 12B and Fig.13 The described method uses a coupling reaction to crosslink the membrane. More specifically, after the membrane is formed, the buffer solution on the second (cis) side 112 of the membrane is exchanged with a similar buffer solution containing a reducing agent. The reducing agent opens the fatty acylamino groups of polymer 7, generating free thiols at the ends of the A block of the copolymer. The reducing agent is washed off. The free thiols are then crosslinked with the maleimide moieties of polymer 8. Fig.26An exemplary reaction product in a film cross-linked as described in Example 9 is schematically shown. Fig.26 As shown, the reaction product of the coupling between the free thiol moiety of polymer 7 and the maleimide moiety of polymer 8 includes sulfosuccinimide formed in the plane at the end of the A block of the block copolymer. Note that the other end of the ABA copolymer is not shown, and in some examples, similar reaction products can be formed in the plane at the end of the other A block, such as in examples where both sides of the membrane are in contact with the reducing agent. In other examples, the reaction product can be formed substantially only at the end of the A block on the first side of the membrane, such as in examples where only that side of the membrane is in contact with the reducing agent.

[0319] Example 10

[0320] In Example 10, polymer 10, dimethylmaleimide-PEO-b-PDMS-b-PEO-dimethylmaleimide ABA polymer is used to form a Figure 3A The structure of the polymer 10 is shown below:

[0321]

[0322] Polymer 10, wherein the A block comprises about 10 PEO RUs and the B block comprises about 41 RUs, wherein the dimethylmaleimide moiety is coupled to the terminal PEO via respective ether bonds.

[0323] With reference such as FIG. 3A to FIG. 3C , FIG. 12A to FIG. 12B and Fig.13 The described approach uses a coupling reaction to crosslink the film. More specifically, after forming the film, the film is exposed to UV light, in response to which the dimethylmaleimide moieties react with each other, resulting in coupling between pairs of copolymer molecules. Fig. 27 An exemplary reaction product in a film cross-linked as described in Example 10 is schematically shown. Fig. 27 As shown, the reaction products of the coupling of the UV-activated dimethylmaleimide moieties of polymer 10 include dimethylmaleimide conjugated products formed in the plane at the ends of the A blocks of the block copolymer. Note that although the reaction products on the other side of the membrane are not shown, in some examples, similar reaction products may be formed in the plane on the other side of the membrane, such as in examples where both sides of the membrane are in contact with the reducing agent. In other examples, the reaction products may be formed substantially only at the ends of one set of A blocks, such as in examples where only one side of the membrane is in contact with the reducing agent.

[0324] Additional Notes

[0325] Although various illustrative examples have been described above, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the invention. The appended claims are intended to cover all such changes and modifications as fall within the true spirit and scope of the invention.

[0326] It should be understood that any corresponding features / examples of each of the aspects of the present disclosure as described herein may be implemented together in any appropriate combination, and any features / examples from any one or more of these aspects may be implemented together with any of the features of the other (multiple) aspects as described herein in any appropriate combination to achieve the benefits as described herein.

Claims

1. A barrier between a first fluid and a second fluid, the barrier comprising: at least one layer, the at least one layer comprising a plurality of amphiphilic molecules, The amphiphilic molecules in the plurality of amphiphilic molecules are cross-linked with each other.

2. The barrier of claim 1, wherein the at least one layer comprises: a first layer comprising a first plurality of amphiphilic molecules; and A second layer comprising a second plurality of amphiphilic molecules in contact with the first plurality of amphiphilic molecules.

3. The barrier according to claim 1, wherein the amphiphilic molecules of the first layer are cross-linked to each other, and The amphiphilic molecules of the second layer are cross-linked to each other.

4. The barrier according to any one of claims 1 to 3, wherein the amphiphilic molecule comprises at least one hydrophobic block coupled to at least one hydrophilic block at an interface.

5. The barrier of claim 4, wherein the amphiphilic molecules are cross-linked to each other at the hydrophilic blocks.

6. A barrier according to claim 4 or claim 5, wherein the amphiphilic molecules are cross-linked to each other at the hydrophobic blocks.

7. The barrier according to any one of claims 4 to 6, wherein the amphiphilic molecules are cross-linked to each other at the interface.

8. The barrier according to any one of claims 1 to 7, wherein the amphiphilic molecules comprise molecules of a diblock copolymer comprising a hydrophobic block coupled to a hydrophilic block.

9. The barrier according to any one of claims 1 to 7, wherein the amphiphilic molecules comprise molecules of a triblock copolymer.

10. The barrier according to claim 9, each molecule of the triblock copolymer comprises a first hydrophobic block and a second hydrophobic block and a hydrophilic block coupled to the first hydrophobic block and the second hydrophobic block and located between the first hydrophobic block and the second hydrophobic block.

11. The barrier according to claim 9, wherein each molecule of the triblock copolymer comprises a first hydrophilic block and a second hydrophilic block and a hydrophobic block coupled to the first hydrophilic block and the second hydrophilic block and located between the first hydrophilic block and the second hydrophilic block.

12. The barrier according to any one of claims 1 to 11, wherein the amphiphilic molecules are cross-linked by the product of a polymerisation reaction.

13. The barrier of claim 12, wherein the product of the polymerization reaction comprises reacted itaconic acid moieties, reacted N-carboxyanhydride moieties, reacted disulfonylpyridyl moieties, reacted N-hydroxysuccinimide (NHS) esters, reacted acrylate moieties, reacted methacrylate moieties, reacted acrylamide moieties, reacted methacrylamide moieties, reacted styrene moieties, reacted maleic acid moieties, reacted carboxylic acid moieties, reacted thiol moieties, reacted allyl moieties, reacted vinyl moieties, reacted propargyl moieties, or reacted maleimide moieties.

14. The barrier according to any one of claims 1 to 11, wherein the amphiphilic molecules are cross-linked via the product of a coupling reaction.

15. The barrier of claim 14, wherein the coupling reaction comprises a thiol-ene click reaction, a thiol-alkyne click reaction, a strain-promoted alkyne-azide cycloaddition, an amide coupling, a thiol / aza-Michael reaction, a [2+2] cycloaddition, a thio-Michael click reaction, a condensation reaction, a [2+2] photocycloaddition, a protein-ligand interaction, a host-guest chemistry, a disulfide formation, an imine formation, or an enamine formation.

16. The barrier of any one of claims 1 to 15, further comprising nanopores within the barrier.

17. The barrier of claim 16, wherein the nanopore comprises α-hemolysin or MspA.

18. A barrier according to any one of claims 1 to 17, the barrier being suspended from a barrier support defining an aperture, the one or more layers being suspended across the aperture.

19. A barrier between a first fluid and a second fluid, the barrier comprising: at least one layer, the at least one layer comprising a plurality of amphiphilic molecules, The amphiphilic molecules contain reactive moieties to undergo cross-linking reactions with each other.

20. The barrier of claim 19, wherein the at least one layer comprises: a first layer comprising a first plurality of said amphiphilic molecules; and A second layer comprising a second plurality of amphiphilic molecules in contact with the first plurality of amphiphilic molecules.

21. A barrier according to claim 19 or claim 20, wherein the reactive moiety is selected from the group consisting of itaconic acid moieties, N-carboxyanhydride moieties, disulfonylpyridyl moieties, N-hydroxysuccinimide (NHS) esters, acrylate moieties, methacrylate moieties, acrylamide moieties, methacrylamide moieties, styrene moieties, maleic acid moieties, carboxylic acid moieties, thiol moieties, allyl moieties, vinyl moieties, propargyl moieties and maleimide moieties.

22. The barrier of any one of claims 19 to 21, wherein the reactive moieties comprise a mixture of moieties that react with each other via a thiol-ene click reaction, a thiol-alkyne click reaction, a strain-promoted alkyne-azide cycloaddition, an amide coupling, a thiol / aza-Michael reaction, a [2+2] cycloaddition, a thio-Michael click reaction, a condensation reaction, a [2+2] photocycloaddition, a protein-ligand interaction, a host-guest chemistry, a disulfide formation, an imine formation, or an enamine formation.

23. The barrier according to any one of claims 19 to 22, wherein the amphiphilic molecule comprises at least one hydrophobic block coupled to at least one hydrophilic block at an interface.

24. The barrier of claim 23, wherein the reactive moiety is located at the hydrophilic block of the corresponding amphiphilic molecule.

25. A barrier according to claim 23 or claim 24, wherein the reactive moiety is located at the hydrophobic block of the corresponding amphiphilic molecule.

26. The barrier according to any one of claims 23 to 25, wherein the reactive moieties are located at the interface of the corresponding amphiphilic molecules.

27. The barrier according to any one of claims 23 to 26, wherein the amphiphilic molecules comprise molecules of a diblock copolymer comprising a hydrophobic block coupled to a hydrophilic block.

28. The barrier of any one of claims 19 to 26, wherein the amphiphilic molecules comprise molecules of a triblock copolymer.

29. The barrier of claim 28, each molecule of the triblock copolymer comprising a first hydrophobic block and a second hydrophobic block and a hydrophilic block coupled to the first hydrophobic block and the second hydrophobic block and located between the first hydrophobic block and the second hydrophobic block.

30. The barrier of claim 28, each molecule of the triblock copolymer comprising a first hydrophilic block and a second hydrophilic block and a hydrophobic block coupled to and located between the first hydrophilic block and the second hydrophilic block.

31. The barrier of any one of claims 19 to 30, further comprising nanopores within the barrier.

32. The barrier of claim 31 , wherein the nanopore comprises α-hemolysin or MspA.

33. A barrier according to any one of claims 19 to 32, the barrier being suspended from a barrier support defining an aperture, the one or more layers being suspended across the aperture.

34. A method of forming a barrier between a first fluid and a second fluid, the method comprising: forming at least one layer, the at least one layer comprising a plurality of amphiphilic molecules, wherein the amphiphilic molecules comprise a reactive moiety; The plurality of amphiphilic molecules are cross-linked to each other using a cross-linking reaction of the reactive moiety.

35. The method of claim 34, wherein forming the at least one layer comprises forming a first layer comprising a first plurality of the amphiphilic molecules, and forming a second layer comprising a second plurality of the amphiphilic molecules.

36. A method according to claim 34 or claim 35, wherein the cross-linking reaction comprises a polymerisation reaction.

37. The method of claim 36, wherein the reactive moiety is selected from the group consisting of itaconic acid moieties, N-carboxyanhydride moieties, disulfonylpyridyl moieties, N-hydroxysuccinimide (NHS) esters, acrylate moieties, methacrylate moieties, acrylamide moieties, methacrylamide moieties, styrene moieties, maleic acid moieties, carboxylic acid moieties, thiol moieties, allyl moieties, vinyl moieties, propargyl moieties, and maleimide moieties.

38. A method according to claim 36 or claim 37, wherein the polymerisation reaction comprises ring-opening polymerisation or step-growth polymerisation.

39. The method of any one of claims 36 to 38, further comprising initiating the polymerization reaction using an initiator.

40. The method of claim 39, wherein the initiator comprises a photoinitiator, a redox system, or photons.

41. The method of claim 40, wherein the photoinitiator is selected from the group consisting of 2,2-dimethoxy-2-phenylacetophenone, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

42. The method of claim 40, wherein the redox system comprises potassium persulfate or ammonium persulfate and N,N,N',N'-tetramethylethylenediamine.

43. The method of claim 34 or claim 35, wherein the cross-linking reaction comprises a coupling reaction.

44. The method of claim 43, wherein the coupling reaction comprises a thiol-ene click reaction, a thiol-alkyne click reaction, a strain-promoted alkyne-azide cycloaddition, an amide coupling, a thiol / aza-Michael reaction, a [2+2] cycloaddition, a thio-Michael click reaction, a condensation reaction, a [2+2] photocycloaddition, a protein-ligand interaction, a host-guest chemistry, a disulfide formation, an imine formation, or an enamine formation.

45. A method according to claim 43 or claim 44, wherein the coupling reaction is initiated using an initiator.

46. ​​The method of claim 45, wherein the initiator comprises a free radical initiator, a redox system, a reducing agent, or photons.

47. The method of claim 46, wherein the free radical initiator comprises 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone or 2,2'-azobis(2-methylpropionamidine) dihydrochloride.

48. The method of claim 46, wherein the redox system comprises potassium persulfate or ammonium persulfate and N,N,N',N'-tetramethylethylenediamine.

49. The method of claim 46, wherein the reducing agent comprises tris(2-carboxyethyl)phosphine, dithiothreitol, sodium ascorbate, or phosphine.

50. The method of any one of claims 43 to 49, wherein the reactive moiety comprises a propargyl moiety, an N-hydroxysuccinimide (NHS) ester, a disulfide pyridyl moiety, an aliphatic amide moiety, a propargyl moiety, an azido moiety, a DBCO moiety, a BCN moiety, an amine moiety, an activated carboxyl moiety, a dimethylmaleimide moiety, or a maleimide moiety.

51. The method of any one of claims 34 to 50, wherein the reactive moiety is located at the hydrophilic block of the amphiphilic molecule.

52. The method of any one of claims 34 to 51, wherein the reactive moiety is located at the interface between the hydrophilic block and the hydrophobic block of the amphiphilic molecule.

53. The method of any one of claims 34 to 52, wherein the reactive moiety is located at the hydrophilic block of the amphiphilic molecule.

54. The method of any one of claims 34 to 53, wherein the amphiphilic molecule has an AB framework.

55. The method of any one of claims 34 to 53, wherein the amphiphilic molecule has an ABA framework.

56. The method of any one of claims 34 to 53, wherein the amphiphilic molecule has a BAB framework.

57. The method of any one of claims 34 to 56, wherein the amphiphilic molecule comprises poly(dimethylsiloxane) (PDMS).

58. The method of any one of claims 34 to 56, wherein the amphiphilic molecule comprises poly(isobutylene) (PIB).

59. The method of any one of claims 34 to 58, wherein the amphiphilic molecule comprises poly(ethylene oxide) (PEO).

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