Methods for the synthesis of nanosheet membranes

Abstract

Disclosed are membrane compositions and methods for preparing membrane compositions.

Description

[0001] Methods for the Synthesis of Nanosheet Membranes

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of priority of U.S. Provisional Application No. 63 / 520,835, filed August 21, 2023, and U.S. Provisional Application No. 63 / 520,832, filed August 21, 2023, each of which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Grant no. DE-SC0023265 awarded by the Department of Energy and Grant no. HDTRA 1-20- 1-0005 awarded by the Department of Defense / Department of Threat Reduction and Grant no. 2137420 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Synthetic polymeric membranes, which are widely used in water purification, gas separations, chemical processing, and bioprocessing, suffer from a ubiquitous trade-off trend: high permeability leads to low selectivity and vice versa. This trade-off manifests due to the structural variations in state-of-the-art membranes, which typically use differences in molecular sizes to affect a separation. In the cases of angstrom-scale separations such as water desalination and gas separations, where nonporous polymeric membranes operate via the solution-diffusion mechanism, the variable size of the free volume elements through which diffusion occurs hampers membrane performance. In porous membranes such as ultrafiltration and microfiltration, this trade-off is primarily due to the broad pore size distribution seen in commercial membranes.

[0008] Biological membranes exhibit high permeability' and high selectivity combinations because, in addition to possessing various transmembrane proteins with well-defined channel pore sizes, they synergistically combine size, charge, van der Waals, and specific binding interactions within the channels to enhance the transport of target species. For example, size exclusion is the major mechanism for water-over-solute selectivity of biological water channel proteins, aquaporins. Water dipole reorientation through a series of specific hydrogen bonding steps is another selectivity mechanism that prevents protons

[0009] 1

[0010] SUBSTITUTE SHEET (RULE 26) from crossing the membrane. Aquaporins have been incorporated into membrane matnces for applications in desalination and water purification because of their ability to maintain high permeability and selectivity. However, high costs, difficulties in fabrication, and low stability associated with membrane protein-based materials have hindered the large-scale applications of aquaporin-based membranes.

[0011] Biological membrane protein channels, synthetic channels, and carbon nanotubes have emerged as promising platforms for the development of separation membranes with precise molecular selectivity. Artificial channels can be synthesized using simple chemistry, and are solvent compatible, thus allowing manufacturing techniques common in polymer processing to be applied. More importantly, flexibility in design of the chemical structures of artificial channels further allows for specific functionalization to tailor their permeability and selectivity. These precisely designed pore structures are ideal for membranes that can overcome the aforementioned permeability-selectivity trade-off of current commercial membranes. Relative to state-of-the-art commercial membranes, biomimetic membranes incorporating these pore structures are expected to exhibit high permeability and selectivity because they possess a high density of channels with a well-defined pore geometry and functionality designed to exclude or pass specific components from complicated mixtures.

[0012] Membrane protein-based biomimetic membranes studied thus far have been limited to small improvements in performance that are much lower than the orders of magnitude enhancement anticipated from early experiments. Current membrane protein-based biomimetic membranes show 2-3 times increases in permeability over commercial membranes with similar or worse selectivity. This has been attributed to the use of vesicular morphologies of channel-reconstituted liposomes and the low protein content in biomimetic matrices used for membrane fabrication.

[0013] In addition, widespread application of membrane protein-based biomimetic membranes developed so far may be limited due to the use of non-scalable fabrication techniques used for membrane synthesis. While interfacial polymerization has been used to make scaled up biomimetic membranes by integrating protein containing vesicles into a polymer layer, these membrane struggle due to low protein loading in the vesicles and a low loading of vesicles into the polymer layer. Designing membranes with high packing densities of channel proteins with uniform pore sizes (e.g., of '0.5-1.5 nm) with less material- and time-intensive synthesis techniques could provide a path to meet the ultimate promise of biomimetic membranes in this important solute size range.

[0014] 2

[0015] SUBSTITUTE SHEET (RULE 26) SUMMARY

[0016] Provided herein are membrane compositions as well as methods of making membrane compositions.

[0017] For example, provided herein are single-step methods of forming membrane compositions. These methods can comprise contacting a membrane support with a solution comprising a matrix polymer and a plurality of nanosheets, thereby depositing the matrix polymer and the plurality of nanosheets on a surface of the membrane support; and crosslinking the matrix polymer and the plurality of nanosheets. In some aspects, these methods can comprise a single contacting step.

[0018] In some aspects, the method can further comprise crosslinking the matrix polymer, the plurality of nanosheets, or a combination thereof to the membrane support.

[0019] In some aspects, contacting the membrane support with the solution comprising the matrix polymer and the plurality of nanosheets can comprise positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing the solution comprising the matrix polymer and the plurality of nanosheets into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the matrix polymer and the plurality of nanosheets on the surface of the membrane support; and introducing a solution comprising one or more crosslinking reagents into the filtration cell under conditions effective to covalently crosslink the plurality of nanosheets to the matrix polymer.

[0020] In some aspects, the matrix polymer and the nanosheets can exhibit opposite charges. For example, in some aspects, the matrix polymer can comprise a positively- charged polymer and the nanosheets are negatively charged. In other aspects, the matrix polymer comprises a negatively charged polymer and the nanosheets are positively charged.

[0021] In other aspects, the matrix polymer and the nanosheets can both be uncharged.

[0022] In other aspects, the matrix polymer can comprise a negatively charged polymer or a positively charged polymer and the nanosheets are uncharged.

[0023] In other aspects, the matrix polymer comprises an uncharged polymer and the nanosheets are positively charged or negatively charged.

[0024] In some aspects, the membrane support can comprises a porous membrane. In some aspects, the membrane support can comprise a polymeric substrate or an inorganic substrate. In certain aspects, the membrane support comprises an ultrafiltration membrane (e.g., a polymeric ultrafiltration membrane).

[0025] 3

[0026] SUBSTITUTE SHEET (RULE 26) In other aspects, the membrane support can comprise a non-porous membrane (e.g., a non-porous polymeric membrane). In certain aspects, the membrane support can comprise a reverse osmosis (RO) membrane. In certain aspects, the membrane support can comprise a nanofiltration membrane.

[0027] In some aspects, the membrane support exhibits a negatively charged surface. In some aspects, the negatively charged surface can be applied by UV-ozone treatment of the membrane surface.

[0028] Each of the nanosheets comprise one or more channels. In some aspects, the channels can comprise a membrane protein, a carbon nanotube, or an artificial channel. In some aspects, each of the plurality of nanosheets can comprise a polymer and a membrane protein. In some aspects, each of the plurality of nanosheets can comprise a polymer, lipid, or a combination thereof, and a membrane protein. In some aspects, the polymer can comprise a block copolymer (e.g., an amphiphilic block copolymer). For example, in some aspects, the block copolymer can comprise one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEO). In some aspects, the block copolymer can comprise one or more hydrophobic blocks of poly caprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEO). In some aspects, the amphiphilic block copolymer comprises a negatively charged moiety, such as a carboxylate. In some aspects, the polymer and membrane protein are present at a mass ratio of from 1000:1 to 1: 1000 (e.g., from 100: 1 to 1: 100 or from 10: 1 to 1 :10).

[0029] In some aspects, the membrane protein can comprise a beta barrel membrane protein. In some aspects, the membrane protein can comprise an alpha helical membrane protein.

[0030] In some aspects, the membrane protein can comprise a porin, a pore-forming toxin, or an aquaporin. In certain aspects, the membrane protein can comprise outer membrane protein F (OmpF), outer membrane protein X (OmpX), a-hemolysin toxin (aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, or aquaporin Z (AqpZ) such as RsAqpZ.

[0031] In some aspects, the matrix polymer comprises an amine group, an amide group, an imide group, an imine group, an azide group, a nitrate group, a nitrite group, a cyanate group, a nitrile group, or a combination thereof. In certain aspects, the matrix polymer comprises a polyamine. In certain aspects, the matrix polymer comprises polyethylenimine.

[0032] SUBSTITUTE SHEET (RULE 26) Also provided herein are membrane compositions prepared by the methods described herein, as well as separation devices comprising membrane compositions prepared by the methods described herein.

[0033] Also provided herein are membrane compositions prepared using molecular crosslinkers as well as methods of making membrane compositions using molecular crosslinkers. For example, provided herein are membrane compositions that comprise a membrane support; and a plurality of nanosheets covalently crosslinked to the membrane support via a molecular crosslinker. In some aspects, the plurality of nanosheets can also be covalently crosslinked to one another via the molecular crosslinker.

[0034] In some aspects, the membrane support can comprises a porous membrane. In some aspects, the membrane support can comprise a polymeric substrate or an inorganic substrate. In certain aspects, the membrane support comprises an ultrafiltration membrane (e.g., a polymeric ultrafiltration membrane).

[0035] In other aspects, the membrane support can comprise a non-porous membrane (e g., a non-porous polymeric membrane). In certain aspects, the membrane support can comprise a reverse osmosis (RO) membrane. In certain aspects, the membrane support can comprise a nanofiltration membrane.

[0036] Each of the nanosheets comprise one or more channels. In some aspects, the channels can comprise a membrane protein, a carbon nanotube, or an artificial channel. In some aspects, each of the plurality of nanosheets can comprise a polymer, a lipid, or a combination thereof, and a membrane protein. In some aspects, the polymer can comprise a block copolymer. For example, in some aspects, the block copolymer can comprise one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEO). For example, in some aspects, the block copolymer can comprise one or more hydrophobic blocks of poly caprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEO). In some aspects, the polymer and membrane protein are present at mass ratio of from 1000:1 to 1: 1000 (e.g., from 100: 1 to 1: 100 or from 10: 1 to 1: 10).

[0037] In some aspects, the membrane protein can comprise a beta barrel membrane protein. In some aspects, the membrane protein can comprise an alpha helical membrane protein.

[0038] In some aspects, the membrane protein can comprise a porin, a pore-forming toxin, or an aquaporin. In certain aspects, the membrane protein can comprise outer membrane protein F (OmpF), outer membrane protein X (OmpX), a-hemolysin toxin (aHL),

[0039] 5

[0040] SUBSTITUTE SHEET (RULE 26) ferrichrome outer membrane transporter (FhuA), AquaporinO, or aquaporin Z (AqpZ) such as Rs AqpZ).

[0041] In some aspects, the molecular crosslinker comprises a small molecule crosslinker. In certain aspects, the small molecule crosslinker can have a molecular weight of 1000 g / mol or less, such as a molecular weight of 800 g / mol or less, a molecular weight of 600 g / mol or less, or a molecular weight of 500 g / mol or less.

[0042] In some aspects, the molecular crosslinker comprises a bifunctional crosslinker. In some aspects, the molecular crosslinker comprises a diamine crosslinker, a diester crosslinker, a dithiol crosslinker, a diazide crosslinker, a dialkyne crosslinker, a divinyl crosslinker, a di(meth)acrylate crosslinker, or a combination thereof.

[0043] Also provided herein are methods of forming a membrane composition. These methods can comprise contacting a membrane support with a molecular crosslinker and a plurality of nanosheets under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support. In some aspects, the plurality of nanosheets are also covalently crosslinked to one another via the molecular crosslinker.

[0044] In some aspects, methods can further comprise activating the membrane support to introduce chemical moieties that can react with the molecular crosslinker to form covalent bonds.

[0045] In some aspects, contacting a membrane support with a molecular crosslinker and a plurality of nanosheets can comprise depositing the plurality of nanosheets on a surface of the membrane support; and contacting the surface with a solution comprising the molecular crosslinker under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support.

[0046] In certain aspects, contacting a membrane support with a molecular crosslinker and a plurality of nanosheets can comprise positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing a solution comprising the plurality of nanosheets dissolved or dispersed in a solvent into the filtration cell so as to contact a surface of the membrane support; applying pressure to the filtration cell to drive the solvent across the membrane support, thereby depositing the plurality of nanosheets on the surface of the membrane support; and introducing a solution comprising the molecular crosslinker into the filtration cell so as to contact a surface of the membrane support under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support.

[0047] 6

[0048] SUBSTITUTE SHEET (RULE 26) In some aspects, the molecular crosslinker comprises a small molecule crosslinker. In certain aspects, the small molecule crosslinker can have a molecular weight of 1000 g / mol or less, such as a molecular weight of 800 g / mol or less, a molecular weight of 600 g / mol or less, or a molecular weight of 500 g / mol or less.

[0049] In some aspects, the molecular crosslinker comprises a bifunctional crosslinker. In some aspects, the molecular crosslinker comprises a diamine crosslinker, a diester crosslinker, a dithiol crosslinker, a diazide crosslinker, a dialkyne crosslinker, a divinyl crosslinker, a di(meth)acry late crosslinker, a dialdehyde crosslinker, or a combination thereof.

[0050] Also provided herein are separation devices comprising the membrane compositions described herein.

[0051] DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a schematic illustration of example methods of fabricating membrane compositions described herein.

[0053] Figure 2A is a plot showing the water permeance of a poly(acrylonitrile) (PAN) ultrafiltration membrane and a PAN membrane after deposition of OmpF nanosheets using the single-step method described herein.

[0054] Figure 2B is a plot showing the water permeance of membrane compositions prepared by depositing 1, 2, and 3 layers OmpF nanosheets on a membrane support using the layer-by-layer deposition methods described in U.S. Patent No. 2022 / 0080364, which is incorporated by reference herein in its entirety.

[0055] Figure 3A is a plot showing the percent of solute rejection for solutes of varying molecular weight from a PAN membrane after deposition of OmpF nanosheets using the single-step method described herein.

[0056] Figure 3B is a plot showing the percent of solute rejection for solutes of varying molecular weight from membrane compositions prepared by depositing 1, 2, and 3 layers OmpF nanosheets on a membrane support using the layer-by-layer deposition methods described in U.S. Patent No. 2022 / 0080364.

[0057] Figure 4 is a schematic illustration of example methods of fabricating membrane compositions described herein.

[0058] Figure 5 is a plot showing the water permeance of a PAN ultrafiltration membrane and a PAN membrane after deposition of polyethyleneimine (PEI) / OmpF nanosheets using the single-step method with EDC / NHS crosslinking.

[0059] 7

[0060] SUBSTITUTE SHEET (RULE 26) Figure 6 is a plot showing the water permeance of a polyethersulfone (PES) ultrafiltration membrane and a PES membrane after deposition of polyethyleneimine (PEI) / OmpF nanosheets using the single-step method with gultaraldehyde crosslinking.

[0061] Figure 7 is a plot showing the water permeance of a cellulose ultrafiltration membrane and a cellulose membrane after deposition of OmpF nanosheets using the single- step method with dopamine crosslinking / coating. The permeance of a membrane prepared using dopamine only is also included as a control.

[0062] Figure 8 is a schematic illustration of the methods of fabricating membrane compositions described herein.

[0063] Figure 9 is a plot showing the water permeance of a poly(acrylomtrile) (PAN) ultrafiltration membrane and a PAN membrane after deposition of 2D OmpF sheets and 2D RsAqpZ sheets (PAN-OmpF-RsAqpZ).

[0064] Figure 10A is a micrograph of a PAN support membrane (10 pm scale, 10,000X mag).

[0065] Figure 1 OB is a micrograph of a PAN membrane after deposition of 2D OmpF sheets and 2D RsAqpZ sheets (PAN-OmpF-RsAqpZ) (10 pm scale, 10,000X mag).

[0066] Figure 11 A is a micrograph of a PAN support membrane (4 pm scale, 30,000X mag).

[0067] Figure 1 IB is a micrograph of a PAN membrane after deposition of 2D OmpF sheets and 2D RsAqpZ sheets (PAN-OmpF-RsAqpZ) (4 pm scale, 30,000X mag).

[0068] Figures 12A-12B are micrographs of a PAN membrane after deposition of 2D OmpF sheets and 2D RsAqpZ sheets (PAN-OmpF-RsAqpZ). Figure 12A includes a 4 pm scale, (30,000X mag) and Figure 12B includes a 1 pm scale, (10,000X mag).

[0069] Figures 13A-13B are micrographs of a PAN membrane after deposition of 2D OmpF sheets and 2D RsAqpZ sheets (PAN-OmpF-RsAqpZ). Figure 13A includes a 10 pm scale, (10,000X mag) and Figure 13B includes a 4 pm scale, (30,000X mag).

[0070] Figure 14 is a plot showing the water permeance of a poly(acrylonitrile) (PAN) ultrafiltration membrane and a PAN membrane after deposition of 2D RsAqpZ sheets (PAN-RsAqpZ).

[0071] DETAILED DESCRIPTION

[0072] Described herein are membrane compositions and methods for preparing membrane compositions. In particular, the methods described herein can provide a simplified route to

[0073] 8

[0074] SUBSTITUTE SHEET (RULE 26) prepare membrane compositions, including biomimetic-membrane filters, from proteincontaining 2D sheets.

[0075] The membrane compositions described herein can be prepared by mixing 2D sheets containing membrane proteins, carbon nanotubes, or their artificial mimics optionally with a cationic polymer (e.g., a polymeric amine such as poly(ethyleneimine) (PEI)). The mixture can then be applied to a membrane support and crosslinked (e.g., using 1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), glutaraldehyde, and / or N-hydroxysuccinimide (NHS)).

[0076] The methods described herein may offer advantages over existing methods for fabricating membrane compositions. For example, the methods described herein can provide for the preparation of highly porous biological or biomimetic channel -based membrane fabrications in a scalable membrane structure prepared using simple processing steps. Further, these membranes are covalently crosslinked, resulting in membranes with improved stability.

[0077] Further, previously developed methods employed an 8-12 step layer-by-layer process to deposit nanoheets sheets onto a substrate. In contrast, in the methods described herein, the cationic polymer and the 2D nanosheets are mixed and applied in a single step, eliminating the multiple layer-by-layer deposition steps and streamlining synthesis. Additionally, existing layer-by-layer processes incorporate polymeric amines as a polyelectrolyte crosslinker. These methods can also be used to form membranes much more efficiently and rapidly than existing methods

[0078] The embodiments of this invention are not limited to particular membranes or methods of using membranes, which can vary greatly without departing from the scope of the invention.

[0079] Definitions

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references

[0081] 9

[0082] SUBSTITUTE SHEET (RULE 26) mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0083] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.

[0084] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1 'A, and 4% This applies regardless of the breadth of the range.

[0085] The term “about,” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to mass, volume, time, temperature, distance, molecular weight, and water permeability. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0086] As used herein, the term “analog” means a molecular derivative of a molecule. The term is synonymous with the terms “structural analog” or “chemical analog.”

[0087] The methods and compositions described herein may comprise, consist essentially of, or consist of the components and ingredients of the present invention as well as other ingredients described herein. As used herein, “consisting essentially of’ means that the methods, systems, apparatuses and compositions may include additional steps, components

[0088] 10

[0089] SUBSTITUTE SHEET (RULE 26) or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, apparatuses, and compositions.

[0090] As used herein, the prefix "oligo-'' refers to a molecular complex comprised of between two and ten monomeric units. For example, oligosaccharides are comprised of between two and ten monosaccharides. Furthermore, unless otherwise specifically limited, the term “oligo-” shall include all possible isomeric configurations of the molecule, including, but are not limited to isotactic, syndiotactic and random symmetries, and combinations thereof. Furthermore, unless otherwise specifically limited, the term “oligo-” shall include all possible geometrical configurations of the molecule.

[0091] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally-occurring amino acids.

[0092] The term “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0093] The terms “water soluble” and “water miscible” as used herein, means that the component is soluble or miscible in water at 25° C. preferably at a concentration of 0.01 wt. %, more preferably at 0. 1 wt. %, and most preferably at 1 wt. %.

[0094] The term “channel” as used herein refers to a pathway formed in or through a medium that allows for movement of fluids, such as liquids and gases.

[0095] The terms “nanosheet,” “two-dimensional nanosheet,” and “2D nanosheet”, as used herein, refer to a material with a thickness on the nanometer scale or less, and lateral dimensions (e.g., a length and a width) that are each larger than the thickness of the material.

[0096] Membrane Compositions and Methods of Making Membrane Compositions in a Single Step

[0097] Described herein are methods of forming membrane compositions. These methods can comprise contacting a membrane support with a solution comprising a matrix polymer

[0098] 11

[0099] SUBSTITUTE SHEET (RULE 26) and a plurality of nanosheets, thereby depositing the matrix polymer and the plurality of nanosheets on a surface of the membrane support; and crosslinking the matrix polymer and the plurality of nanosheets. In some embodiments, these methods can comprise a single contacting step (i.e., a single step in which a solution comprising both the matrix polymer and the plurality of nanosheets is applied to the membrane support).

[0100] Depending on the surface chemistry of the membrane support, the polymer, the nanosheets, and the cross-linking method chosen, the matrix polymer and / or the nanosheets can also be crosslinked to the membrane support. In some aspects, the method can further comprise crosslinking the matrix polymer, the plurality of nanosheets, or a combination thereof to the membrane support.

[0101] In some embodiments, contacting the membrane support with the solution comprising the matrix polymer and the plurality of nanosheets can comprise positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing the solution comprising the matrix polymer and the plurality of nanosheets into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the matrix polymer and the plurality of nanosheets on the surface of the membrane support; and introducing a solution comprising one or more crosslinking reagents into the filtration cell under conditions effective to covalently crosslink the plurality of nanosheets to the matrix polymer. Other types of filtration cells can be used, if desired, provided these cells are operated in a "‘deadend” mode where the only path for liquid is through the membrane. The membrane compositions can similarly be formed by performing these contacting steps using a membrane support formed as a membrane module (e.g., in a flat sheet, a spiral-wound, a hollow fiber, or a plate-and-frame configuration).

[0102] Also described herein are method of forming membrane compositions that comprise contacting a membrane support with a solution comprising a plurality of nanosheets, thereby depositing the plurality of nanosheets on a surface of the membrane support; and contacting a membrane support with a solution comprising a matrix polymer, thereby physically immobilizing the plurality of nanosheets on a surface of the membrane support within the matrix polymer; wherein each of the nanosheets comprise one or more channels. In some embodiments, the matrix polymer and the plurality of nanosheets are present in the same solution, and the method comprises a single contacting step.

[0103] 12

[0104] SUBSTITUTE SHEET (RULE 26) In some embodiments, contacting the membrane support with the solution comprising the matrix polymer and the solution comprising the plurality of nanosheets comprises positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing the solution comprising the plurality of nanosheets into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the plurality of nanosheets on the surface of the membrane support; introducing a solution comprising a matrix polymer into the filtration cell so as to contact a surface of the membrane support; and applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the matrix polymer on the surface of the membrane support so that the plurality of nanosheets are physically immobilized on the surface of the membrane support within the matrix polymer. In other embodiments, contacting the membrane support with the solution comprising the matrix polymer and the solution comprising the plurality of nanosheets comprises positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing the solution comprising the plurality of nanosheets and the matnx polymer into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the plurality of nanosheets and the matrix polymer on the surface of the membrane support, wherein the plurality of nanosheets are physically immobilized on the surface of the membrane support within the matrix polymer. The membrane compositions can similarly be formed by performing these contacting steps using a membrane support formed as a membrane module (e.g., in a flat sheet, a spiral-wound, a hollow fiber, or a plate-and-frame configuration).

[0105] In some embodiments, the matrix polymer and the nanosheets can exhibit opposite charges. For example, in some embodiments, the matrix polymer can comprise a positively charged polymer and the nanosheets are negatively charged. In other embodiments, the matrix polymer comprises a negatively charged polymer and the nanosheets are positively charged.

[0106] In other embodiments, the matrix polymer and the nanosheets can both be uncharged.

[0107] In other embodiments, the matrix polymer can comprise a negatively charged polymer or a positively charged polymer and the nanosheets are uncharged.

[0108] 13

[0109] SUBSTITUTE SHEET (RULE 26) In other embodiments, the matrix polymer comprises an uncharged polymer and the nanosheets are positively charged or negatively charged.

[0110] In some embodiments, the membrane support can comprise a porous membrane. In some embodiments, the membrane support can comprise a polymeric substrate or an inorganic substrate. In certain embodiments, the membrane support comprises an ultrafiltration membrane (e.g., a polymeric ultrafiltration membrane).

[0111] In other embodiments, the membrane support can comprise a non-porous membrane (e.g., a non-porous polymeric membrane). In certain embodiments, the membrane support can comprise a reverse osmosis (RO) membrane. In certain embodiments, the membrane support can comprise a nanofiltration membrane.

[0112] In some embodiments, the membrane support exhibits a negatively charged surface. In some embodiments, the negatively charged surface can be applied by UV-ozone treatment. Examples of suitable membrane supports are discussed in more detail below.

[0113] Each of the nanosheets comprise one or more channels. In some embodiments, the channels can comprise a membrane protein, a carbon nanotube, or an artificial channel. In some embodiments, each of the plurality of nanosheets can comprise a polymer, a lipid, or a combination thereof, and a membrane protein. Examples of suitable nanosheets, as well as methods of forming nanosheets, are discussed in more detail below.

[0114] In some embodiments, the polymer can comprise a block copolymer (e.g., an amphiphilic block copolymer). For example, in some embodiments, the block copolymer can comprise one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEO), or one or more hydrophobic blocks of poly caprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEO). In some embodiments, the amphiphilic block copolymer comprises a negatively charged moiety, such as a carboxylate. In some embodiments, the polymer and membrane protein are present at mass ratio of from 1000: 1 to 1: 1000 (e.g., from 100: 1 to 1 : 100 or from 10:1 to 1: 10). Examples of suitable polymers are discussed in more detail below.

[0115] In some embodiments, the membrane protein can comprise a beta barrel membrane protein. In some embodiments, the membrane protein can comprise an alpha helical membrane protein. In some embodiments, the membrane protein can comprise a porin, a pore-forming toxin, or an aquaporin. In certain embodiments, the membrane protein can comprise outer membrane protein F (OmpF), outer membrane protein X (OmpX), a- hemolysin toxin (aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, or

[0116] 14

[0117] SUBSTITUTE SHEET (RULE 26) aquaporin Z (AqpZ) such as RsAqpZ. Examples of suitable proteins are discussed in more detail below.

[0118] In some embodiments, the matrix polymer can comprise a positively charged polymer. In some embodiments, the matrix poly mer comprises an amine group, an amide group, an imide group, an imine group, an azide group, a nitrate group, a nitrite group, a cyanate group, a nitrile group, or a combination thereof. In certain embodiments, the matrix polymer comprises a polyamine. In certain embodiments, the matrix polymer comprises polyethylenimine.

[0119] In some embodiments, the membrane compositions described herein can have specific solute selectivity, high permeabilities, or a combination thereof. The improved solute selectivity and high permeabilities can be an improvement over existing commercial membranes.

[0120] In some embodiments, the membrane compositions provide enhanced permeability. Preferably, the water permeability of the membrane compositions can be, for example, at least about 10 LMH bar1, at least about 25 LMH bar at least about 50 LMH bar1, at least about 75 LMH bar1, at least about 100 LMH bar at least about 150 LMH bar1, at least about 200 LMH bar1, at least about 250 LMH bar1, at least about 300 LMH bar '. at least about 500 LMH bar1, at least about 750 LMH bar1, at least about 1000 LMH bar1, at least about 1500 LMH bar1, at least about 2000 LMH bar or more.

[0121] In some embodiments, the membrane compositions provide high selectivity. In certain embodiments, the membrane compositions can exclude molecules larger than 25 Da, 50 Da, 75 Da, 100 Da, 150 Da, 200 Da, 250 Da, 300 Da, 350 Da, 400 Da, 450 Da, 500 Da, 550 Da, 600 Da, 650 Da, 700 Da, 750 Da, 800 Da, 850 Da, 900 Da, 950 Da, or 1000 Da.

[0122] The membranes can be suitable for use in precision separations. Thus, the membrane compositions can be used in a variety of separations, biomimetic devices, sensors, drug delivery devices, etc. Precision separations can be performed with the use of the membrane compositions in filtration devices, including, but not limited to, masks, air purifiers, water filters, desalination devices, and biomimetic devices. The compositions described herein can provide substantial energy savings in applications ranging from water treatment to small molecule bioseparations.

[0123] Examples of particular applications in which the membrane compositions described herein may be utilized include water vapor transport, water-borne pollutant rejection, resource recovery, and ion-ion separations. In certain embodiments, the membrane compositions described herein are utilized to perform solute-water separations in the

[0124] 15

[0125] SUBSTITUTE SHEET (RULE 26) angstrom to nanometer size range (e.g., ion-ion separations or the separation of small molecule in solution).

[0126] Membrane Compositions and Methods of Making Membrane Compositions using Molecular Crosslinkers

[0127] Described herein are membrane compositions that comprise a membrane support; and a plurality of nanosheets covalently crosslinked to the membrane support via a molecular crosslinker. In some embodiments, the plurality of nanosheets can also be covalently crosslinked to one another via the molecular crosslinker.

[0128] In some embodiments, the membrane support can comprise a porous membrane. In some embodiments, the membrane support can comprise a polymeric substrate or an inorganic substrate. In certain embodiments, the membrane support comprises an ultrafiltration membrane (e.g., a polymeric ultrafiltration membrane).

[0129] In other embodiments, the membrane support can comprise a non-porous membrane (e.g., a non-porous polymeric membrane). In certain embodiments, the membrane support can comprise a reverse osmosis (RO) membrane. In certain embodiments, the membrane support can comprise a nanofiltration membrane. Examples of suitable membranes are discussed in more detail below.

[0130] Each of the nanosheets comprise one or more channels. In some embodiments, the channels can comprise a membrane protein, a carbon nanotube, or an artificial channel. In some embodiments, each of the plurality of nanosheets can comprise a polymer, a lipid, or a combination thereof, and a membrane protein. Examples of suitable nanosheets, as well as methods of forming nanosheets, are discussed in more detail below.

[0131] In some embodiments, the polymer can comprise a block copolymer. For example, in some embodiments, the block copolymer can comprise one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEG), one or more hydrophobic blocks of poly caprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEG). In some embodiments, the polymer and membrane protein are present at mass ratio of from 1000: 1 to 1: 1000 (e.g., from 100: 1 to 1: 100 or from 10:1 to 1: 10). Examples of suitable polymers are discussed in more detail below.

[0132] In some embodiments, the membrane protein can comprise a beta barrel membrane protein. In some embodiments, the membrane protein can comprise an alpha helical membrane protein. In some embodiments, the membrane protein can comprise a porin, a pore-forming toxin, or an aquaporin. In certain embodiments, the membrane protein can comprise outer membrane protein F (OmpF), outer membrane protein X (OmpX), a-

[0133] 16

[0134] SUBSTITUTE SHEET (RULE 26) hemolysin toxin (otHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, or aquaporin Z (AqpZ) such as RsAqpZ. Examples of suitable proteins are discussed in more detail below.

[0135] In some embodiments, the molecular crosslinker comprises a small molecule crosslinker. In certain embodiments, the small molecule crosslinker can have a molecular weight of 1000 g / mol or less, such as a molecular weight of 800 g / mol or less, a molecular weight of 600 g / mol or less, or a molecular weight of 500 g / mol or less.

[0136] In some embodiments, the molecular crosslinker comprises a bifunctional crosslinker. In some embodiments, the molecular crosslinker comprises a diamine crosslinker, a diester crosslinker, a dithiol crosslinker, a diazide crosslinker, a dialkyne crosslinker, a divinyl crosslinker, a di(meth)acrylate crosslinker, a dialdehyde crosslinker (e.g., glutaraldehyde), or a combination thereof. In other embodiments, the molecular crosslinker can comprise a polyfunctional crosslinker (e.g., a tnfunctional crosslinker or a tetrafunctional crosslinker).

[0137] Also provided herein are methods of forming membrane composition. These methods can comprise contacting a membrane support with a molecular crosslinker and a plurality of nanosheets under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support. In some embodiments, the plurality of nanosheets are also covalently crosslinked to one another via the molecular crosslinker.

[0138] In some embodiments, methods can further comprise activating the membrane support to introduce chemical moieties that can react with the molecular crosslinker to form covalent bonds.

[0139] In some embodiments, contacting a membrane support with a molecular crosslinker and a plurality of nanosheets can comprise depositing the plurality of nanosheets on a surface of the membrane support; and contacting the surface with a solution comprising the molecular crosslinker under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support.

[0140] In certain embodiments, contacting a membrane support with a molecular crosslinker and a plurality of nanosheets can comprise positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing a solution comprising the plurality of nanosheets dissolved or dispersed in a solvent into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive the solvent across the membrane support, thereby depositing the plurality of nanosheets on the surface of the

[0141] 17

[0142] SUBSTITUTE SHEET (RULE 26) membrane support; and introducing a solution comprising the molecular crosslinker into the filtration cell so as to contact a surface of the membrane support under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support. The membrane compositions can similarly be formed by performing these contacting steps using a membrane support formed as a membrane module (e.g., in a flat sheet, a spiral-wound, a hollow fiber, or a plate-and-frame configuration).

[0143] In some embodiments, the molecular crosslinker comprises a small molecule crosslinker. In certain embodiments, the small molecule crosslinker can have a molecular weight of 1000 g / mol or less, such as a molecular weight of 800 g / mol or less, a molecular weight of 600 g / mol or less, or a molecular weight of 500 g / mol or less.

[0144] In some embodiments, the molecular crosslinker comprises a bifunctional crosslinker. In some embodiments, the molecular crosslinker comprises a diamine crosslinker, a diester crosslinker, a dithiol crosslinker, a diazide crosslinker, a dialkyne crosslinker, a divinyl crosslinker, a di(meth)acrylate crosslinker, a dialdehyde crosslinker (e.g., glutaraldehyde), or a combination thereof. In other embodiments, the molecular crosslinker can comprise a polyfunctional crosslinker (e.g., a trifunctional crosslinker or a tetrafunctional crosslinker).

[0145] In some embodiments, the membrane compositions described herein can have specific solute selectivity, high permeabilities, or a combination thereof. The improved solute selectivity and high permeabilities can be an improvement over existing commercial membranes.

[0146] In some embodiments, the membrane compositions provide enhanced permeability. Preferably, the water permeability of the membrane compositions can be, for example, at least about 10 LMH bar1, at least about 25 LMH bar at least about 50 LMH bar1, at least about 75 LMH bar1, at least about 100 LMH bar at least about 150 LMH bar1, at least about 200 LMH bar1, at least about 250 LMH bar1, at least about 300 LMH bar '. at least about 500 LMH bar1, at least about 750 LMH bar1, at least about 1000 LMH bar1, at least about 1500 LMH bar1, at least about 2000 LMH bar h or more.

[0147] In some embodiments, the membrane compositions provide high selectivity. In certain embodiments, the membrane compositions can exclude molecules larger than 25 Da, 50 Da, 75 Da, 100 Da, 150 Da, 200 Da, 250 Da, 300 Da, 350 Da, 400 Da, 450 Da, 500 Da, 550 Da, 600 Da, 650 Da, 700 Da, 750 Da, 800 Da, 850 Da, 900 Da, 950 Da, or 1000 Da.

[0148] The membranes can be suitable for use in precision separations. Thus, the membrane compositions can be used in a variety of separations, biomimetic devices, sensors, drug

[0149] 18

[0150] SUBSTITUTE SHEET (RULE 26) delivery devices, etc. Precision separations can be performed with the use of the membrane compositions in filtration devices, including, but not limited to, masks, air purifiers, water filters, desalination devices, and biomimetic devices. The compositions described herein can provide substantial energy savings in applications ranging from water treatment to small molecule bioseparations.

[0151] Examples of particular applications in which the membrane compositions described herein may be utilized include water vapor transport, water-borne pollutant rejection, resource recovery, and ion-ion separations. In certain embodiments, the membrane compositions described herein are utilized to perform solute-water separations in the angstrom to nanometer size range (e.g., ion-ion separations or the separation of small molecule in solution).

[0152] Membrane Supports

[0153] The compositions and methods described herein can comprise a membrane support.

[0154] In some embodiments, the membrane support can comprise a porous support. The porous structure can have a plurality of pores. In some examples, the plurality of pores can have an average pore diameter from about 10 nm and about 500 pm, such as from about 15 nm and about 100 pm, or from about 20 nm and about 1 pm.

[0155] In other embodiments, the membrane support can comprise a non-porous membrane (e.g., a non-porous polymeric membrane). In certain embodiments, the membrane support can comprise a reverse osmosis (RO) membrane. In certain embodiments, the membrane support can comprise a nanofiltration membrane.

[0156] In some embodiments, the membrane support can comprise a polymeric substrate and / or an inorganic substrate. Example polymeric substrates include, but are not limited to, a thermoplastic, a thermoset, or a combination thereof.

[0157] In some examples, the membrane support can comprise a synthetic polymer. Examples of synthetic polymers include polysulfones, poly ethersulfones, poly vinylidene difluoride (PVDF), polypropylene, acrylic polymers; poly(methyl methacrylate), polyamide (Nylon), polyimide polytetrafluoroethylene (PTFE), polyetherimide, polyacrylonitrile, polyethylene, polycarbonate, polytetrafluoroethylene, poly(dimethylsiloxane), polystyrene, and polyphenylene oxide. In certain examples, the polymeric substrate can comprise poly ethersulfone or polycarbonate.

[0158] In some embodiments, the membrane support may comprise a natural polymer or modified natural polymer. Examples of natural poly mer and modified natural polymer

[0159] 19

[0160] SUBSTITUTE SHEET (RULE 26) polymers include cellulose, cellulose esters, cellulose nitrate, cellulose acetate, and regenerated cellulose.

[0161] In some embodiments, the membrane support may comprise an inorganic material. Examples of appropriate inorganic materials include, but are not limited to, aluminum oxide (AI2O3), metal oxide / ceramic; silicon carbide (SiC), zirconium oxide, silicon dioxide, and titanium dioxide. In an exemplary embodiment, the inorganic substrate is aluminum oxide.

[0162] In some embodiments, a surface of the membrane support can be negatively charged.

[0163] Matrix Polymers

[0164] The methods and compositions comprise a matrix polymer. In some embodiments, the matrix polymer can comprise a positively charged polymer.

[0165] Example matrix polymers include those with a functional group such as an amine group, an amide group, an imide group, an imine group, an azide group, a nitrate group, a nitrite group, a cyanate group, a nitrile group, or a combination thereof.

[0166] Specific examples of the matrix polymers include polyethyleneimine, polyallylamine, polyvinylamine, polyvinylpyridine, polyethyleneimine-epichlorohydrine reaction products, polyamide-polyamine resins, polyamide-epichlorohydrine resins, chitosan compounds, cationized starch, polyaminesulfone, polyvinylimidazole, polyamidine, dicyanamide-polyalkylene polyamine condensation products, polyalkylene polyamine-dicyandiamide ammonium salt condensation products, dicyandiamide-formalin condensation products, polymers and copolymers of diallyldimethylammonium chloride, vinylpyrrolidone-vinylimidazole copolymers, polymers and copolymers of vinylbenzyltrimethylammonium chloride, polymers and copolymers of dimethylaminoethyl (meth)acrylate, polymers and copolymers of (meth)acryloyloxyalkyltrialkylammonium chloride, polymers and copolymers of (meth)acryloyloxyalkyldialkylbenzylammonium chloride, and acid neutralization products thereof.

[0167] Examples of matrix polymers that have an amino group include polyethyleneimine, polyallylamine, and polyvinylamine. In some embodiments, the matrix polymer can comprise polyethyleneimine or polyallylamine. In certain embodiments, the matrix polymer can comprise polyethyleneimine.

[0168] Nanosheets and Methods of Preparing the Same

[0169] The compositions and methods described herein can comprise nanosheets comprise one or more channels. In some embodiments, the channels can comprise a membrane protein, a carbon nanotube, or an artificial channel. In some embodiments, each of the

[0170] 20

[0171] SUBSTITUTE SHEET (RULE 26) plurality of nanosheets can comprise a polymer, a lipid, or a combination thereof, and a membrane protein.

[0172] Suitable nanosheets can be prepared by dissolving a polymer in solvent, dissolving a protein in solvent, and combining the two to form a combined solution. In some embodiments, the polymer can be dissolved in solvent, then the protein can be added and dissolved in the solution with the polymer to form a combined solution. In an alternative embodiment, the protein can be dissolved in solvent, then the polymer can be dissolved in the solution with the protein to form a combined solution. In a further embodiment, the polymer and protein can be dissolved separately in solvent and then combined to form a combined solution. In still a further embodiment, the protein and polymer can be dissolved together in solvent to form a combined solution.

[0173] In some embodiments, the polymer and membrane protein are present at mass ratio of from 1000: 1 to 1: 1000 (e.g., from 100: 1 to 1 :100 or from 10: 1 to 1: 10). In some embodiments, the polymer and membrane protein are in a mass ratio of between about 10:1 and about 1 : 10, between about 9: 1 and about 1:9, between about 8:1 and about 1:8, between about 7: 1 and about 1 :7, between about 6: 1 and about 1:6, between about 5: 1 and about 1 :5, between about 4:1 and about 1:4, between about 3: 1 and about 1 :3, between about 2:1 and about 1 :2, at about 1 : 1. In some embodiments, the mass ratio of the polymer to membrane protein is between about 1: 10 and about 1:2.

[0174] In some embodiments, the combined solution is mixed. In certain embodiments, the combined solution is mixed homogeneously. Any suitable method of mixing can be performed. Preferred methods of mixing include, but are not limited to, sonicating, manual mixing, automatic mixing, stirring, or a combination thereof.

[0175] The combined solution can be applied to a substrate. The substrate can be of any suitable size for preparation of the desired membrane size. The substrate can be of a suitable for preparation of a membrane. Preferably the substrates can comprise glass, plastic, composite, metal, or a mixture or combination thereof. The combined solution can be applied to a substrate by any suitable method, including, but not limited to, spraying, pouring, extruding, squirting, or otherwise applying. Optionally, the combined solution can be spread on the substrate.

[0176] Once applied to the substrate, the solvent can be evaporated from the combined solution. Any suitable means of evaporating can be applied. Example evaporation techniques include, but are not limited to, air drying, heating, vacuum drying, or a combination thereof. If using heat as an evaporation technique, it is preferably applied at a

[0177] 21

[0178] SUBSTITUTE SHEET (RULE 26) temperature of less than the melting point of the polymer. In certain embodiments, the method of evaporation can be heating and vacuum drying.

[0179] Once dried, a nanosheet comprising a polymer and a membrane protein can be removed from the substrate. Optionally, a buffer can be added to the dried nanosheet and / or two-dimensional crystal. In an embodiment, the nanosheet can be used to prepare a membrane. The nanosheet comprising a polymer and a membrane protein can be incorporated into a membrane using the methods described herein. The membrane can be incorporated into a separation device.

[0180] Membrane Proteins

[0181] As discussed above, the nanosheets used in the compositions and methods described herein can comprise a membrane protein. One or more types of membrane proteins may be used depending on the effect that is desired in the final membrane. Preferably the membrane protein comprises a pore or a channel. Membrane proteins suitable for inclusion in the membranes are for instance selected from, but not limited to, membrane proteins found in The Transporter Classification Database (TCDB; www.tcdb.org). The TCDB is an International Union of Biochemistry and Molecular Biology (lUBMB)-approved classification system for membrane transport proteins. For example, suitable native, recombinant, and engineered membrane proteins may include but are not limited to ABC transporters, ATPases, G-protein coupled receptors, holins, ion channels, major intrinsic proteins (including aquaporin water channels), mechanosensitive channels, outer membrane pore-forming proteins (porins), pore-forming toxins, rhodopsins, and the like. Additional pores or channels may include mutated or engineered pores or channels with increased thermal or solvent stability, and performance properties such as “always open” pores or channels. The embodiments of this invention are not limited to particular membrane protein, which can vary and are understood by skilled artisans.

[0182] In some embodiments, the membrane protein is a beta barrel membrane protein or an alpha helical membrane protein. Although known to be less stable than beta barrel membrane proteins, alpha helical membrane proteins also maintain their structure when exposed to solvents during the short processing time utilized. In some embodiments, the membrane protein is a ponn, a pore-forming toxin, or an aquaporin.

[0183] Porins are beta barrel membrane proteins present in the outer membrane of gramnegative bacteria and some gram-positive Mycobacteria, the outer membrane of mitochondria, and the outer chloroplast membrane. In an exemplary embodiment, the porin is outer membrane protein F (OmpF) from E. coli. In another exemplary embodiment, the

[0184] 22

[0185] SUBSTITUTE SHEET (RULE 26) porin is FhuA from E. coll that originally forms part of a larger pore complex for active ferrichrome-iron and antibiotic transports. FhuA AC / A4L is engineered with its central alpha-helical plug removed to function as a passive pore. All known porins are useful in the invention.

[0186] Pore-forming toxins (also known as pore-forming proteins) are produced by bacteria as well as other organisms including earthworms, which produce the pore-forming toxin lysenin. Pore-forming toxins may be beta barrel pore-forming toxins (including a- hemolysin, Panton-Valentine leucocidin, Clostridial Epsilon-toxin) or alpha helical poreforming toxins (including haemolysin E, actinoporins, Corynebactenal porin B). In an exemplary embodiment, the pore-forming toxin is a-hemolysin (o.HL). a self-assembled structure created by Staphylococcus aureus to porate cell membranes.

[0187] Aquaporins (AQPs) are integral membrane proteins from a larger family of major intrinsic proteins (MIPs) that form pores in the membrane of biological cells, mainly facilitating transport of water between cells. Aquaporin proteins are composed of a bundle of six transmembrane a-helices. All known aquaporin water channels (including but not limited to AQPO, AQP1, AqpZ, AQP4, SoPIP2;l, NtAQPl, AQP9, AqpX) are useful in the invention. In an exemplary embodiment, the aquaporin is AqpZ from Rhodobacter sphaeroides.

[0188] Preferably, the membrane protein comprises between about 10 wt. % and about 90 wt. % of the nanosheet or two-dimensional crystal, more preferably between about 15 wt. % and about 85 wt. % of the nanosheet or two-dimensional crystal, most preferably between about 20 wt. % and about 80 wt. % of the nanosheet or two-dimensional crystal.

[0189] Preferably, the membrane protein comprises between about 0. 1 wt. % and about 60 wt. % of the combined solution, more preferably between about 0.5 wt. % and about 55 wt. % of the combined solution, most preferably between about 1 wt. % and about 50 wt. % of the combined solution.

[0190] Polymers

[0191] As discussed above, the nanosheets used in the compositions and methods described herein can comprise a polymer. Example polymers include, but are not limited to, a thermoplastic, a thermoset, or a combination thereof. In some embodiments, the polymer can be a block copolymer.

[0192] Various types of amphiphilic copolymers can be used. In one embodiment, the copolymer is an ABA copolymer, where A is hydrophilic and B is hydrophobic where A is the same or different hydrophilic segments and B is a hydrophobic segment. Thus, the term

[0193] 23

[0194] SUBSTITUTE SHEET (RULE 26) “ABA copolymer” includes an ABC copolymer, where the hydrophilic segments A and C are different. AB copolymers can also be used.

[0195] The block copolymer includes at least one segment B that includes a hydrophobic polymer. Any of a number of hydrophobic polymers can be used, such as, but not limited to, polysiloxane such as poly dimethylsiloxane and poly diphenylsiloxane, perfluoropolyether, polystyrene, polyoxypropylene, polyvinylacetate, polyoxybutylene, polyisoprene, polybutadiene, polyvinylchloride, polyalkylacrylate (PAA), polyalkylmethacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylates, polyacrylates, polysulfones, polyvinylethers, and polypropylene oxide), and copolymers thereof.

[0196] In some embodiments, the hydrophobic segment contains a predominant amount of hydrophobic monomers. A hydrophobic monomer is a monomer that typically gives a homopolymer that is insoluble in water and can absorb less than 10% by weight of water.

[0197] Suitable hydrophobic monomers are C1-C18 alkyl and C3-C18 cycloalkyl acrylates and methacrylates. C3-C18 alkylacrylamides and -methacrylamides, acry lonitrile, methacrylonitrile, vinyl C1-C18 alkanoates, C2-C18 alkenes, C2-C18 haloalkenes, styrene, (lower alkyl)styrene, C4-C12 alkyl vinyl ethers, C2-C10 perfluoro-alkyl acry lates and methacrylates and correspondingly partially fluorinated acrylates and methacrylates, C3 through C12 perfluoroalkylethylthiocarbonylaminoethyl acrylates and methacrylates, acryloxy- and methacryloxyalkylsiloxanes, N-vinylcarbazole, Cl through C12 alkyl esters of maleic acid, fumaric acid, itaconic acid, mesaconic acid, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl valerate, chloroprene, vinyl chloride, vinylidene chloride, vinyltoluene, vinyl ethyl ether, perfluorohexyl ethylthiocarbonylaminoethyl methacrylate, isobomyl methacrylate, trifluoroethyl methacrylate, hexafluoroisopropyl methacrylate, hexafluorobutyl methacrylate, tristrimethylsilyloxysilylpropyl methacrylate (TRIS), butadiene, dimethylsiloxane, and 3 -methacryloxy propylpentamethyldisiloxane.

[0198] In some embodiments, the hydrophobic polymer is one which displays a relatively high oxygen diffusion rate there through, such as, but not limited to, polysiloxanes, perfluoroalkyl ethers, specific unsaturated polymers, and polysulfones. In one embodiment, the hydrophobic polymer is a polysiloxane block having terminal alkylene groups.

[0199] In some embodiments, the hydrophobic polymer includes a perfluoroalkyl-polyether block. In other embodiments, the hydrophobic polymer includes an unsaturated polymer, such as a polymer of a conjugated aliphatic or alicyclic diene, which may be substituted by halogen or lower alkyl, a polymer of an alkyne or dialkyne, which may be substituted by

[0200] 24

[0201] SUBSTITUTE SHEET (RULE 26) lower alkyl or trimethyl silyl a copolymer of a conjugated diene and a hydrophilic or hydrophobic vinylic monomer, and also partially hydrated derivatives of these compounds.

[0202] Specific examples of polymers of conjugated dienes are cis-, trans-, iso- or syndiotactic poly-l,2-butadiene, poly-1, 4-butadiene or polyisoprene, poly-pentenamer, poly chloroprene and polypiperylen. Other examples of copolymers are butadiene- or isoprene-copolymers with hydrophilic or hydrophobic vinylic monomers, such as acrylonitrile, styrene, acrylic acid or hydroxy ethylmethacrylate. An example of a poly alkyne is poly-1 -trimethylsilyl-propyne. In some embodiments, examples of polymers included unsaturated polymers are syndiotactic poly-1, 2-butadiene, poly- 1, 4-butadiene and polyisoprene. An especially preferred unsaturated polymer is poly-1 -trimethylsilyl-propyne. Another especially preferred unsaturated polymer is poly- 1, 4-butadiene. The hydrophobic polymer may include a single type of polymer or more than one type of polymer, such as two or more of those discussed above. The mean molecular weight of one segment B is in the range from about 500 to about 50,000, preferably in the range from about 800 to about 15,000, more preferably in the range of about 1,000 to 12,000, particularly preferably in the range from about 5,000 to about 12,000. In certain embodiments, the mean molecular weight of one segment B is in the range of abut 1,000 to about 2,000.

[0203] In some embodiments in addition to the hydrophobic segment B, the amphiphilic segmented copolymer includes at least one segment A which includes at least one hydrophilic polymer, such as, but not limited to, polyoxazoline, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, poly(meth)acrylic acid, polyethylene oxide-co-polypropyleneoxide block copolymers, poly(vinylether), poly(N,N-dimethylacrylamide), polyacrylic acid, polyacyl alkylene imine, polyhydroxyalkylacrylates such as hydroxyethyl methacrylate (HEMA), hydroxy ethyl acrylate, and hydroxypropyl acrylate, polyols, and copolymeric mixtures of two or more of the above mentioned polymers, natural polymers such as polysaccharides and polypeptides, and copolymers thereof, and polyionic molecules such as polyallylammonium, polyethyleneimine, polyvinylbenzyltrimethylammonium, polyaniline, sulfonated polyaniline, polypyrrole, and polypyridinium, polythiophene-acetic acids, polystyrenesulfonic acids, zwitterionic molecules, and salts and copolymers thereof.

[0204] In some embodiments, the hydrophilic segment preferably contains a predominant amount of hydrophilic monomers. A hydrophilic comonomer is a monomer that typically gives a homopolymer that is soluble in water or can absorb at least 10% by weight of water.

[0205] 25

[0206] SUBSTITUTE SHEET (RULE 26) Suitable hydrophilic monomers are hydroxyl-substituted lower alkyl acrylates and methacr lates, acrylamide, methacrylamide, (lower alkyl) acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylated acrylates and methacrylates, polyethyleneglycol-mono methacrylates and polyethyleneglycolmonomethylether methacrylates, hydroxyl-substituted (lower alkyl)acrylamides and methacrylamides, hydroxyl-substituted lower alkyl vinyl ethers, sodium vinylsulfonate, sodium styrenesulfonate, 2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl-2- pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4'-dialkyloxazolin-5-one, 2- and 4-vinylpyridine, vinylically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, amino(lower alkyl)-(where the term amino also includes quaternary ammonium), mono(lower alkylamino)(lower alkyl) and di(lower alkylamino)(lower alkyl) acrylates and methacrylates, allyl alcohol, 3-trimethylammonium 2-hydroxypropylmethacrylate chloride (Blemer.QA, for example from Nippon Oil), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethylmethacrylamide, glycerol methacrylate, and N-(l,l- dimethyl-3-oxobutyl)acrylamide.

[0207] In some embodiments, the segment A includes a polymer displaying a relatively high water or ion diffusion rate there through. Specific examples of hydrophilic monomers from which such polymers can be made are cyclic imino ethers, vinyl ethers, cyclic ethers including epoxides, cyclic unsaturated ethers, N-substituted aziridines, beta-lactones and beta-lactams. Further suitable monomers include ketene acetals, vinyl acetals and phosphoranes. Suitable cyclic imino ethers include 2-oxazoline. If a 2-oxazoline having an alkenyl group in 2 position is used as hydrophilic monomer, a polymerizable unsaturated group is provided within segment A (in a side chain) of the amphiphilic segmented copolymer to serve as the polymerizable unsaturated group necessary for the final polymerization to obtain a polymeric product or as an additional polymerizable unsaturated group which offers the possibility of direct crosslinking in the preparation of the polymer. In some embodiments, the cyclic imino ether is 2-methyloxazoline. The most preferred vinyl ethers are methyl vinyl ether, ethyl vinyl ether and methoxy ethyl vinyl ether.

[0208] In some embodiments, the mean molecular weight of one segment A is in the range from about 200 to about 50,000, from about 500 to about 50,000, from about 800 to about 15,000, from about 1,000 to 12,000, particularly from about 5,000 to about 12,000.

[0209] Preferred block copolymers, include, but are not limited to, an amphiphilic diblock or triblock block copolymer comprising one or more hydrophobic blocks selected from the group consisting of polybutadiene (PB), poly dimethylsiloxane (PDMS), polypropylene

[0210] 26

[0211] SUBSTITUTE SHEET (RULE 26) (PP), polypropylene oxide (PPO), poly ethylethylene (PEE), polyisobutylene (PIB), polyisoprene (PI), poly caprolactone (PCL), polystyrene (PS), fluorinated polymers, and polymethylmethacrylate (PMMA); and one or more hydrophilic blocks selected from the group consisting of polymethyloxazoline (PMOXA), polyethyloxazoline (PEtOXA), and polyethylene oxide (PEO). In particular embodiments of methods herein, the block copolymer comprises one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEO). In particular embodiments of methods herein, the block copolymer comprises one or more hydrophobic blocks of poly caprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEO).

[0212] Preferably, the polymer comprises between about 10 wt. % and about 90 wt. % of the nanosheet or two-dimensional crystal, more preferably between about 15 wt. % and about 85 wt. % of the nanosheet or two-dimensional crystal, most preferably between about 20 wt. % and about 80 wt. % of the nanosheet or two-dimensional crystal.

[0213] Preferably, the polymer comprises between about 0.1 wt. % and about 60 wt. % of the combined solution, more preferably between about 0.5 wt. % and about 55 wt. % of the combined solution, most preferably between about 1 wt. % and about 50 wt. % of the combined solution.

[0214] Preferably, the polymer comprises between about 0.01 wt. % and about 20 wt. % of the combined solution, more preferably between about 0.1 wt. % and about 15 wt. % of the combined solution, most preferably between about 0.5 wt. % and about 10 wt. % of the combined solution.

[0215] In some embodiments, the polymer comprises between about 0.001 wt. % and about 20 wt. % of the combined solution, more preferably about 0.005 wt. % and about 20 wt. % of the combined solution, more preferably between about 0.01 wt. % and about 15 wt. % of the combined solution, most preferably between about 0.05 wt. % and about 10 wt. % of the combined solution.

[0216] Lipids

[0217] As discussed above, the nanosheets used in the compositions and methods described herein can comprise a lipid. Examples of suitable lipids include cationic lipids, neutral lipids, PEGylated lipids, and ionizable lipids.

[0218] Examples of cationic lipids include, but are not limited to, DOTMA: [ 1 -(2,3- sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA (2,3-dioleyloxy-N-[2-(spermine carboxamido)ethyl]-

[0219] 27

[0220] SUBSTITUTE SHEET (RULE 26) N.N-dimethyl-l -propanaminium trifluoroacetate), DORIE (N-[l-(2,3-dioleyloxypropyl)]- N,N-dimethyl-N-hydroxy ethylammonium bromide), DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: 0,0- ditetradecanoyl-N-. alpha. -lrimelhylammonioacelyl)dielhanolamine chloride, CLIP 1: rac- [(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac- [2(2,3 -dihexadecyloxypropyloxy methyloxy )ethy 1 ] -trimethylammonium, CLIP9 : rac- [2(2,3- dihexadecyloxy propyloxy succinyloxy)ethyl] -trimethylammonium, oligofectamine, lipids described in U.S. Patent No. 5,049,386, N-[l-(2,3-dioleyloxypropyl)]-N,N-dimethyl-N- hydroxy ethylammonium bromide (DORIE), 2,3-dioleyloxy-N-[2-(spermine carboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), and the like as disclosed in International Publication Nos. WO91 / 16024 and WO97 / 019675;

[0221] DLinDMA and the like as disclosed in International Publication No. WO2005 / 121348; and DLin-K-DMA and the like as disclosed in International Publication No. W02009 / 086558; and (3R,4R)-3,4-bis((Z)-Hexadec-9-enyloxy)-l-methylpyrrolidine, and N-Methyl-N,N- bis(2-((Z)-octadec-6- enyloxy)ethyl)amine and the like as disclosed in International Publication No. WO2011 / 13636, or any combination thereof.

[0222] Examples of neutral lipids include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), cholesterol, or any combination thereof. In some embodiments, the one or more neutral lipids can include cholesterol.

[0223] Examples of PEGylated lipids include, but are not limited to, a PEG- ditetradecylacetamide, a PEG-myristoyl diglyceride, a PEG-diacylglycerol, a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, PEG-DMG, PEG-DSPE, or any combinations thereof. In some embodiments, the one or more PEGylated lipids comprise 1 ,2-dimyristoyl-sn-glycerol (DMG-PEG).

[0224] Examples of ionizable lipids include, but are not limited to, N,N-dimethyl-2,3- di oleyloxypropylamine (DODMA), [(4-hy droxybutyl)azanediyl] di(hexane-6, 1 -diy l)bis(2- hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino [octanoate (SM-102), DLin-MC3-DMA, DLin-KC2-DMA, 1- (2,3-bis(((9Z,12Z)-octadeca-9,12-dien-l-yl)oxy)propyl)pyrrolidine (A066), or any combination thereof. In some embodiments, the one or more ionizable lipids comprise N,N- dimethyl-2,3-dioleyloxypropylamine (DODMA).

[0225] 28

[0226] SUBSTITUTE SHEET (RULE 26) Solvents

[0227] As discussed above, the nanosheets used in the compositions and methods described herein can employ a solvent. Preferably, the solvent is an organic solvent. Preferred solvents include nonpolar solvents, polar protic solvents, and mixtures thereof. In some embodiments, the solvent is a water miscible solvent. Preferred solvents have a boiling point of less than 80° C., less than 75° C., or less than 70° C. Preferred solvents include, but are not limited to, chloroform, decane, dodecane, heptane, hexane, methanol, or a mixture thereof. The solvent used herein can be of a single solvent or a mixture of many different solvents.

[0228] In some embodiments, the solvent comprises between about 0.1 wt. % and about 99 wt. % of the combined solution, more preferably between about 0.5 wt. % and about 95 wt. % of the combined solution, most preferably between about 1 wt. % and about 90 wt. % of the combined solution.

[0229] Buffers

[0230] As discussed above, the nanosheets used in the compositions and methods described herein can optionally employ a buffer. Preferably the buffer is an aqueous buffer. Example buffers include, but are not limited to, Tris (tns(hydroxymethyl)aminomethane), HEPES (N-[2-hydroxyethyl]piperazine-N'-(2-ethanesulphonic) acid), MES (2-(N- morpholino)ethanesulfonic acid), PBS (phosphate buffered saline), MOPS (3-[N- morpholino]propanesulphonic acid), or a mixture thereof. Additional buffers suitable for use in the disclosed methods include Bis-tris (2-bis[2-hydroxyethyl]amino-2- hydroxymethyl-l,3-propanediol), ADA (N-[2-acetamido]-2-iminodiacetic acid), ACES (2- [2-acetamino]-2-aminoethanesulphonic acid), PIPES (1,4-piperazinediethanesulphonic acid), MOPSO (3-[N-morpholino]-2-hydroxypropanesulphonic acid), Bis-tris propane (1,3 bis[tris(hydroxymethyl)methylaminopropane]), BES (N,N-bis[2-hydroxyethyl]-2- aminoethanesulphonic acid), TES (2-[2 -hydroxy- 1,1- bis(hydroxymethyl)ethylamino]ethanesulphonic acid), DIPSO (3-N,N-bis[2- hydroxyethyl]amino-2-hydroxypropanesulphonic) acid), MOBS (4-N- morpholinobutanesulphonic acid), TAPSO (3 [N-tris-hydroxymethyl-methylamino]-2- hydroxypropanesulphomc acid), Tris (2-amino-2-[hydroxymethyl]-l,3-propanediol), HEPPSO (N-[2-hydroxyethyl]piperazine-N'-[2-hydroxypropanesulphonic]acid), POPSO (piperazine-N,N'-bis[2-hydroxypropanesulphonic]acid), TEA (triethanolamine), EPPS (N- [2-hydroxyethyl]-piperazine-N'-[3-propanesulphonic]acid), Tricine (N- tris[hydroxymethyl]methylglycine), Glycylglycine (diglycine), Bicine (N,N-Bis(2-

[0231] 29

[0232] SUBSTITUTE SHEET (RULE 26) hydroxyethyl)glycine), HEPBS (N-[2 -hydroxy ethyl]piperazine-N'-[4- butanesulphonic]acid), TAPS (N-tris[hydroxymethyl]methyl-3- aminopropanesulphonic]acid), AMPD (2-amino-2-methyl-l,3-propanediol), TABS (N- tris[hydroxymethyl]methyl-4-aminobutanesulphonic acid), AMPSO (3-[(l,l-dimethyl-2- hydroxyethyl)amino]-2-hydroxypropanesulphonic acid), CHES (2-(N- cyclohexylamino)ethanesulphonic acid), CAPSO (3-[cyclohexylamino]-2-hydroxy-l- propanesulphonic acid), AMP (2-amino-2-methyl-l -propanol), CAPS (3-cyclohexylamino- 1-propanesulphonic acid), or CABS (4-[cyclohexylamino]-l-butanesulphonic acid).

[0233] In some embodiments, the buffer comprises between about 0.01 wt. % and about 20 wt. % of the nanosheet or two-dimensional crystal, more preferably between about 0. 1 wt. % and about 15 wt. % of the nanosheet or two-dimensional crystal, most preferably between about 0.5 wt. % and about 10 wt. % of the nanosheet or two-dimensional crystal.

[0234] Crosslinkers

[0235] As discussed above, the nanosheets used in the compositions and methods described herein can employ a crosslinker. Example crosslinkers are capable of crosslinking an amine group to the carboxylic acid groups present on the membrane support and / or nanosheets. Example crosslinkers include, but are not limited to, 1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), glutaraldehyde, and N-Hydroxysuccinimide (NHS).

[0236] EXAMPLES

[0237] The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non- critical parameters which can be changed or modified to yield essentially the same results. Aspects of these methods (e.g., reaction times, pH values, types and volumes of 2D sheets used, support membrane used, etc.) can be varied from trial to trial. All steps were performed at room temperature.

[0238] Example 1: Single-Step Method for the Synthesis of Nanosheet Membranes Procedure for the Preparation of Nanosheets

[0239] Suitable 2D nanosheets can be prepared using the methods described in U.S. 8,378,003 to Manish et al., U.S. Patent Application Publication No. 2014 / 0051785 to

[0240] 30

[0241] SUBSTITUTE SHEET (RULE 26) Kumar et al., and U.S. 11,235,286 to Kumar et al., each of which are incorporated herein by reference in its entirety.

[0242] PEI / OmpF Deposition onto a PAN Membrane with EDC / NHS Crosslinking

[0243] Example membrane compositions were prepared from 2D nanosheets using the procedure described below. All steps were performed at room temperature.

[0244] Step 1. A poly(acrylonitrile) (PAN) ultrafiltration membrane was loaded into a dead-end filtration cell (membrane area 3.5 cm2). DI water was passed through the membrane to verify the water permeance.

[0245] Step 2. A polyethyleneimine (PEI) solution was prepared by dissolving 100 mg / L PEI in 20 mM HEPES buffer (pH 7.0). In these examples, the PEI was branched with a molecular weight (M.N.) of -60,000 Da.

[0246] Step 3. 120 pL of 2D nanosheet solution (e.g., outer membrane protein F (OmpF) nanosheet solution) was added to 9 mL of the PEI solution prepared in Step 2. The nanosheets were prepared using Outer Membrane Protein F (OmpF) and carboxyl- terminated poly(butadiene-b-ethylene oxide) (PB-PEO-COOH).

[0247] Step 4. DI water was removed from the dead-end cell and the solution prepared in Step 3 was added to the dead-end cell and the membrane was soaked in this solution (no applied pressure) for 20 minutes. The dead-end cell was then pressurized to ~20 psi until all of the solution had passed through the membrane (approximately 46 minutes).

[0248] Step 5. 12 mL of a crosslinking solution containing 4 mM 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10 mM N- hydroxysuccinimide (NHS) in 20 mM HEPES buffer (pH 7.2) was prepared.

[0249] Step 6. The solution from Step 5 was added to the dead-end cell and the membrane was soaked in this solution (no applied pressure) for 18 hours. The dead-end cell was then pressurized to -20 psi until all of the solution had passed through the membrane (approximately 64 minutes).

[0250] Step 7. The dead-end cell was then flushed with a volume of DI water before storage or testing.

[0251] Using the procedure above, a membrane composition was prepared by adding outer membrane protein F (OmpF) 2D nanosheets to a PAN support membrane.

[0252] Figure 2 A and Figure 5 are plots showing the water permeance of the membrane composition prepared above. Figure 3 A is a plot showing the percent of solute rejection for solutes of varying molecular weight from the membrane composition prepared above.

[0253] 31

[0254] SUBSTITUTE SHEET (RULE 26) For purposes of comparison, Figure 2B and Figure 3B show the water permeance (Figure 2B) and percent of solute rejection for solutes of varying molecular weight (Figure 3B) from membrane compositions prepared by depositing 1, 2, and 3 layers OmpF nanosheets on a membrane support using the layer-by-layer deposition methods described in U.S. Patent No. 2022 / 0080364.

[0255] Unexpectedly, membrane compositions prepared using the single-step methods described herein exhibited similar behavior to membrane compositions prepared using significantly more complex, layer-by-layer deposition processes.

[0256] Example 2. PEI / OmpF Deposition onto a Polyethersulfone (PES) Membrane with Glutaraldehyde Crosslinking

[0257] Example membrane compositions were prepared from 2D nanosheets using the procedure described below. All steps were performed at room temperature.

[0258] Step 1. A polyethersulfone (PES) ultrafiltration membrane was loaded into a deadend filtration cell (membrane area 3.5 cm2). DI water was passed through the membrane to verify the water permeance.

[0259] Step 2. A polyethyleneimine (PEI) solution was prepared by dissolving 200 mg / L PEI in water and adjusting the pH to 7.0. In these examples, the PEI was branched with a molecular weight (M.N.) of -60,000 Da.

[0260] Step 3. 120 pL of 2D nanosheet solution (e.g., outer membrane protein F (OmpF) nanosheet solution) was added to 9 mL of the PEI solution prepared in Step 2. The nanosheets were prepared using Outer Membrane Protein F (OmpF) and alcohol-terminated poly(butadiene-b-ethylene oxide) (PB-PEO-OH).

[0261] Step 4. DI water was removed from the dead-end cell and the solution prepared in Step 3 was added to the dead-end cell. The dead-end cell was then pressurized to -6-20 psi until all of the solution had passed through the membrane (approximately 44 minutes). 5 mL of deionized water was then added to the dead-end cell. The dead-end cell was then pressurized at -21 psi until all of the water had permeated through the membrane (approximately 22 minutes).

[0262] Step 5. A crosslinking solution containing 4.5% by volume glutaraldehyde in water was prepared.

[0263] Step 6. -10 mL of the solution from Step 5 was added to the dead-end cell and the membrane was soaked in this solution (no applied pressure) for -30 minutes. The dead-end cell was then pressurized to -20 psi until all of the solution had passed through the membrane (approximately 59 minutes). Deionized water was then added to the dead-end

[0264] 32

[0265] SUBSTITUTE SHEET (RULE 26) cell. The dead-end cell was then pressurized at ~20 psi until at least a few mL of the water had permeated through the membrane.

[0266] Step 7. The dead-end cell was then flushed with a volume of DI water before storage or testing.

[0267] Using the procedure above, a membrane composition was prepared by adding outer membrane protein F (OmpF) 2D nanosheets to a PES support membrane. Figure 6 is a plot showing the water permeance of the membrane composition prepared above.

[0268] Example 3. PEI / OmpF Deposition onto a Cellulose Membrane with Dopamine Crosslinking

[0269] Example membrane compositions were prepared from 2D nanosheets using the procedure described below. All steps were performed at room temperature.

[0270] Step 1. A cellulose ultrafiltration membrane was loaded into a dead-end filtration cell (membrane area 3.5 cm2). DI water was passed through the membrane to verify the water permeance.

[0271] Step 2. A dopamine solution was prepared by dissolving 1 g / L dopamine hydrochloride, 20 mg / L PEI, and 10 rnM tris(hydroxymethyl)aminomethane hydrochloride (Tris HC1) in water. The pH was adjusted to 8.5 and the solution was stirred for ~1 hour before application to the membrane surface.

[0272] Step 3. 120 pL of 2D nanosheet solution (e.g., outer membrane protein F (OmpF) nanosheet solution) was added to 9 mL of water. The nanosheets were prepared using Outer Membrane Protein F (OmpF) and amine-terminated poly(caprolactone-b-ethylene oxide) (PB-PEO-NH2).

[0273] Step 4. DI water was removed from the dead-end cell and ~9 mL of the solution prepared in Step 3 was added to the dead-end cell. The dead-end cell was then pressurized to ~3-4 psi until all of the solution had passed through the membrane (approximately 9 minutes).

[0274] Step 5. ~5 mL of the solution from Step 2 was added to the dead-end cell and the dead-end cell was then pressurized to ~3-4 psi until all of the solution had passed through the membrane (approximately 10 minutes).

[0275] Step 7. The dead-end cell was then flushed with a volume of DI water before storage or testing.

[0276] Using the procedure above, a membrane composition was prepared by adding outer membrane protein F (OmpF) 2D nanosheets to a cellulose support membrane. Figure 7 is a plot showing the water permeance of the membrane composition prepared above.

[0277] 33

[0278] SUBSTITUTE SHEET (RULE 26) Example 4: Synthesis of Nanosheet Membranes Using Molecular Crosslinkers — Adding OmpF 2D sheets and Aquaporin (RsAqpZ) 2D Sheets to a PAN Support Membrane.

[0279] General Procedure for the Preparation of Nanosheets

[0280] Suitable 2D nanosheets can be prepared using the methods described in U.S. 8,378,003 to Manish et al., U.S. Patent Application Publication No. 2014 / 0051785 to Kumar et al., and U.S. 11,235,286 to Kumar et al., each of which are incorporated herein by reference in its entirety.

[0281] General Procedure for the Preparation of Membrane Compositions

[0282] A general procedure for the preparation membrane compositions is described below. Aspects of these methods (e.g., reaction times, pH values, types and volumes of 2D sheets used, support membrane used, etc.) can be varied from trial to trial. All steps were performed at room temperature.

[0283] Step 1. A poly(acrylonitrile) (PAN) ultrafiltration membrane was loaded into a dead-end filtration cell (membrane area 3.5 cm2). DI water was passed through the membrane to verify the water permeance.

[0284] Step 2. DI water was removed from the dead-end cell and a solution containing 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) was added. 12 mL of solution comprising 4 mM EDC and 10 mM NHS in 2 mM MES buffer (pH 4.9) was used. The PAN membrane was soaked in this solution for -3 hours.

[0285] Step 3. The solution from step 2 was removed from the dead-end cell. A solution comprising m-phenylenediamine (MPD) was then added to the dead-end cell. 12 mL of solution comprising 10 mM MPD in 2 mM HEPES buffer (pH 7.2-7.4) was used. The PAN membrane was soaked in this solution for -20 hours.

[0286] Step 4. A solution containing EDC and NHS (4 mM EDC and 10 mM NHS in 2 mM HEPES buffer, pH 7.2) was prepared.

[0287] Step 5. 90 uL of 2D nanosheet solution was added to 9 mL of the solution prepared in step 4. The solution was agitated for -1-2.5 hours to disperse the nanosheets in the solution.

[0288] Step 6. Any remaining solution was removed from the dead-end cell. 3 mL of the solution prepared in step 5 was added to the dead-end cell and the membrane was soaked in this solution (no applied pressure) for -25 minutes. The dead-end cell was then pressurized to -20 psi until all of the solution had passed through the membrane. This process was then

[0289] 34

[0290] SUBSTITUTE SHEET (RULE 26) repeated two times without soaking (i.e., the solution from step 5 was added and the deadend cell was immediately pressurized).

[0291] Step 7. 12 mL of the MPD solution (10 mM MPD in 2 mM HEPES buffer, pH 7.2- 7.4) was then added to the filtration cell and the membrane was soaked in this solution (no applied pressure) for up to 16 hours. The dead-end cell was then pressurized to -20 psi until all of the solution had passed through the membrane.

[0292] Step 8. The dead-end cell was then flushed with a volume of DI water before storage or testing.

[0293] If desired, steps 4-7 can be repeated to add multiple types of 2D sheets to the same membrane.

[0294] Using the general procedure above, a membrane composition was prepared by adding outer membrane protein F (OmpF) 2D nanosheets and Aquaporin (RsAqpZ) 2D nanosheets to a PAN support membrane.

[0295] Figures 10A and 11 A are micrographs showing a PAN support membrane. Figures 10B, 11B, 12A-12B, and 13A-13B are micrographs showing PAN membranes after deposition of 2D OmpF sheets and 2D RsAqpZ sheets (PAN-OmpF-RsAqpZ). As shown in Figures 10B, 11B, 12A-12B, and 13A-13B, excellent surface coverage was observed using the general procedure outlined above.

[0296] Figure 9 is a plot showing the water permeance of a poly(acrylonitrile) (PAN) ultrafiltration membrane and a PAN membrane after deposition of 2D OmpF sheets and 2D RsAqpZ sheets (PAN-OmpF-RsAqpZ). As illustrated in Figure 9, following deposition of 2D OmpF sheets and 2D RsAqpZ sheets on the PAN membrane, the water permeance decreased by -30% (26.5%).

[0297] Example 5: Synthesis of Nanosheet Membranes Using Molecular Crosslinkers — Adding Aquaporin (RsAqpZ) 2D sheets to a PAN support membrane.

[0298] Using the general procedure described in Example 4, a membrane composition was prepared by adding Aquaporin (RsAqpZ) 2D nanosheets to a PAN support membrane.

[0299] Figure 14 is a plot showing the water permeance of a poly(acrylonitrile) (PAN) ultrafiltration membrane and a PAN membrane after deposition of 2D RsAqpZ sheets (PAN-RsAqpZ). As illustrated in Figure 14, following deposition of RsAqpZ sheets on the PAN membrane, the water permeance decreased by -90% (91.6%).

[0300] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations

[0301] 35

[0302] SUBSTITUTE SHEET (RULE 26) of a few aspects of the claims. Any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, components, compositions, and method steps disclosed herein are specifically described, other combinations of the compounds, components, compositions, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

[0303] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0304] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0305] SUBSTITUTE SHEET (RULE 26)

Claims

WHAT IS CLAIMED IS:

1. A method of forming a membrane composition, the method comprising contacting a membrane support with a solution comprising a matrix polymer and a plurality of nanosheets, thereby depositing the matrix polymer and the plurality of nanosheets on a surface of the membrane support; and crosslinking the matrix polymer and the plurality of nanosheets, wherein each of the nanosheets comprise one or more channels.

2. The method of claim 1, wherein contacting the membrane support with the solution comprising the matrix polymer and the plurality of nanosheets comprises positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing the solution comprising the matrix polymer and the plurality of nanosheets into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the matrix polymer and the plurality of nanosheets on the surface of the membrane support; and introducing a solution comprising one or more crosslinking reagents into the filtration cell under conditions effective to covalently crosslink the plurality of nanosheets to the matrix polymer.

3. The method of any of claims 1-2, wherein the method comprises a single contacting step.

4. The method of any of claims 1-3, wherein the matrix polymer and the nanosheets exhibit opposite charges.

5. The method of claim 4, wherein the matrix polymer comprises a positively charged polymer and the nanosheets are negatively charged.SUBSTITUTE SHEET (RULE 26)6. The method of claim 4, wherein the matrix polymer comprises a negatively charged polymer and the nanosheets are positively charged.

7. The method of any of claims 1-3, wherein the matrix polymer and the nanosheets are both uncharged.

8. The method of any of claims 1-3, wherein the matrix polymer comprises a negatively charged polymer or a positively charged polymer and the nanosheets are uncharged.

9. The method of any of claims 1-3, wherein the matrix polymer comprises an uncharged polymer and the nanosheets are positively charged or negatively charged.

10. The method of any of claims 1-9, wherein the membrane support exhibits a negatively charged surface.

11. The method of claim 10, wherein the negatively charged surface is applied by UV- ozone treatment.

12. The method of any of claims 1-11, wherein the membrane support comprises a porous membrane.

13. The method of any of claims 1-12, wherein the membrane support comprises a polymeric substrate or an inorganic substrate.

14. The method of any of claims 1-13, wherein the membrane support comprises an ultrafdtration membrane, a nanofiltration membrane, or a reverse osmosis membrane.

15. The method of any of claims 1-14, wherein the channels comprise a membrane protein, a carbon nanotube, or an artificial channel.SUBSTITUTE SHEET (RULE 26)16. The method of any of claims 1-15, wherein each of the plurality of nanosheets comprise a polymer, a lipid, or a combination thereof, and a membrane protein.

17. The method of claim 16, wherein the polymer comprises an amphiphilic block copolymer.

18. The method of claim 17, wherein said amphiphilic block copolymer comprises one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEO), or one or more hydrophobic blocks of polycaprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEO).

19. The method of any of claims 17-18, wherein the amphiphilic block copolymer comprises a negatively charged moiety, such as a carboxylate, a neutrally charged moiety, such as an alcohol, or a positively charged moiety, such as an amine.

20. The method of any of claims 15-19, wherein the membrane protein comprises a beta barrel membrane protein.

21. The method of any of claims 15-20, wherein the membrane protein comprises an alpha helical membrane protein.

22. The method of any of claims 15-21, wherein the membrane protein comprises a porin, a pore-forming toxin, or an aquaporin.

23. The method of any of claims 15-22, wherein the membrane protein comprises outer membrane protein F (OmpF), outer membrane protein X (OmpX), a-hemolysin toxin (aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, or aquaporin Z (AqpZ) such as RsAqpZ.SUBSTITUTE SHEET (RULE 26)24. The method of any of claims 16-23, wherein the polymer and membrane protein are present at mass ratio of from 1000:1 to 1 : 1000, such as from 100:1 to 1: 100 or from 10:1 to 1 :10.

25. The method of any of claims 1-24, wherein the matrix polymer comprises an amine group, an amide group, an imide group, an imine group, an azide group, a nitrate group, a nitrite group, a cyanate group, a nitrile group, or a combination thereof.

26. The method of any of claims 1-25, wherein the matrix polymer comprises a polyamine.

27. The method of any of claims 1-26, wherein the matrix polymer comprises polyethylenimine.

28. The method of any of claims 1-27, wherein the method further comprising crosslinking the matrix polymer, the plurality of nanosheets, or a combination thereof to the membrane support.

29. A membrane composition prepared by the method of any of claims 1-28.

30. A separation device comprising the membrane composition of claim 29.

31. A membrane composition comprising a membrane support; and a plurality of nanosheets covalently crosslinked to the membrane support via a molecular crosslinker, wherein each of the nanosheets comprise one or more channels.

32. The composition of claim 31, wherein the membrane support comprises a porous membrane.

33. The composition of any of claims 31-32, wherein the membrane support comprises a polymeric substrate or an inorganic substrate.40SUBSTITUTE SHEET (RULE 26)34. The composition of any of claims 31-33, wherein the membrane support comprises an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane.

35. The composition of any of claims 31-34, wherein the channels comprise a membrane protein, a carbon nanotube, or an artificial channel.

36. The composition of any of claims 31-35, wherein each of the plurality of nanosheets comprise a polymer, a lipid, or a combination thereof, and a membrane protein.

37. The composition of claim 36, wherein the polymer comprises a block copolymer.

38. The composition of claim 37, wherein said amphiphilic block copolymer comprises one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEO), or one or more hydrophobic blocks of polycaprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEO).

39. The composition of any of claims 35-38, wherein the membrane protein comprises a beta barrel membrane protein.

40. The composition of any of claims 35-38, wherein the membrane protein comprises an alpha helical membrane protein.

41. The composition of any of claims 35-40, wherein the membrane protein comprises a porin, a pore-forming toxin, or an aquaporin.

42. The composition of any of claims 35-41, wherein the membrane protein comprises outer membrane protein F (OmpF), outer membrane protein X (OmpX), a-hemolysin toxin (aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, or aquaporin Z (AqpZ) such as Rs AqpZ.SUBSTITUTE SHEET (RULE 26)43. The composition of any of claims 36-42, wherein the polymer and membrane protein are present at mass ratio of from from 1000: 1 to 1 : 1000, such as from 100: 1 to 1 : 100 or from 10: 1 to 1 : 10.

44. The composition of any of claims 31-43, wherein the molecular crosslinker comprises a small molecule crosslinker.

45. The composition of any of claim 44, wherein the small molecule crosslinker has a molecular weight of 1000 g / mol or less, such as a molecular weight of 800 g / mol or less, a molecular weight of 600 g / mol or less, or a molecular weight of 500 g / mol or less.

46. The composition of any of claims 31-45, wherein the plurality of nanosheets are also covalently crosslinked to one another via a molecular crosslinker.

47. The composition of any of claims 31-46, wherein the molecular crosslinker comprises a bifunctional crosslinker.

48. The composition of any of claims 31-47, wherein the molecular crosslinker comprises a diamine crosslinker, a diester crosslinker, a dithiol crosslinker, a diazide crosslinker, a dialkyne crosslinker, a divinyl crosslinker, a di(meth)acrylate crosslinker, a dialdehyde crosslinker, or a combination thereof.

49. A separation device comprising the membrane composition of any of claims 31-48.

50. A method of forming a membrane composition, the method comprising contacting a membrane support with a molecular crosslinker and a plurality of nanosheets under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support, wherein each of the nanosheets comprise one or more channels.SUBSTITUTE SHEET (RULE 26)51. The method of claim 50, wherein contacting a membrane support with a molecular crosslinker and a plurality of nanosheets comprises depositing the plurality of nanosheets on a surface of the membrane support; and contacting the surface with a solution comprising the molecular crosslinker under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support.

52. The method of any of claims 50-51, wherein contacting a membrane support with a molecular crosslinker and a plurality of nanosheets comprises positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing a solution comprising the plurality of nanosheets dissolved or dispersed in a solvent into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive the solvent across the membrane support, thereby depositing the plurality of nanosheets on the surface of the membrane support; and introducing a solution comprising the molecular crosslinker into the filtration cell so as to contact a surface of the membrane support under conditions effective to covalently crosslink the plurality of nanosheets to the membrane support.

53. The method of any of claims 50-52, wherein the plurality of nanosheets are also covalently crosslinked to one another via a molecular crosslinker.

54. The composition of any of claims 50-53, wherein the molecular crosslinker comprises a bifunctional crosslinker.

55. The composition of any of claims 50-54, wherein the molecular crosslinker comprises a diamine crosslinker, a diester crosslinker, a dithiol crosslinker, a diazide crosslinker, a dialkyne crosslinker, a divinyl crosslinker, a di(meth)acrylate crosslinker, a dialdehyde crosslinker, or a combination thereof.SUBSTITUTE SHEET (RULE 26)56. The composition of any of claims 50-55, further comprising activating the membrane support to introduce chemical moieties that can react with the molecular crosslinker to form covalent bonds.

57. A method of forming a membrane composition, the method comprising contacting a membrane support with a solution comprising a plurality of nanosheets, thereby depositing the plurality of nanosheets on a surface of the membrane support; and contacting a membrane support with a solution comprising a matrix polymer, thereby physically immobilizing the plurality of nanosheets on a surface of the membrane support within the matrix polymer; wherein each of the nanosheets comprise one or more channels.

58. The method of claim 57, wherein contacting the membrane support with the solution comprising the matrix polymer and the solution comprising plurality of nanosheets comprises positioning the membrane support within a filtration cell, such as a dead-end filtration cell or other filtration cell operating in a normal flow (dead-end) mode; introducing the solution comprising the plurality of nanosheets into the filtration cell so as to contact a surface of the membrane support; applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the plurality of nanosheets on the surface of the membrane support; introducing a solution comprising a matrix polymer into the filtration cell so as to contact a surface of the membrane support; and applying pressure to filtration cell to drive solvent across the membrane support, thereby depositing the matrix polymer on the surface of the membrane support so that the plurality of nanosheets are physically immobilized on the surface of the membrane support within the matrix polymer.

59. The method of any of claims 57-58, wherein the matrix polymer and the plurality of nanosheets are present in the same solution, and wherein the method comprises a single contacting step.SUBSTITUTE SHEET (RULE 26)60. The method of any of claims 57-59, wherein the matrix polymer and the nanosheets exhibit opposite charges.

61. The method of claim 60, wherein the matrix polymer comprises a positively charged polymer and the nanosheets are negatively charged.

62. The method of claim 60, wherein the matrix polymer comprises a negatively charged polymer and the nanosheets are positively charged.

63. The method of any of claims 57-59, wherein the matrix polymer and the nanosheets are both uncharged.

64. The method of any of claims 57-59, wherein the matrix polymer comprises a negatively charged polymer or a positively charged polymer and the nanosheets are uncharged.

65. The method of any of claims 57-59, wherein the matrix polymer comprises an uncharged polymer and the nanosheets are positively charged or negatively charged.

66. The method of any of claims 57-65, wherein the membrane support exhibits a negatively charged surface.

67. The method of claim 66, wherein the negatively charged surface is applied by UV- ozone treatment.

68. The method of any of claims 57-67, wherein the membrane support comprises a porous membrane.

69. The method of any of claims 57-68, wherein the membrane support comprises a polymeric substrate or an inorganic substrate.SUBSTITUTE SHEET (RULE 26)70. The method of any of claims 57-69, wherein the membrane support comprises an ultrafiltration membrane, a nanofiltration membrane, or a reverse osmosis membrane.

71. The method of any of claims 57-70, wherein the channels comprise a membrane protein, a carbon nanotube, or an artificial channel.

72. The method of any of claims 57-71, wherein each of the plurality of nanosheets comprise a polymer, a lipid, or a combination thereof, and a membrane protein.

73. The method of claim 72, wherein the polymer comprises an amphiphilic block copolymer.

74. The method of claim 73, wherein said amphiphilic block copolymer comprises one or more hydrophobic blocks of polybutadiene (PB) and one or more hydrophilic blocks of polyethylene oxide (PEO), or one or more hydrophobic blocks of polycaprolactone (PC) and one or more hydrophilic blocks of polyethylene oxide (PEO).

75. The method of any of claims 73-74, wherein the amphiphilic block copolymer comprises a negatively charged moiety, such as a carboxylate, a neutrally charged moiety, such as an alcohol, or a positively charged moiety, such as an amine.

76. The method of any of claims 71-75, wherein the membrane protein comprises a beta barrel membrane protein.

77. The method of any of claims 71-76, wherein the membrane protein comprises an alpha helical membrane protein.

78. The method of any of claims 71-77, wherein the membrane protein comprises a porin, a pore-forming toxin, or an aquaporin.SUBSTITUTE SHEET (RULE 26)79. The method of any of claims 71-78, wherein the membrane protein comprises outer membrane protein F (OmpF), outer membrane protein X (OmpX), a-hemolysin toxin (aHL), ferrichrome outer membrane transporter (FhuA), AquaporinO, or aquaporin Z (AqpZ) such as RsAqpZ.

80. The method of any of claims 72-79, wherein the polymer and membrane protein are present at mass ratio of from 1000:1 to 1 : 1000, such as from 100:1 to 1: 100 or from 10:1 to 1 :10.

81. The method of any of claims 57-80, wherein the matrix polymer comprises an amine group, an amide group, an imide group, an imine group, an azide group, a nitrate group, a nitrite group, a cyanate group, a nitrile group, or a combination thereof.

82. The method of any of claims 57-81, wherein the matrix polymer comprises a polyamine.

83. The method of any of claims 57-82, wherein the matrix polymer comprises polyethylenimine.

84. The method of any of claims 57-83, wherein the method further comprising crosslinking the matrix polymer, the plurality of nanosheets, or a combination thereof to the membrane support.

85. A membrane composition prepared by the method of any of claims 57-84.

86. A separation device comprising the membrane composition of claim 85.SUBSTITUTE SHEET (RULE 26)

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