Nanopore systems and methods for single-molecule polymer profiling

Abstract

The present invention relates to means and methods for analyzing target analytes using nanopore-based sensors, and more specifically to methods, nanopore systems, and devices for single-molecule profiling of polymers, e.g., polypeptides or polysaccharides. A method is provided for dissociating a non-nucleic acid polymer analyte through nanopores, wherein the nanopores are contained in a membrane separating the fluid chamber of the nanopore system into cis and trans sides, and comprising adding the analyte to the cis side and enabling the dissociation, wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) resulting from a net ionic current flow from cis to trans, preferably the cis-to-trans EOF resulting from a net ionic current flow from cis to trans exceeding a total ionic current flow of 0.2 or less than -0.2.

Description

[Background technology]

[0001] cross reference This application claims the benefits of European Application No. EP22204589.0, filed on 28 October 2022, which is incorporated herein by reference in its entirety.

[0002] Characterizing and identifying analytes is a crucial aspect of scientific research. These scientific studies can have a significant impact on both clinical and scientific endeavors. [Overview of the project]

[0003] In one embodiment, the Disclosure provides a nanopore system comprising a fluid chamber and a membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side and a trans side; and a non-nucleic acid polymer analyte comprising a linear length greater than the channel length of the nanopores; and a method comprising dislocating the non-nucleic acid polymer analyte from the cis side to the trans side of the fluid chamber, wherein the non-nucleic acid polymer analyte comprises elongated structures, the nanopore system having a cis-to-trans electroosmotic force arising from a net ionic current flow from cis to trans, and the cis-to-trans electroosmotic force dislocates the non-nucleic acid polymer analyte through the nanopores against an electrophoretic force acting in the opposite direction to the cis-to-trans electroosmotic force.

[0004] In some embodiments, the electroosmotic force is at least 10% greater than the electrophoretic force. In some embodiments, the electroosmotic force is at least 50% greater than the electrophoretic force. In some embodiments, the electroosmotic force is at least 100% greater than the electrophoretic force. In some embodiments, the cis side of the fluid chamber contains a first solution, and the transform side of the fluid chamber contains a second solution. In some embodiments, the first solution contains a solute at a first concentration, and the second solution contains a solute at a second concentration. In some embodiments, the solute contains ions or osmolite. In some embodiments, the difference between the first concentration and the second concentration of the solute is configured to generate an electroosmotic force from cis to transform in the presence of an applied potential.

[0005] In some embodiments, the non-nucleoside polymer analyte is an unmodified (label-free) non-nucleoside polymer analyte. In some embodiments, the analyte is an unmodified analyte. In some cases, the unmodified analyte may be the wild-type form of the analyte. In some cases, the unmodified analyte may not contain any additional molecules coupled to the unmodified analyte. In some embodiments, the analyte is a label-free or tag-free analyte. In some cases, a label-free analyte may include an analyte that is not coupled to any peptide label, any protein label, any nucleic acid label, any sugar label, any lipid label, or any combination thereof. In some cases, a tag-free analyte may include an analyte that is not coupled to any peptide tag, any protein tag, any nucleic acid tag, any sugar tag, any tag label, or any combination thereof. In some embodiments, the ends of the non-nucleoside polymer analyte lack a three-dimensional structure. In some embodiments, at least a portion of the non-nucleoside polymer analyte is denatured. In some embodiments, the linear length of the non-nucleic acid polymer analyte is greater than the channel length of the nanopores traversing the membrane when the non-nucleic acid polymer analyte is elongated. In some embodiments, the non-nucleic acid polymer analyte contains at least about 25 repeating units. In some embodiments, the non-nucleic acid polymer analyte contains peptide units, sugar units, water-soluble plastic monomers, or any combination thereof. In some embodiments, the non-nucleic acid polymer analyte contains polypeptides, polysaccharides, or water-soluble plastics.

[0006] In some embodiments, the non-nucleic acid polymer analyte comprises a polypeptide of at least 30 peptide units. In some embodiments, the at least 30 peptide units include positively or negatively charged peptide units. In some embodiments, the polypeptide is in a denatured state. In some embodiments, the polypeptide is provided in a folded state.

[0007] In some embodiments, the method further includes measuring a signal generated by dislocating a non-nucleic acid polymer analyte through nanopores.

[0008] In some embodiments, the measurement involves measuring a signal about the state of (a) open channels in the nanopores, (b) capture of non-nucleoside polymer analytes by the nanopores, or (c) passage of non-nucleoside polymer analytes through the nanopores. In some embodiments, the measurement involves detecting the difference between states (a), (b), and (c). In some embodiments, the signal includes an ionic current, a change in ionic current, or a derivative thereof.

[0009] In some embodiments, the linear length of the non-nucleic acid polymer analyte is at least 1 kDa. In some embodiments, the linear length of the non-nucleic acid polymer analyte is up to 4,000 kDa. In some embodiments, the linear length of the non-nucleic acid polymer analyte is at least twice as long as the channel length of the nanopore. In some embodiments, the linear length of the non-nucleic acid polymer analyte is up to twice as long as the channel length of the nanopore. In some embodiments, the linear length of the non-nucleic acid polymer analyte is at least 3 nanometers.

[0010] In some embodiments, the electroosmotic force from cis to transform includes a net ionic current flow from cis to transform. In some embodiments, the electroosmotic force from cis to transform is regulated by pH, type of salt, salt concentration, osmotic pressure across the system membrane, modification of nanopores, or any combination thereof. In some embodiments, the electroosmotic force from cis to transform is regulated by modification of the charge of the nanopores. In some embodiments, the electroosmotic force from cis to transform is regulated by an asymmetric salt distribution between the cis side and the transform side of the membrane. In some embodiments, the nanopores have an ion selectivity P(+) / P(-) greater than 2.0. In some embodiments, the nanopores have an ion selectivity P(+) / P(-) less than 0.50.

[0011] In some embodiments, the nanopore system further includes a pair of electrodes. In some embodiments, the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the net electroosmotic current relative to the applied voltage is greater than about 0.10 pA / mV. In some embodiments, the nanopores include internal pore constriction of about 0.5 nanometers to about 2 nanometers (nm).

[0012] In some embodiments, the nanopores include alpha-helical oligomeric pore structures. In some embodiments, the nanopores include beta-barrel oligomeric pore structures. In some embodiments, the nanopores include recombinant nanopores. In some embodiments, the nanopores include erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, their modified variants, or their ion-selective variants.

[0013] In some embodiments, the nanopores include biological nanopores. In some embodiments, the biological nanopores are modified to restrict the passage of one or more ions through the channels of the nanopores. In some embodiments, the biological nanopores restrict the passage of one or more ions through the channels of the nanopores by modifying the charge of the channels of the nanopores. In some embodiments, the net charge of the channels is negative. In some embodiments, the net charge of the channels is positive.

[0014] In some embodiments, the nanopores include mutant CytK nanopores. In some embodiments, the mutant CytK nanopores include one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions include K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof. In some embodiments, one or more amino acid substitutions include K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof. In some embodiments, the mutant CytK nanopores include (a) K128D and K155D, (b) K128D, K155D, and T116D, (c) T147D or S151D, (d) K128D, K155D, and S120D, (e) Q122D, T147D, or S155D, (f) K128D, K155D, Q145D, and S151D, and (g) one of the amino acid substitution combinations of those combinations. In some embodiments, the mutant CytK nanopores include one or more of the following amino acid substitution combinations: (a) S120D, G122D, or K155D; (b) S120D in combination with K128F / K128D; (c) Q122D or S151D; (d) K128D or K128F; (e) S120D, K115D, and Q122D; (f) K128F, S120D, and G122D; (g) K128F, S120D, G122D, and K155D; and combinations thereof.

[0015] In another embodiment, the present disclosure provides a system comprising: a fluid chamber; a membrane comprising nanopores, the membrane being configured to separate the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, the first and second solutions being configured to displace a non-nucleic acid polymer analyte across the nanopores using electroosmotic flow, the non-nucleic acid polymer analyte comprising elongated structures, the non-nucleic acid polymer analyte comprising a linear length greater than the channel length of the nanopores; and a pair of electrodes comprising a first electrode and a second electrode, the first electrode being positioned on the cis side of the fluid chamber and the second electrode being positioned on the trans side of the fluid chamber, the pair of electrodes being configured to generate electrophoretic force acting in the opposite direction to the electroosmotic flow.

[0016] In another embodiment, the Disclosure provides a system comprising: a fluid chamber; a membrane comprising nanopores, the membrane being configured to separate the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, the first and second solutions being configured to displace a non-nucleoside polymer analyte using electroosmotic flow; a pair of electrodes comprising a first electrode and a second electrode; and a controller operably coupled to the fluid chamber, the nanopores, and the pair of electrodes, wherein the controller uses the pair of electrodes to generate electrophoretic force acting in the opposite direction to the electroosmotic flow displacing the non-nucleoside polymer analyte through the nanopores; detecting one or more signals related to at least one feature of the non-nucleoside polymer analyte during or after the displacement of the non-nucleoside polymer analyte through the nanopores, wherein the non-nucleoside polymer analyte comprises a linear length greater than the channel length of the nanopores.

[0017] In some embodiments, the controller uses the pair of electrodes to detect one or more signals associated with at least one feature of the non-nucleic acid polymer analyte. In some embodiments, the electroosmotic flow is greater than the electrophoretic force.

[0018] In some embodiments, the electroosmotic flow is at least 10% greater than the electrophoretic force. In some embodiments, the electroosmotic flow is at least 50% greater than the electrophoretic force. In some embodiments, the electroosmotic flow is at least 100% greater than the electrophoretic force. In some embodiments, the first solution contains a solute at a first concentration, and the second solution contains a solute at a second concentration.

[0019] In some embodiments, the solute comprises ions or osmolite. In some embodiments, the difference between a first concentration and a second concentration of the solute is configured to generate an electroosmotic flow in the presence of an applied potential.

[0020] In some embodiments, the electroosmotic flow includes a net ionic current flow from cis to transform. In some embodiments, the electroosmotic flow is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the system, modification of nanopores, or any combination thereof. In some embodiments, the electroosmotic flow is regulated by modification of the charge of the nanopores. In some embodiments, the electroosmotic flow is regulated by an asymmetric salt distribution between the cis side and the transform side of the membrane. In some embodiments, the nanopores have an ion selectivity P(+) / P(-) greater than 2.0. In some embodiments, the nanopores have an ion selectivity P(+) / P(-) less than 0.50.

[0021] In some embodiments, a pair of electrodes may be configured to provide an applied voltage. The applied voltage may be across the membrane. The applied voltage may result in electrophoretic force. In some embodiments, a pair of electrodes may be configured to provide electrophoretic force across the membrane. A pair of electrodes may be configured to measure a signal.

[0022] In some embodiments, the nanopores include alpha-helical oligomeric pore structures. In some embodiments, the nanopores include beta-barrel oligomeric pore structures. In some embodiments, the nanopores include recombinant nanopores. In some embodiments, the nanopores include erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, their modified variants, or their ion-selective variants.

[0023] In some embodiments, the nanopores include biological nanopores. In some embodiments, the biological nanopores are modified to restrict the passage of one or more ions through the channels of the nanopores. In some embodiments, the biological nanopores restrict the passage of one or more ions through the channels of the nanopores by modifying the charge of the channels of the nanopores. In some embodiments, the net charge of the channels is negative. In some embodiments, the net charge of the channels is positive.

[0024] In some embodiments, the nanopores include mutant CytK nanopores. In some embodiments, the mutant CytK nanopores include one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions include K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof. In some embodiments, one or more amino acid substitutions include K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof. In some embodiments, the mutant CytK nanopores include (a) K128D and K155D, (b) K128D, K155D, and T116D, (c) T147D or S151D, (d) K128D, K155D, and S120D, (e) Q122D, T147D, or S155D, (f) K128D, K155D, Q145D, and S151D, and (g) one of the amino acid substitution combinations of those combinations. In some embodiments, the mutant CytK nanopores include one or more of the following amino acid substitution combinations: (a) S120D, G122D, or K155D; (b) S120D in combination with K128F / K128D; (c) Q122D or S151D; (d) K128D or K128F; (e) S120D, K115D, and Q122D; (f) K128F, S120D, and G122D; (g) K128F, S120D, G122D, and K155D; and combinations thereof.

[0025] In some embodiments, the non-nucleoside polymer analyte is an unmodified (label-free) non-nucleoside polymer analyte. In some embodiments, the ends of the non-nucleoside polymer analyte lack a three-dimensional structure. In some embodiments, at least a portion of the non-nucleoside polymer analyte is modified. In some embodiments, the linear length of the non-nucleoside polymer analyte is greater than the channel length of the nanopores traversing the membrane, if the non-nucleoside polymer analyte is elongated. In some embodiments, the non-nucleoside polymer analyte contains at least about 25 repeating units.

[0026] In some embodiments, the non-nucleic acid polymer analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof. In some embodiments, the non-nucleic acid polymer analyte comprises polypeptides, polysaccharides, or water-soluble plastics. In some embodiments, the non-nucleic acid polymer analyte comprises a polypeptide of at least 30 peptide units. In some embodiments, at least 30 peptide units include positively or negatively charged peptide units. In some embodiments, the polypeptide is in a denatured state. In some embodiments, the polypeptide is provided in a folded state. In some embodiments, the linear length of the non-nucleic acid polymer analyte is at least 1 kDa. In some embodiments, the linear length of the non-nucleic acid polymer analyte is up to 4,000 kDa. In some embodiments, the linear length of the non-nucleic acid polymer analyte is at least twice as large as the channel length of the nanopore. In some embodiments, the linear length of the non-nucleic acid polymer analyte is up to twice as large as the channel length of the nanopore.

[0027] In another aspect, the disclosure provides a device including an array of systems that include any of the systems disclosed herein.

[0028] In another aspect, the disclosure provides the use of any of the methods, kits, or devices disclosed herein for characterizing at least one feature of a non-nucleic acid polymer analyte.

[0029] In another aspect, the disclosure provides the use of any of the systems disclosed herein for characterizing at least one feature of a non-nucleic acid polymer analyte.

[0030] In another embodiment, the Disclosure provides the use of any of the methods, kits, or devices disclosed herein for the detection and analysis of one or more non-nucleic acid polymer analytes at the single-molecule level.

[0031] In another aspect, the present disclosure provides the use of any of the systems disclosed herein for the detection and analysis of one or more non-nucleic acid polymer analytes at the single-molecule level.

[0032] In another aspect, the Disclosure provides the use of any of the methods, kits, or devices disclosed herein for the detection and analysis of one or more polypeptides.

[0033] In another aspect, the Disclosure provides the use of any of the systems disclosed herein for the detection and analysis of one or more polypeptides.

[0034] Another aspect of this disclosure provides a non-temporary computer-readable medium containing machine-executable code that, when executed by one or more computer processors, implements any of the methods described herein or elsewhere.

[0035] Another aspect of this disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory contains machine-executable code that, when executed by the one or more computer processors, implements any of the methods described herein or elsewhere.

[0036] Another aspect of the present disclosure is a method for dislocating a non-nucleic acid polymer analyte through nanopores, wherein the nanopores are contained within a membrane separating a fluid chamber of the nanopore system into cis and trans sides, the polymer analyte is added to the cis side, and the dislocation of the polymer analyte is enabled, wherein the length of the elongated polymer analyte is greater than the longitudinal axis of the central channel of the nanopore perpendicular to the membrane. The nanopore system provides a method in which a cis-to-trans electroosmotic force (EOF) arises from a net ionic current flow from cis to trans, and this cis-to-trans EOF overcomes the trans-to-cis electrophoretic force (EPF) acting on the polymer analyte.

[0037] In some embodiments, the polymer analyte is an unmodified (label-free) analyte.

[0038] In some embodiments of any one of the prior embodiments, the polymer ends are not structured, and preferably the polymer is modified or partially modified.

[0039] In some embodiments of any one of the prior embodiments, the polymer analyte includes at least 25 repeating units, preferably at least 35 repeating units, and more preferably at least 45 repeating units.

[0040] In some embodiments of any one of the prior embodiments, the polymer analyte is synthetic, semi-synthetic, or of biological origin, e.g., a biopolymer, and preferably comprises or consists of peptide units, sugar units, and water-soluble plastic monomers, and any combination thereof. In some embodiments, the polymer analyte is a polypeptide, polysaccharide, or water-soluble plastic such as PEG, or a PEGylated polypeptide. In some embodiments, the polymer analyte is a polypeptide of at least 30 peptide units, and contains positively and negatively charged residues. In some embodiments, the polypeptide is in a denatured / expanded state, and preferably, the polypeptide is added in a pre-denatured state.

[0041] In some embodiments of any one of the prior embodiments, (c) further comprises measuring a change in ionic current caused by dislocation of a target polymer through nanopores, preferably, operation (c) comprises measuring a change in current for states of (i) open channel, (ii) polymer capture by nanopores, and (iii) polymer passage from (ii) through nanopores, more preferably, measuring comprises detecting the difference between states (i), (ii), and (iii).

[0042] Another aspect of the present disclosure provides a nanopore system for dislocating polymer analytes through nanopores, wherein the system comprises nanopores contained in a membrane separating the fluid chamber of the nanopore system into a cis side and a trans side, the analyte being added to the cis side, and the nanopore system having a cis-to-trans electroosmotic force (EOF) resulting from a net ionic current flow from cis to trans, the cis-to-trans EOF overcoming the trans-to-cis electrophoretic force (EPF) acting on the polymer analyte.

[0043] In some embodiments of any one of the prior embodiments, the nanopore system has a cis-to-transformer EOF resulting from a net cis-to-transformer ion current flow exceeding a total ion current flow of greater than 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, more preferably greater than 0.35 or less than -0.35.

[0044] In some embodiments of any one of the prior embodiments, the cis-to-trans EOF is controlled by modifying the charge of the nanopores (e.g., genetically engineered) or any combination thereof, by adjusting the pH, type and / or concentration of salts and / or osmotic pressure across the membrane of the nanopore system.

[0045] In some embodiments of any one of the prior embodiments, the cis-to-trans EOF is regulated by modification of the nanopores and / or by an asymmetric salt distribution between the cis and trans sides of the chamber.

[0046] In some embodiments of any one of the prior embodiments, the nanopore system has an ion selectivity P greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, most preferably greater than 3.0 or less than 0.33. (+) / P (-) It has.

[0047] In some embodiments of any one of the prior embodiments, the system has an ion selectivity P(+) / P(-) greater than 2.0, preferably greater than 2.5, and more preferably greater than 3.0, and a negative voltage is applied to the transformer side, and preferably the system includes cation-selective (mutant) nanopores.

[0048] In some embodiments of any one of the prior embodiments, the nanopores are biological nanopores, preferably biological nanopores having internal pore constriction in the range of 0.5 to 2 nm.

[0049] In some embodiments of any one of the prior embodiments, the nanopores are alpha-helical or beta-barrel oligomeric pore-forming toxins or porins, and preferably the nanopores are selected from the group consisting of erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, phage-derived portal proteins, and modified variants thereof, or ion-selective variants thereof.

[0050] In some embodiments of any one of the prior embodiments, the nanopores include biological nanopores that have been modified, for example by genetic engineering, to provide desired ion selectivity, and preferably the ion-selective nanopores are modified to have a net charge in the region facing the lumen greater than 21, preferably greater than 28, more preferably greater than 35, and most preferably, the net charge is negative.

[0051] In some embodiments of any one of the prior embodiments, the nanopores are mutant CytK nanopores containing one or more amino acid substitutions selected from the group consisting of K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, and S151D, and the numbering corresponds to CytK amino acids available in UniProt with accession number A0A2S1A9G3_9BACI. Preferably, the CytK mutant nanopores include one of the following combinations of amino acid substitutions: K128D and K155D; K128D, K155D, and T116D (optionally further comprising T147D and / or S151D); K128D, K155D, and S120D (optionally further comprising Q122D, T147D, and / or S155D); and K128D, K155D, Q145D, and S151D.

[0052] Another aspect of this disclosure provides an analytical device including an array of nanopore systems according to any one of the prior embodiments. Another aspect of the present disclosure provides the use of a method, nanopore system, or device according to any one of the prior embodiments for characterizing at least one feature of a target polymer, preferably at the single-molecule level, for the detection and analysis of one or more target polymers, and more preferably for the detection and analysis of one or more target polypeptides.

[0053] Additional aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As realized, other different embodiments of the present disclosure are possible, and some of its details are modifiable in various obvious ways without departing from the present disclosure. Accordingly, the drawings and description should be considered illustrative and not limiting in nature.

[0054] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. To the extent that any publications and patents or patent applications incorporated by reference conflict with any disclosures contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material. [Modes for carrying out the invention]

[0055] While various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous modifications, alterations, and substitutions are conceivable without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0056] If a value is stated as a range, it will be understood that such disclosure includes the disclosure of all possible subranges within that range, as well as the disclosure of any specific numerical value that falls within that range, regardless of whether a particular numerical value or subrange is explicitly stated.

[0057] The terms "a," "an," and "the," as used herein, generally refer to singular and plural references, respectively, unless the context explicitly indicates otherwise. Any reference to "or" herein is intended to encompass "and / or" unless otherwise stated.

[0058] Whenever the terms “at least,” “greater than,” or “greater than or equal to” precede the first number in a sequence of two or more numbers, the terms “at least,” “greater than,” or “greater than or equal to” apply to each of the numbers in that sequence. For example, 1, 2, or 3 or more is equal to 1 or more, 2 or more, or 3 or more.

[0059] Whenever the terms “no more than,” “less than,” or “less than or equal to” precede the first of two or more consecutive numbers, the terms “no more than,” “less than,” or “less than or equal to” apply to each of those consecutive numbers. For example, 3, 2, or 1 or less is equal to 3 or less, 2 or less, or 1 or less.

[0060] In some embodiments, methods for analyzing analytes are provided.

[0061] One aspect of this disclosure provides a method comprising providing a nanopore system. The nanopore system may include a fluid chamber. The fluid chamber can be separated into a cis side and a trans side. The method may further include providing a non-nucleoside polymer analyte. In some cases, the non-nucleoside polymer analyte may include a linear length greater than the channel length of the nanopores. The method may further include dissociating the non-nucleoside polymer analyte. In some cases, the non-nucleoside polymer analyte can be dissociated from the cis side to the trans side of the fluid chamber. The non-nucleoside polymer analyte may include elongated structures. In some cases, the nanopore system may have a cis-to-trans electroosmotic force. The electroosmotic force may include a net ionic current flow from cis to trans. In some cases, the cis-to-trans electroosmotic force can dissociate the non-nucleoside polymer analyte through the nanopores against the electrophoretic force. In some cases, the electrophoretic case can act in the opposite direction to the cis-to-trans electroosmotic force.

[0062] One aspect of the present disclosure provides a system comprising a nanopore system. The nanopore system may include a fluid chamber. The system may also include a membrane. In some cases, the membrane may include nanopores. In some cases, the membrane can separate the fluid chamber into a cis side and a trans side. In some cases, the cis side may contain a first solution. In some cases, the trans side may contain a second solution. In some cases, the cis side may contain a first solution and the trans side may contain a second solution. The system may further include a non-nucleoside polymer analyte. The first and second solutions may be configured to displace the non-nucleoside polymer analyte across the nanopores using electroosmotic flow. In some cases, the non-nucleoside polymer analyte may have an elongated structure. In some cases, the non-nucleoside polymer analyte may have a linear length greater than the channel length of the nanopores. The system may further include a pair of electrodes. In some cases, the pair of electrodes may include a first electrode and a second electrode. The first electrode may be located on the cis side of the fluid chamber. The second electrode may be located on the transformer side of the fluid chamber. The first electrode may be located on the cis side of the fluid chamber, and the second electrode may be located on the transformer side of the fluid chamber. In some cases, the pair of electrodes may be configured to generate electrophoretic force. In some cases, the electrophoretic force may act in the opposite direction to the electroosmotic flow.

[0063] In some embodiments, changes in ion current can be measured while the analyte displaces through the nanopores. In some cases, changes in ion current can be measured by a voltage-based chip. In some cases, the voltage-based chip can measure changes in voltage and / or current across the nanopores. In some cases, the voltage-based chip may be a transformer electrode.

[0064] The characterization method may involve measuring the flow of ionic current through the pores, typically by measuring the current. Alternatively, the flow of ions through the pores may be measured optically, as disclosed by Heron et al: J.Am.Chem.Soc.9 Vol.131, No.5, 2009. Therefore, the apparatus may also include an electrical circuit capable of applying a potential and measuring the electrical signal across the membrane and pores. The characterization method may be performed using a patch clamp or a voltage clamp. The characterization method may involve the use of a voltage clamp. In some embodiments, a spacer may be attached to the analyte.

[0065] In some embodiments, the analytes include polymer analytes. The analytes may include nucleic acid polymer analytes or non-nucleic acid polymer analytes. The analytes may be synthetic, semi-synthetic, or of biological origin. For example, synthetic analytes may include analytes constructed by non-biological chemical processes, such as polyethylene glycol (PEG), synthetically constructed peptides of proteins, or synthetically constructed DNA molecules. Biological analytes may include analytes produced by biological processes, such as proteins produced by cells or by systems using cellular (or cell-derived) components (e.g., enzyme in vitro translation systems). Semi-synthetic analytes may include those of biological and non-biological origin, such as moieties made from biologically produced proteins conjugated to PEG molecules. Possible electrical measurements include current measurement, impedance measurement, tunneling, electron tunneling measurement (Ivanov AP et al., Nano Lett. 201 1 Jan 12; 1 l(l): 279-85), FET measurement (International Application WO2005 / 124888), voltage FET measurement, or any combination thereof. In some embodiments, the signal may be an electron tunnel across a solid-state nanopore or a voltage FET measurement across a solid-state nanopore.

[0066] The characterization method may involve measuring the flow of ionic current through the pores by measuring the current. Alternatively, the flow of ions through the pores may be measured optically, as disclosed by Heron et al: J.Am.Chem.Soc.9 Vol.131, No.5, 2009. Therefore, the apparatus may also include an electrical circuit capable of applying a potential and measuring the electrical signal across the membrane and pores. The characterization method may be performed using patch clamps or voltage clamps. The characterization method preferably involves the use of voltage clamps.

[0067] The characterization method may be performed on an array of wells or nanopore channels, each array containing 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells or nanopore channels.

[0068] This characterization method may involve measuring the current flowing through the pores. The method is typically performed using a voltage applied across the membrane and pores. The voltage used is typically +2V to -2V, and typically -400mV to +400mV. The voltage used is preferably within a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, 20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. More preferably, the voltage used is in the range of mV to 240mV, and most preferably in the range of 120mV to 220mV. By using an increased applied potential, it is possible to increase the discrimination between different nucleotides in each pore.

[0069] In some embodiments, the analyte includes a protein or peptide. The protein or peptide may be in a folded state, an unfolded state, or an intermediate state between these. The folded state includes a state in which the polymer is in a low-energy state so that the protein or peptide maintains a two-dimensional or three-dimensional structure. This low-energy state may be based on the interaction of the amino acids of the peptide or protein with each other. The unfolded state may include a state in which the polymer is in a high-energy state so that the protein or peptide does not maintain a two-dimensional or three-dimensional structure. An intermediate state between the folded and unfolded states may be an energy state in which one or more parts of the peptide or protein can maintain a two-dimensional or three-dimensional structure, while other parts of the peptide or protein cannot.

[0070] In some embodiments, the analyte may include a non-nucleic acid polymer analyte. In some embodiments, a portion of the non-nucleic acid polymer analyte may include nucleic acid molecules. In some cases, a portion of the non-nucleic acid polymer analyte may be 0% to about 100% of the non-nucleic acid polymer analyte. In some cases, a portion of the non-nucleic acid polymer analyte may be at least about 0%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, or up to approximately 0% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be approximately 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% of the non-nucleic acid polymer analyte.

[0071] In some embodiments, a portion of the non-nucleic acid polymer analyte may contain oligosaccharide molecules. In some cases, a portion of the non-nucleic acid polymer analyte may be 0% to about 100% of the non-nucleic acid polymer analyte. In some cases, a portion of the non-nucleic acid polymer analyte may be at least about 0%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, or up to approximately 0% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be approximately 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% of the non-nucleic acid polymer analyte.

[0072] The analyte may include the contour length. In some embodiments, the contour length may include the length of the analyte when it is not fully unfolded. In some cases, the analyte may be 1% to about 100% unfolded. In some cases, the analyte may be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% unfolded. In some cases, the analytes may be spread to a maximum of approximately 100%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than 1%. In some cases, the analyte may be unfolded to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some cases, the linear length of the analyte may be that of the analyte when 100% unfolded. The analyte may include the linear length. In some embodiments, the contour length of the analyte may be the linear length of the analyte. The linear length may be the length of the analyte in its unfolded state. In some embodiments, the linear length of the analyte may be about 3 nanometers (nm) to about 5It may be 000nm. In some embodiments, the linear length of the analyte is approximately 3-5nm, approximately 5nm-10nm, approximately 10nm-15nm, approximately 15nm-20nm, approximately 20nm-25nm, approximately 25nm-30nm, approximately 30nm-35nm, approximately 35nm-40nm, approximately 40nm-45nm, approximately 45nm-50nm, approximately 50nm-55nm, approximately 55nm-60nm, approximately 60nm-65nm, approximately 65nm-70nm, approximately 70nm-75nm, approximately 75nm-80nm, approximately 80nm-85nm, approximately 85nm-90nm, approximately 90nm-95nm, and approximately 9 5nm~about 100nm, about 100nm~about 150nm, about 150nm~about 200nm, about 200nm~about 250nm, about 250nm~about 300nm, about 300nm to about 350nm, about 350nm to about 400nm, about 400nm to about 450nm, about 450nm to about 500nm, Approximately 500nm to approximately 550nm, approximately 550nm to approximately 600nm, approximately 600nm to approximately 650nm, approximately 650nm to approximately 700nm, approximately 700nm ~750nm, approximately 750nm ~ approximately 800nm, approximately 800nm ​​~ approximately 850nm, approximately 850nm ~ approximately 900nm, approximately 900nm ~ approximately 950n m, about 950nm to about 1,000nm, about 1,000nm to about 1,100nm, about 1,100nm to about 1,200nm, about 1,200n m ~ approx. 1,300nm, approx. 1,300nm ~ approx. 1,400nm, approx. 1,400nm ~ approx. 1,500nm, approx. 1,500nm ~ approx. 1,600 nm, about 1,600nm to about 1,700nm, about 1,700nm to about 1,800nm, about 1,800nm ​​to about 1,900nm, about 1,90 0nm ~ approx. 2,000nm, approx. 2,000nm ~ approx. 2,100nm, approx. 2,100nm ~ approx. 2,200nm, approx. 2,200nm ~ approx. 2,3 00nm, about 2,300nm to about 2,400nm, about 2,400 to about 2,500nm, about 2,500nm to about 2,600nm, about 2,6 00nm ~ approx. 2,700nm, approx. 2,700nm ~ approx. 2,800nm, approx. 2,800nm ​​~ approx. 2,900nm, approx. 2,900nm ~ approx. 3, 000nm, about 3,000nm to about 3,100nm, about 3,200nm to about 3,300nm, about 3,300nm to about 3,400nm, about 3 , 400nm ~ approx. 3,500nm, approx. 3,500 ~ approx. 3,600nm, approx. 3,600nm ~ approx. 3,700nm, approx. 3,700nm ~ approx. 3,It could be 800nm, approximately 3,800nm ​​to 3,900nm, approximately 3,900nm to 4,000nm, approximately 4,000nm to 4,100nm, approximately 4,100nm to 4,200nm, approximately 4,200nm to 4,300nm, approximately 4,300nm to 4,400nm, approximately 4,400nm to 4,500nm, approximately 4,500nm to 4,600nm, approximately 4,600nm to 4,700nm, approximately 4,700nm to 4,800nm, approximately 4,800nm ​​to 4,900nm, or approximately 4,900nm to 5,000nm.

[0073] By some means, the linear length of the analyte is at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 10 nm, at least about 15 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, and less All are approximately 100nm, at least approximately 110nm, at least approximately 120nm, at least approximately 130nm, at least approximately 140nm, at least approximately 150nm, at least approximately 160nm, at least approximately 170nm, at least approximately 180nm, at least approximately 190nm, at least approximately 200nm, at least approximately 210nm, at least approximately 220nm, at least approximately 230nm, at least approximately 240nm, at least approximately 250nm, at least approximately 260nm, at least approximately 270nm, at least approximately 280nm, at least approximately 290nm, at least approximately 300nm, and less All are approximately 310nm, at least approximately 320nm, at least approximately 330nm, at least approximately 340nm, at least approximately 350nm, at least approximately 360nm, at least approximately 370nm, at least approximately 380nm, at least approximately 390nm, at least approximately 400nm, at least approximately 410nm, at least approximately 420nm, at least approximately 430nm, at least approximately 440nm, at least approximately 450nm, at least approximately 460nm, at least approximately 470nm, at least approximately 480nm, at least approximately 490nm, at least approximately 500nm, at least approximately 510nm, and less At least approximately 520nm, at least approximately 530nm, at least approximately 540nm, at least approximately 550nm, at least approximately 560nm, at least approximately 570nm, at least approximately 580nm, at least approximately 590nm, at least approximately 600nm, at least approximately 610nm, at least approximately 620nm, at least approximately 630nm, at least approximately 640nm, at least approximately 650nm, at least approximately 660nm, at least approximately 670nm, at least approximately 680nm, at least approximately 690nm, at least approximately 700nm, at least approximately 710nm, at least approximately 720nm,At least approximately 730nm, at least approximately 740nm, at least approximately 750nm, at least approximately 760nm, at least approximately 770nm, at least approximately 780nm, at least approximately 790nm, at least approximately 800nm, at least approximately 810nm, at least approximately 820nm, at least approximately 830nm, at least approximately 840nm, at least approximately 850nm, at least approximately 860nm, at least approximately 870nm, at least approximately 880nm, at least approximately 890nm, at least approximately 900nm, at least approximately 910nm, At least approximately 920nm, at least approximately 930nm, at least approximately 940nm, at least approximately 950nm, at least approximately 960nm, at least approximately 970nm, at least approximately 980nm, at least approximately 990nm, at least approximately 1,000nm, at least approximately 1,100nm, at least approximately 1,200nm, at least approximately 1,300nm, at least approximately 1,400nm, at least approximately 1,500nm, at least approximately 1,600nm, at least approximately 1,700nm, at least approximately 1,800nm, at least approximately 1,9 00nm, at least about 2,000nm, at least about 2,100nm, at least about 2,200nm, at least about 2,300nm, at least about 2,400nm, at least about 2,500nm, at least about 2,600nm, at least about 2,700nm, at least about 2,800nm, at least about 2,900nm, at least about 3,000nm, at least about 3,100nm, at least about 3,200nm, at least about 3,300nm, at least about 3,400nm, at least about 3,500nm, At least approximately 3,600 nm, at least approximately 3,700 nm, at least approximately 3,800 nm, at least approximately 3,900 nm, at least approximately 4,000 nm, at least approximately 4,100 nm, at least approximately 4,200 nm, at least approximately 4,300 nm, at least approximately 4,400 nm, at least approximately 4,500 nm, at least approximately 4,600 nm, at least approximately 4,700 nm, at least approximately 4,800 nm, at least approximately 4,900 nm, at least approximately 5,000 nm, or may exceed approximately 5,000 nm.

[0074] In some embodiments, the linear length of the analyte is approximately 5,000 nm, 4,900 nm, 4,800 nm, 4,700 nm, 4,600 nm, 4,500 nm, 4,400 nm, 4,300 nm, 4,200 nm, 4,100 nm, 4,000 nm, 3,900 nm, 3,800 nm, 3,700 nm, 3,600 nm, 3,500 nm, 3,400 nm, 3,300 nm, and 3,200 nm. Up to approximately 3,100nm, up to approximately 3,000nm, up to approximately 2,900nm, up to approximately 2,800nm, up to approximately 2,700nm, up to approximately 2,600nm, up to approximately 2,500nm, up to approximately 2,400nm, up to approximately 2,300nm, up to approximately 2,200nm, up to approximately 2,100nm, up to approximately 2,000nm, up to approximately 1,900nm, up to approximately 1,800nm, up to approximately 1,700nm, up to approximately 1,600nm, up to approximately 1,500nm, up to approximately 1,400nm, up to approximately 1,300nm, up to approximately 1,200nm, up to approximately 1,100nm, up to approximately 1,100nm, Largest is approximately 1,000nm, maximum is approximately 990nm, maximum is approximately 980nm, maximum is approximately 970nm, maximum is approximately 960nm, maximum is approximately 950nm, maximum is approximately 940nm, maximum is approximately 930nm, maximum is approximately 920nm, maximum is approximately 910nm, maximum is approximately 900nm, maximum is approximately 890nm, maximum is approximately 880nm, maximum is approximately 870nm, maximum is approximately 860nm, maximum is approximately 850nm, maximum is approximately 840nm, maximum is approximately 830nm, maximum is approximately 820nm, maximum is approximately 810nm, maximum is approximately 800nm, maximum is approximately 790nm, maximum is approximately 780nm, maximum is approximately 770nm, maximum is approximately 760nm. Up to approximately 750nm, up to approximately 740nm, up to approximately 730nm, up to approximately 720nm, up to approximately 710nm, up to approximately 700nm, up to approximately 690nm, up to approximately 680nm, up to approximately 670nm, up to approximately 660nm, up to approximately 650nm, up to approximately 640nm, up to approximately 630nm, up to approximately 620nm, up to approximately 610nm, up to approximately 600nm, up to approximately 590nm, up to approximately 580nm, up to approximately 570nm, up to approximately 560nm, up to approximately 550nm, up to approximately 540nm, up to approximately 530nm, up to approximately 520nm, up to approximately 510nm,Up to approximately 500nm, up to approximately 490nm, up to approximately 480nm, up to approximately 470nm, up to approximately 460nm, up to approximately 450nm, up to approximately 440nm, up to approximately 430nm, up to approximately 420nm, up to approximately 410nm, up to approximately 400nm, up to approximately 390nm, up to approximately 380nm, up to approximately 370nm, up to approximately 360nm, up to approximately 350nm, up to approximately 340nm, up to approximately 330nm, up to approximately 320nm, up to approximately 310nm, up to approximately 300nm, up to approximately 290nm, up to approximately 280nm, up to approximately 270nm, up to approximately 260nm, up to approximately 250nm, up to approximately 240nm, up to approximately 230nm, up to approximately 220nm, up to approximately 210nm, The maximum wavelength is approximately 200nm, the maximum is approximately 190nm, the maximum is approximately 180nm, the maximum is approximately 170nm, the maximum is approximately 160nm, the maximum is approximately 150nm, the maximum is approximately 140nm, the maximum is approximately 130nm, the maximum is approximately 120nm, the maximum is approximately 110nm, the maximum is approximately 100nm, the maximum is approximately 95nm, the maximum is approximately 90nm, the maximum is approximately 85nm, the maximum is approximately 80nm, the maximum is approximately 75nm, the maximum is approximately 70nm, the maximum is approximately 65nm, the maximum is approximately 60nm, the maximum is approximately 55nm, the maximum is approximately 50nm, the maximum is approximately 45nm, the maximum is approximately 40nm, the maximum is approximately 35nm, the maximum is approximately 30nm, the maximum is approximately 25nm, the maximum is approximately 20nm, the maximum is approximately 15nm, the maximum is approximately 10nm, the maximum is approximately 5nm, the maximum is approximately 3nm, or possibly less than approximately 3nm.

[0075] In some embodiments, the linear length of the analyte is approximately 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, and 2 50nm, about 260nm, about 270nm, about 280nm, about 290, about 300nm, about 310nm, about 320nm, about 330nm, about 34 0nm, about 350nm, about 360nm, about 370nm, about 380nm, about 390nm, about 400nm, about 410nm, about 420nm, about 43 0nm, about 440nm, about 450nm, about 460nm, about 470nm, about 480nm, about 490nm, about 500nm, about 510nm, about 5 20nm, approximately 530nm, approximately 540nm, approximately 550nm, approximately 560nm, approximately 570nm, approximately 580nm, approximately 590nm, approximately 600nm, approximately 6 10nm, about 620nm, about 630nm, about 640nm, about 650nm, about 660nm, about 670nm, about 680nm, about 690nm, about 700nm, about 710nm, about 720nm, about 730nm, about 740nm, about 750nm, about 760nm, about 770nm, about 780nm, Approximately 790nm, approximately 800nm, approximately 810nm, approximately 820nm, approximately 830nm, approximately 840nm, approximately 850nm, approximately 860nm, approximately 870nm , about 880nm, about 890nm, about 900nm, about 910nm, about 920nm, about 930nm, about 940nm, about 950nm, about 960nm , about 970nm, about 980nm, about 990nm, about 1,000nm, about 1,100nm, about 1,200nm, about 1,300nm, about 1,4 00nm, approx. 1,500nm, approx. 1,600nm, approx. 1,700nm, approx. 1,800nm, approx. 1,900nm, approx. 2,000nm, approx. 2,1 00nm, approx. 2,200nm, approx. 2,300nm, approx. 2,400nm, approx. 2,500nm, approx. 2,600nm, approx. 2,700nm, approx. 2, 800nm, approx. 2,900nm, approx. 3,000nm, approx. 3,100nm, approx. 3,200nm, approx. 3,300nm, approx. 3,400nm, approx. 3,It could be 500nm, approximately 3,600nm, approximately 3,700nm, approximately 3,800nm, approximately 3,900nm, approximately 4,000nm, approximately 4,100nm, approximately 4,200nm, approximately 4,300nm, approximately 4,400nm, approximately 4,500nm, approximately 4,600nm, approximately 4,700nm, approximately 4,800nm, approximately 4,900nm, or approximately 5,000nm.

[0076] The unfolded analyte may or may not contain secondary structural elements. These secondary structural elements may include α-helices, β-helices, coils, or β-sheets. Helices may be counterclockwise or clockwise. The analyte may be unfolded, fully or partially. The contour length may be the length of the polymer analyte when its two ends fully extend from each other, or, in some cases, the contour length of the analyte when its two ends do not fully extend from each other. In some embodiments, the analyte may include structured portions, unstructured portions, modified portions, partially modified portions, or a combination thereof. In some embodiments, the analyte may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or at least about 6 ends. In some embodiments, the ends of the analyte may include structured portions, unstructured portions, modified portions, partially modified portions, or a combination thereof.

[0077] The analyte may contain repeating units. In some embodiments, the analyte may contain about 2 to about 100 repeating units. In some cases, the analyte may contain approximately 2 to 5 repeating units, approximately 5 to 10 repeating units, approximately 10 to 15 repeating units, approximately 15 to 20 repeating units, approximately 20 to 25 repeating units, approximately 25 to 30 repeating units, approximately 30 to 35 repeating units, approximately 35 to 40 repeating units, approximately 40 to 45 repeating units, approximately 45 to 50 repeating units, approximately 50 to 55 repeating units, approximately 55 to 60 repeating units, approximately 60 to 65 repeating units, approximately 65 to 70 repeating units, approximately 70 to 75 repeating units, approximately 75 to 80 repeating units, approximately 80 to 85 repeating units, approximately 85 to 90 repeating units, approximately 90 to 95 repeating units, or approximately 95 to 100 repeating units.

[0078] In some embodiments, the analyte may include at least about 2 repeating units, at least about 3 repeating units, at least about 4 repeating units, at least about 5 repeating units, at least about 10 repeating units, at least about 15 repeating units, at least about 20 repeating units, at least about 25 repeating units, at least about 30 repeating units, at least about 35 repeating units, at least about 40 repeating units, at least about 45 repeating units, at least about 50 repeating units, at least about 55 repeating units, at least about 60 repeating units, at least about 65 repeating units, at least about 70 repeating units, at least about 75 repeating units, at least about 80 repeating units, at least about 85 repeating units, at least about 90 repeating units, at least about 95 repeating units, at least about 100 repeating units, or more than 100 repeating units. In some embodiments, the analytes may include up to about 100 repeating units, up to about 95 repeating units, up to about 90 repeating units, up to about 85 repeating units, up to about 80 repeating units, up to about 75 repeating units, up to about 70 repeating units, up to about 65 repeating units, up to about 60 repeating units, up to about 55 repeating units, up to about 50 repeating units, up to about 45 repeating units, up to about 40 repeating units, up to about 35 repeating units, up to about 30 repeating units, up to about 25 repeating units, up to about 20 repeating units, up to about 15 repeating units, up to about 10 repeating units, up to about 5 repeating units, up to about 4 repeating units, up to about 3 repeating units, up to about 2 repeating units, or less than 2 repeating units.In some embodiments, the analytes may include approximately 2 repeating units, approximately 3 repeating units, approximately 4 repeating units, approximately 5 repeating units, approximately 10 repeating units, approximately 15 repeating units, approximately 20 repeating units, approximately 25 repeating units, approximately 30 repeating units, approximately 35 repeating units, approximately 40 repeating units, approximately 45 repeating units, approximately 50 repeating units, approximately 55 repeating units, approximately 60 repeating units, approximately 65 repeating units, approximately 70 repeating units, at least approximately 75 repeating units, at least approximately 80 repeating units, approximately 85 repeating units, approximately 90 repeating units, approximately 95 repeating units, or approximately 100 repeating units.

[0079] The units may include peptide units, sugar units, lipid units, nucleotides, water-soluble plastic monomers, or combinations thereof. The analyte may include polypeptides, polysaccharides, lipids, nucleic acids, water-soluble plastics, or combinations thereof. In some embodiments, the analyte may include a charge. The charge may be positive or negative. The charge may be distributed uniformly or non-uniformly throughout the analyte. In some embodiments, the charge may be the result of an amino acid residue. The amino acid residue may be native or mutant residue. In some cases, the mutant residue may be a point mutation in the analyte. In some cases, the mutant residue may be a residue different from the wild-type sequence of the analyte. In some embodiments, the analyte may include a peptide. The peptide may include a polypeptide or a protein. The protein may be a full-length protein or a cleaved protein. A cleaved protein (e.g., a peptide) may be a protein that is shorter than when the protein was first made. For example, a protein may become shorter due to cleavage (e.g., by peptidase) or degradation (e.g., due to acidic or basic conditions). The protein may include a sequence that is a native protein sequence or a modified protein sequence. Sequences can be modified by mutation, deletion, or insertion. Sequences may also be combinations of sequences. For example, a first natural sequence can be added to or inserted into a second natural sequence to form a third sequence which is a combination of the first and second sequences.

[0080] In another embodiment, the present disclosure provides a system for determining one or more features of an analyte. In some embodiments, the system includes a fluid chamber. In some embodiments, the system includes a membrane. The membrane can divide the fluid chamber into two or more sides. The membrane can divide the fluid chamber into a cis side and a transform side. The cis side may contain a fluid solution. The transform side may contain a fluid solution. The fluid solution may be configured to provide an electroosmotic flow (also called electroosmotic force). The electroosmotic force may act across the membrane. In some embodiments, the membrane includes nanopores. In some embodiments, a pair of electrodes is provided. The pair of electrodes may be arranged by one electrode on the cis side of the fluid chamber and the other electrode on the transform side of the fluid chamber. In some embodiments, electrophoretic force is provided.

[0081] In other embodiments, the disclosure provides methods for determining one or more characteristics of an analyte. In some embodiments, the method involves dislocating the analyte through nanopores. Dislocation may be assisted by electroosmosis, electrophoresis, or a combination thereof. Dislocation may be opposed to electroosmosis, electrophoresis, or a combination thereof. In some embodiments, the analyte is in a pre-modified state before dislocation. In some embodiments, the analyte may dislocate through nanopores in an elongated form. In some cases, the elongated form of the analyte may not include a three-dimensional structure. In some cases, the elongated form of the analyte may be a completely linear structure. In some cases, the elongated form of the analyte may be a linear structure. In some cases, the analyte may dislocate through nanopores in a folded structure. In some cases, the analyte in a folded structure may include a non-elongated analyte. In some cases, the non-elongated structure may not include any three-dimensional structure in the analyte. In some cases, the analyte having a folded structure may include a three-dimensional structure. In some embodiments, the method includes measuring a signal. The signal may be caused by or influenced by the dislocation of the analyte. In some embodiments, one or more analytes dislocate. The signal of one or more dislocated analytes may be measured.

[0082] In some embodiments, analyte dislocations through nanopores occur in the cis-to-trans direction. In some embodiments, analyte dislocations through nanopores occur in the trans-to-cis direction. In some embodiments, analyte dislocations through nanopores occur in the direction of electroosmotic flow (EOF). In some embodiments, analyte dislocations through nanopores occur in the opposite direction to electrophoretic flow (EPF). In some embodiments, analyte dislocations through nanopores occur in the direction of EOF or in the opposite direction to EPF.

[0083] In some embodiments, EOF may be greater than EPF. In some cases, EOF is about 0.1% to about 500% greater than EPF. In some cases, EOF is about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, and about 80%. ~approximately 85%, approximately 85%~approximately 90%, approximately 90%~approximately 95%, approximately 95%~approximately 100%, approximately 100%~approximately 110%, approximately 110%~approximately 120%, approximately 120%~approximately 130%, approximately 130%~approximately 140%, approximately 140%~approximately 150%, approximately 150%~approximately 160%, approximately 160%~approximately 170%, approximately 170%~approximately 180%, approximately 180%~approximately 190%, approximately 190%~approximately 200%, approximately 200%~approximately 210%, approximately 210%~ Approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% to approximately 360%, Approximately 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or 490% to 500% longer and larger.

[0084] In some cases, EOF is at least approximately 0.1%, at least approximately 0.5%, at least approximately 1%, at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, at least approximately 110%, at least approximately 120%, at least approximately 130%, at least approximately 140%, at least approximately 150%, at least approximately 160%, at least approximately 170%, at least approximately 180%, at least approximately 190%, and at least approximately 2%. 00%, may be greater than or greater than 500%, or at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%.

[0085] In some cases, EOF is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, up to approximately 390%, up to approximately 380%, and up to approximately 370% compared to EPF. Up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to Approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to It may be approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1% larger, or up to less than 0.1% larger.

[0086] In some cases, EOF is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and 2% higher than EPF. 00%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, or may be approximately 500% larger.

[0087] In some embodiments, analyte dislocations through the nanopores occur in the direction of the EOF. In some embodiments, analyte dislocations through the nanopores occur in the direction of the EPF. In some embodiments, analyte dislocations through the nanopores occur in the direction of the EOF or the EPF.

[0088] Alternatively, in some embodiments, analyte dislocations through the nanopores occur in the direction of the EPF. In some embodiments, analyte dislocations through the nanopores occur in the direction opposite to the EOF. In some embodiments, analyte dislocations through the nanopores occur in the direction of the EPF or in the direction opposite to the EOF.

[0089] Alternatively, in some embodiments, EPF may be greater than EOF. In some embodiments, EPF may be greater than EOF. In some cases, EPF is about 0.1% to about 500% greater than EOF. In some cases, EPF is about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, and about 80%. ~approximately 85%, approximately 85%~approximately 90%, approximately 90%~approximately 95%, approximately 95%~approximately 100%, approximately 100%~approximately 110%, approximately 110%~approximately 120%, approximately 120%~approximately 130%, approximately 130%~approximately 140%, approximately 140%~approximately 150%, approximately 150%~approximately 160%, approximately 160%~approximately 170%, approximately 170%~approximately 180%, approximately 180%~approximately 190%, approximately 190%~approximately 200%, approximately 200%~approximately 210%, approximately 210%~ Approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% to approximately 360%, Approximately 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or 490% to 500% longer and larger.

[0090] In some cases, EPF is at least approximately 0.1%, at least approximately 0.5%, at least approximately 1%, at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, at least approximately 110%, at least approximately 120%, at least approximately 130%, at least approximately 140%, at least approximately 150%, at least approximately 160%, at least approximately 170%, at least approximately 180%, at least approximately 190%, and at least approximately 2%. 00%, may be greater than or greater than 500%, or at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%.

[0091] In some cases, EPF is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, up to approximately 390%, up to approximately 380%, and up to approximately 370% compared to EOF. Up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to Approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to It may be approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1% larger, or up to less than 0.1% larger.

[0092] In some cases, EPF is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and 2% higher than EOF. 00%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, or may be approximately 500% larger.

[0093] In some embodiments, analyte dislocation through nanopores may occur in the absence of one or more auxiliary proteins. In some embodiments, analyte dislocation through nanopores may occur in the presence of an endofiltration (EOF). In some cases, analyte dislocation may occur in the presence of an EOF and in the absence of one or more auxiliary proteins. In some cases, one or more auxiliary proteins may be capable of moving the analyte through the nanopores. In some cases, one or more auxiliary proteins may include translocases, helicases, unfoldases, DNA polymerases, RNA polymerases, topoisomerases, or any combination thereof.

[0094] In some embodiments, nanopores include biological nanopores or solid-state nanopores. Biological nanopores may include mutations in a portion of the biological nanopore. Mutations may include insertions, substitutions, deletions, or combinations thereof. In some embodiments, nanopores may include recombinant nanopores. In some cases, recombinant nanopores may include components from one or more different types of nanopores. In some embodiments, nanopores may be modified to restrict the passage of one or more ions through the channels of the nanopore. In some cases, nanopores may restrict the passage of one or more ions through the channels of the nanopore by modifying the charge of the channels of the nanopore. In some cases, nanopores may be modified to have net negatively charged channels. In some cases, net negatively charged channels may restrict the passage of one or more anions through the channels of the nanopore. In some cases, nanopores may be modified to have net positively charged channels. In some cases, a net positively charged channel may restrict the passage of one or more cations through the channel in the nanopore. In some cases, the charge of the nanopore may be altered at the cis inlet of the channel. In some cases, the charge of the nanopore may be altered at the trans inlet of the channel. In some cases, the charge of the nanopore may be altered within the central channel of the channel.

[0095] Nanopores may include certain geometric shapes. Geometric shapes may include toroidal shapes comprising rings or channels. Toroidal shapes may include toroidal polyhedral shapes comprising rings or channels. Rings may include one or more proteins forming the nanopore. Rings may include cross-sectional geometric shapes similar to those of one or more proteins forming the nanopore. Rings may be wider on the cis side than on the trans side, or wider on the trans side than on the cis side. Rings may include portions comprising conical geometric shapes, cylindrical geometric shapes, amorphous geometric shapes, or combinations thereof. Channels may include the central portion of the nanopore geometric shape that does not contain proteins or peptides. Channels may allow molecules to pass through the nanopore (e.g., through the channel). Channels may restrict molecules from passing through the nanopore. The restriction may be based on the width of the channel or the charge of the channel. Channels may include channel length. The channel length may be the length of the channel measured along the longitudinal axis of the channel, perpendicular to the toroidal ring shape of the nanopore geometry. The channel length can be measured as the distance along the longitudinal axis of the channel between the furthest points of the nanopore along the longitudinal axis of the channel. In some embodiments, the channel may have a cis-side starting point and a trans-side ending point of the nanopore, or a trans-side starting point and a cis-side ending point of the nanopore. In some embodiments, the channel length is smaller than the linear length or contour length of the analyte. In some embodiments, the channel length is larger than the linear length or contour length of the analyte. In some embodiments, the channel includes channel lengths of about 2 to 40 nm. In some embodiments, the channel includes channel lengths of about 2 to 5 nm, about 5 to 10 nm, about 10 to 15 nm, about 15 to 20 nm, about 20 to 25 nm, about 25 to 30 nm, about 30 to 35 nm, or about 35 to 40 nm.In some cases, the channel includes channel lengths of at least approximately 2 nm, at least approximately 3 nm, at least approximately 4 nm, at least approximately 5 nm, at least approximately 6 nm, at least approximately 7 nm, at least approximately 8 nm, at least approximately 9 nm, at least approximately 10 nm, at least approximately 11 nm, at least approximately 12 nm, at least approximately 13 nm, at least approximately 14 nm, at least approximately 15 nm, at least approximately 16 nm, at least approximately 17 nm, at least approximately 18 nm, at least approximately 19 nm, at least approximately 20 nm, at least approximately 21 nm, at least approximately 22 nm, at least approximately 23 nm, at least approximately 24 nm, at least approximately 25 nm, at least approximately 26 nm, at least approximately 27 nm, at least approximately 28 nm, at least approximately 29 nm, at least approximately 30 nm, at least approximately 31 nm, at least approximately 32 nm, at least approximately 33 nm, at least approximately 34 nm, at least approximately 35 nm, at least approximately 36 nm, at least approximately 37 nm, at least approximately 38 nm, at least approximately 39 nm, at least approximately 40 nm, or greater than 40 nm. In some cases, the channels are approximately 40nm, 39nm, 38nm, 37nm, 36nm, 35nm, 34nm, 33nm, 32nm, 31nm, 30nm, 29nm, 28nm, 27nm, 26nm, 25nm, 24nm, 23nm, and 22nm. Includes channel lengths of approximately 21nm, 20nm, 19nm, 18nm, 17nm, 16nm, 15nm, 14nm, 13nm, 12nm, 11nm, 10nm, 9nm, 8nm, 7nm, 6nm, 5nm, 4nm, 3nm, 2nm, or less than 2nm.In some cases, the channel includes channels of approximately 2nm, 3nm, 4nm, 5nm, 6nm, 7nm, 8nm, 9nm, 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm, 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm, or 40nm.

[0096] In some embodiments, the internal nanopore channel may include a lumen. In some cases, the lumen of the nanopore channel may be about 0.5 nm to about 10 nm. In some cases, the lumen of the nanopore channel may be at least about 0.5 nm, at least about 1.0 nm, at least about 1.5 nm, at least about 2.0 nm, at least about 2.5 nm, at least about 3.0 nm, at least about 3.5 nm, at least about 4.0 nm, at least about 4.5 nm, at least about 5.0 nm, at least about 5.5 nm, at least about 6.0 nm, at least about 6.5 nm, at least about 7.0 nm, at least about 7.5 nm, at least about 8.0 nm, at least about 8.5 nm, at least about 9.0 nm, at least about 9.5 nm, at least about 10.0 nm, or greater than 10.0 nm. In some cases, the lumen of the nanopore channel may be up to approximately 10.0 nm, up to approximately 9.5 nm, up to approximately 9.0 nm, up to approximately 8.5 nm, up to approximately 8.0 nm, up to approximately 7.5 nm, up to approximately 7.0 nm, up to approximately 6.5 nm, up to approximately 6.0 nm, up to approximately 5.5 nm, up to approximately 5.0 nm, up to approximately 4.5 nm, up to approximately 4.0 nm, up to approximately 3.5 nm, up to approximately 3.0 nm, up to approximately 2.5 nm, up to approximately 2.0 nm, up to approximately 1.5 nm, up to approximately 1.0 nm, up to approximately 0.5 nm, or less than 0.5 nm. In some cases, the lumen of the nanopore channel may be approximately 0.5 nm, 1.0 nm, 1.5 nm, 2.0 nm, 2.5 nm, 3.0 nm, 3.5 nm, 4.0 nm, 4.5 nm, 5.0 nm, 5.5 nm, 6.0 nm, 6.5 nm, 7.0 nm, 7.5 nm, 8.0 nm, 8.5 nm, 9.0 nm, 9.5 nm, or 10.0 nm.

[0097] In some embodiments, the internal nanopore channel may contain one or more constrictions. In some cases, the internal nanopore channel may contain about 1 to about 50 constrictions. In some cases, the internal nanopore channel may contain at least about 1 constriction, at least about 5 constrictions, at least about 10 constrictions, at least about 15 constrictions, at least about 20 constrictions, at least about 25 constrictions, at least about 30 constrictions, at least about 25 constrictions, at least about 30 constrictions, at least about 35 constrictions, at least about 40 constrictions, at least about 45 constrictions, at least about 50 constrictions, or more than 50 constrictions. In some cases, the internal nanopore channel may contain up to about 50 constrictions, up to about 45 constrictions, up to about 40 constrictions, up to about 35 constrictions, up to about 30 constrictions, up to about 25 constrictions, up to about 20 constrictions, up to about 15 constrictions, up to about 10 constrictions, up to about 5 constrictions, up to about 1 constriction, or less than 1 constriction. In some cases, the internal nanopore channel may contain about 1 constriction, about 5 constrictions, about 10 constrictions, about 15 constrictions, about 20 constrictions, about 25 constrictions, about 30 constrictions, about 35 constrictions, about 40 constrictions, about 45 constrictions, or about 50 constrictions.

[0098] In some embodiments, one or more constrictions may be in size from about 0.2 nm to about 2 nm. In some cases, one or more constrictions may be in size from at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about 1.0 nm, at least about 1.1 nm, at least about 1.2 nm, at least about 1.3 nm, at least about 1.4 nm, at least about 1.5 nm, at least about 1.6 nm, at least about 1.7 nm, at least about 1.8 nm, at least about 1.9 nm, at least about 2.0 nm, or greater than 2.0 nm. In some cases, one or more constrictions may be up to approximately 2.0 nm, up to approximately 1.9 nm, up to approximately 1.8 nm, up to approximately 1.7 nm, up to approximately 1.6 nm, up to approximately 1.5 nm, up to approximately 1.4 nm, up to approximately 1.3 nm, up to approximately 1.2 nm, up to approximately 1.1 nm, up to approximately 1.0 nm, up to approximately 0.9 nm, up to approximately 0.8 nm, up to approximately 0.7 nm, up to approximately 0.6 nm, up to approximately 0.5 nm, up to approximately 0.4 nm, up to approximately 0.3 nm, up to approximately 0.2 nm, or less than 0.2 nm in size. In some cases, one or more constrictions may be approximately 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1.0 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, or 2.0 nm in size.

[0099] In some embodiments, the analyte is longer than the length of the channels in the nanopores. In some embodiments, the analyte may be at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 55 times, at least about 60 times, at least about 65 times, at least about 70 times, at least about 75 times, at least about 80 times, at least about 85 times, at least about 90 times, at least about 95 times, at least about 100 times, or greater than about 100 times the length of the channels in the nanopores. In some embodiments, the analyte is approximately 100 times the channel length of the nanopores, up to approximately 95 times, up to approximately 90 times, up to approximately 80 times, up to approximately 75 times, up to approximately 70 times, up to approximately 65 times, up to approximately 60 times, up to approximately 55 times, up to approximately 50 times, up to approximately 45 times, up to approximately 40 times, up to approximately 35 times, up to approximately 30 times, up to approximately 25 times, up to approximately It can be 20 times, up to approximately 19 times, up to approximately 18 times, up to approximately 17 times, up to approximately 16 times, up to approximately 15 times, up to approximately 14 times, up to approximately 13 times, up to approximately 12 times, up to approximately 11 times, up to approximately 10 times, up to approximately 9 times, up to approximately 8 times, up to approximately 7 times, up to approximately 6 times, up to approximately 5 times, up to approximately 4 times, up to approximately 3 times, up to approximately 2 times, or less than approximately 2 times.

[0100] In some embodiments, the analyte may be approximately 2 to 100 times the channel length of the nanopore. In some embodiments, the analyte may be approximately 2 to 5 times, 2 to 10 times, 2 to 20 times, 2 to 30 times, 2 to 40 times, 2 to 50 times, 2 to 60 times, 2 to 70 times, 2 to 80 times, 2 to 90 times, 2 to 100 times, 5 to 10 times, 5 to 20 times, 5 to 30 times, 5 to 40 times, and 5 to 50 times. , approximately 5 times to approximately 60 times, approximately 5 times to approximately 70 times, approximately 5 times to approximately 80 times, approximately 5 times to approximately 90 times, approximately 5 times to approximately 100 times, approximately 10 times to approximately 20 times, approximately 10 times to approximately 30 times, approximately 10 times to approximately 40 times, approximately 10 times to approximately 50 times, approximately 10 times to approximately 60 times, approximately 10 times to approximately 70 times, approximately 10 times to approximately 80 times, approximately 10 times to approximately 90 times, approximately 10 times to approximately 100 times, approximately 20 times to approximately 30 times, approximately 20 times to approximately 40 times, approximately 20 times to approximately 50 times, approximately 20 times 20x to 70x, 20x to 80x, 20x to 90x, 20x to 100x, 30x to 40x, 30x to 50x, 30x to 60x, 30x to 70x 0x, approx. 30x to approx. 80x, approx. 30x to approx. 90x, approx. 30x to approx. 100x, approx. 40x to approx. 50x, approx. 40x to approx. 60x, approx. 0x to approximately 100x, approximately 50x to approximately 60x, approximately 50x to approximately 70x, approximately 50x to approximately 80x, approximately 50x to approximately 90x, approximately 50x to approximately 100x, approximately 60x to approximately 70x, approximately 60x to approximately 80x, approximately 60x to approximately 90x, approximately 60x to approximately 100x, approximately 70x to approximately 80x, approximately 70x to approximately 90x, approximately 70x to approximately 100x, approximately 80x to approximately 90x, approximately 80x to approximately 100x, or approximately 90x to approximately 100x.

[0101] In some embodiments, the analyte may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times the channel length of the nanopore.

[0102] In some embodiments, the linear length of the analyte is greater than the channel length of the nanopore. In some cases, the linear length of the analyte is about 0.1% to about 500% longer than the channel length of the nanopore. In some embodiments, the linear length of the analyte is about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, and about 75%. ~80%, 80%~85%, 85%~90%, 90%~95%, 95%~100%, 100%~110%, 110%~120%, 120%~130%, 130%~140%, 140%~150%, 150%~160%, 160%~170%, 170%~180%, 180%~190%, 190%~200%, 200%~210 %, approximately 210%~220%, approximately 220%~230%, approximately 230%~240%, approximately 240%~250%, approximately 250%~260%, approximately 260%~270%, approximately 270%~280%, approximately 280%~290%, approximately 290%~300%, approximately 300%~310%, approximately 310%~320%, approximately 320%~330%, approximately 330%~340%, approximately 340%~350%, approximately 350%~ 360%, approximately 360% to 370%, approximately 370% to 380%, approximately 380% to 390%, approximately 390% to 400%, approximately 400% to 410%, approximately 410% to 420%, approximately 420% to 430%, approximately 430% to 440%, approximately 440% to 450%, approximately 450% to 460%, approximately 460% to 470%, approximately 470% to 480%, approximately 480% to 490%, or approximately 490% to 500% longer.

[0103] In some cases, the linear length of the analyte is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 1 90%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or longer than 500%.

[0104] In some cases, the linear length of the analyte is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, up to approximately 390%, and up to approximately 380%. %, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, maximum Large: approximately 210%, Maximum: approximately 200%, Maximum: approximately 190%, Maximum: approximately 180%, Maximum: approximately 170%, Maximum: approximately 160%, Maximum: approximately 150%, Maximum: approximately 140%, Maximum: approximately 130%, Maximum: approximately 120%, Maximum: approximately 110%, Maximum: approximately 100%, Maximum: approximately 95%, Maximum: approximately 90%, Maximum: approximately 85%, Maximum: approximately 80%, Maximum: approximately 75% Up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or less than 0.1% longer.

[0105] In some cases, the linear length of the analyte is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, and 180% of the channel length of the nanopore. Approximately 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or approximately 500% longer.

[0106] In some embodiments, the linear length of the analyte is about 1 nm to about 5,000 nm longer than the channel length of the nanopore. In some embodiments, the linear length of the analyte is about 0.1 nm to about 0.5 nm, about 0.5 nm to about 1 nm, about 1 nm to about 3 nm, about 3 to about 5 nm, about 5 nm to about 10 nm, about 10 nm to about 15 nm, about 15 nm to about 20 nm, about 20 nm to about 25 nm, about 25 nm to about 30 nm, about 30 nm to about 35 nm, about 35 nm to about 40 nm, about 40 nm to about 45 nm, about 45 nm to about 50 nm, about 50 nm to about 55 nm, about 55 nm to about 60 nm, about 60 nm to about 65 nm, about 65 nm to about 70 nm, and about 70 nm longer than the channel length of the nanopore. m ~ about 75nm, about 75nm - about 80nm, about 80nm - about 85nm, about 85nm - about 90nm, about 90nm - about 95nm, about 95 nm ~ approx. 100nm, approx. 100nm ~ approx. 150nm, approx. 150nm ~ approx. 200nm, approx. 200nm ~ approx. 250nm, approx. 250nm ~ Approx. 300nm, approx. 300nm ~ approx. 350nm, approx. 350nm ~ approx. 400nm, approx. 400nm ~ approx. 450nm, approx. 450nm ~ approx. 50 0nm, about 500nm to about 550nm, about 550nm to about 600nm, about 600nm to about 650nm, about 650nm to about 700nm, Approximately 700nm to approximately 750nm, approximately 750nm to approximately 800nm, approximately 800nm ​​to approximately 850nm, approximately 850nm to approximately 900nm, approximately 90 0nm ~ approx. 950nm, approx. 950nm ~ approx. 1,000nm, approx. 1,000nm ~ approx. 1,100nm, approx. 1,100nm ~ approx. 1,20 0nm, approx. 1,200nm ~ approx. 1,300nm, approx. 1,300nm ~ approx. 1,400nm, approx. 1,400nm ~ approx. 1,500nm, approx. 1,500nm to approx. 1,600nm, approx. 1,600nm to approx. 1,700nm, approx. 1,700nm to approx. 1,800nm, approx. 1,800n m ~ approx. 1,900nm, approx. 1,900nm ~ approx. 2,000nm, approx. 2,000nm ~ approx. 2,100nm, approx. 2,100nm ~ approx. 2, 200nm, approximately 2,200nm to approximately 2,300nm, approximately 2,300nm to approximately 2,400nm, approximately 2,400 to approximately 2,500nm, approximately 2,500nm to approx. 2,600nm, approx. 2,600nm to approx. 2,700nm, approx. 2,700nm to approx. 2,800nm, approx. 2,800 nm ~ approx. 2,900nm, approx. 2,900nm ~ approx. 3,000nm, approx. 3,000nm ~ approx. 3,100nm, approx. 3,200nm ~ approx. 3,300nm, about 3,300nm to about 3,400nm, about 3,400nm to about 3,500nm, about 3,500 to about 3,600nm, about 3,600nm to about 3,700nm, about 3, 700nm to approx. 3,800nm, approx. 3,800nm ​​to approx. 3,900nm, approx. 3,900nm to approx. 4,000nm, approx. 4,000nm to approx. 4,100nm, approx. 4,100 to approx. 200nm, approximately 4,200nm to approximately 4,300nm, approximately 4,300nm to approximately 4,400nm, approximately 4,400nm to approximately 4,500nm, approximately 4,500nm to approximately 4,600nm, approximately 4,600nm to approximately 4,700nm, approximately 4,700nm to approximately 4,800nm, approximately 4,800nm ​​to approximately 4,900nm, or even longer, approximately 4,900nm to approximately 5,000nm.

[0107] In some embodiments, the analyte may be located on the cis side of the nanopore system. In some embodiments, the analyte may be located on the trans side of the nanopore system. In some embodiments, the analyte may be located within the channels of the nanopores. In some embodiments, the analyte may be located on the cis side of the nanopore system and simultaneously within the channels of the nanopores. In some embodiments, the analyte may be located on the trans side of the nanopore system and simultaneously within the channels of the nanopores. In some embodiments, the analyte may be located on the cis side of the nanopore system, within the channels of the nanopores, and simultaneously on the trans side of the nanopore system.

[0108] In some embodiments, the linear length of the analyte is at least about 0.1 nm, at least about 0.5 nm, at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 10 nm, at least about 15 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, At least approximately 75nm, at least approximately 80nm, at least approximately 85nm, at least approximately 90nm, at least approximately 95nm, at least approximately 100nm, at least approximately 110nm, at least approximately 120nm, at least approximately 130nm, at least approximately 140nm, at least approximately 150nm, at least approximately 160nm, at least approximately 170nm, at least approximately 180nm, at least approximately 190nm, at least approximately 200nm, at least approximately 210nm, at least approximately 220nm, at least approximately 230nm, at least approximately 240nm, at least approximately 250nm, less All are approximately 260nm, at least approximately 270nm, at least approximately 280nm, at least approximately 290nm, at least approximately 300nm, at least approximately 310nm, at least approximately 320nm, at least approximately 330nm, at least approximately 340nm, at least approximately 350nm, at least approximately 360nm, at least approximately 370nm, at least approximately 380nm, at least approximately 390nm, at least approximately 400nm, at least approximately 410nm, at least approximately 420nm, at least approximately 430nm, at least approximately 440nm, at least approximately 450nm, at least approximately 460nm, and less At least approximately 470nm, at least approximately 480nm, at least approximately 490nm, at least approximately 500nm, at least approximately 510nm, at least approximately 520nm, at least approximately 530nm, at least approximately 540nm, at least approximately 550nm, at least approximately 560nm, at least approximately 570nm, at least approximately 580nm, at least approximately 590nm, at least approximately 600nm, at least approximately 610nm, at least approximately 620nm, at least approximately 630nm, at least approximately 640nm, at least approximately 650nm, at least approximately 660nm, at least approximately 670nm,At least approximately 680nm, at least approximately 690nm, at least approximately 700nm, at least approximately 710nm, at least approximately 720nm, at least approximately 730nm, at least approximately 740nm, at least approximately 750nm, at least approximately 760nm, at least approximately 770nm, at least approximately 780nm, at least approximately 790nm, at least approximately 800nm, at least approximately 810nm, at least approximately 820nm, at least approximately 830nm, at least approximately 840nm, at least approximately 850nm, at least approximately 860nm, at least approximately 870nm, and at least Also approximately 880nm, at least approximately 890nm, at least approximately 900nm, at least approximately 910nm, at least approximately 920nm, at least approximately 930nm, at least approximately 940nm, at least approximately 950nm, at least approximately 960nm, at least approximately 970nm, at least approximately 980nm, at least approximately 990nm, at least approximately 1,000nm, at least approximately 1,100nm, at least approximately 1,200nm, at least approximately 1,300nm, at least approximately 1,400nm, at least approximately 1,500nm, at least approximately 1,600nm, at least approximately 1 700nm, at least about 1,800nm, at least about 1,900nm, at least about 2,000nm, at least about 2,100nm, at least about 2,200nm, at least about 2,300nm, at least about 2,400nm, at least about 2,500nm, at least about 2,600nm, at least about 2,700nm, at least about 2,800nm, at least about 2,900nm, at least about 3,000nm, at least about 3,100nm, at least about 3,200nm, at least about 3,300nm, at least about 3,400nm , at least about 3,500 nm, at least about 3,600 nm, at least about 3,700 nm, at least about 3,800 nm, at least about 3,900 nm, at least about 4,000 nm, at least about 4,100 nm, at least about 4,200 nm, at least about 4,300 nm, at least about 4,400 nm, at least about 4,500 nm, at least about 4,600 nm, at least about 4,700 nm, at least about 4,800 nm, at least about 4,900 nm, at least about 5,000 nm, or longer than 5,000 nm.

[0109] In some embodiments, the linear length of the analyte is greater than the channel length of the nanopore by up to approximately 5,000 nm, up to approximately 4,900 nm, up to approximately 4,800 nm, up to approximately 4,700 nm, up to approximately 4,600 nm, up to approximately 4,500 nm, up to approximately 4,400 nm, up to approximately 4,300 nm, up to approximately 4,200 nm, up to approximately 4,100 nm, up to approximately 4,000 nm, up to approximately 3,900 nm, up to approximately 3,800 nm, up to approximately 3,700 nm, up to approximately 3,600 nm, up to approximately 3,500 nm, up to approximately 3,400 nm, and up to approximately 3,300 nm. Up to approximately 3,200nm, up to approximately 3,100nm, up to approximately 3,000nm, up to approximately 2,900nm, up to approximately 2,800nm, up to approximately 2,700nm, up to approximately 2,600nm, up to approximately 2,500nm, up to approximately 2,400nm, up to approximately 2,300nm, up to approximately 2,200nm, up to approximately 2,100nm, up to approximately 2,000nm, up to approximately 1,900nm, up to approximately 1,800nm, up to approximately 1,700nm, up to approximately 1,600nm, up to approximately 1,500nm, up to approximately 1,400nm, up to approximately 1,300nm, up to approximately 1,200nm, Up to approximately 1,100, up to approximately 1,000nm, up to approximately 990, up to approximately 980nm, up to approximately 970nm, up to approximately 960nm, up to approximately 950nm, up to approximately 940nm, up to approximately 930nm, up to approximately 920nm, up to approximately 910nm, up to approximately 900nm, up to approximately 890nm, up to approximately 880nm, up to approximately 870nm, up to approximately 860nm, up to approximately 850nm, up to approximately 840nm, up to approximately 830nm, up to approximately 820nm, up to approximately 810nm, up to approximately 800nm, up to approximately 790nm, up to approximately 780nm, up to approximately 770nm, Up to approximately 760nm, up to approximately 750nm, up to approximately 740nm, up to approximately 730nm, up to approximately 720nm, up to approximately 710nm, up to approximately 700nm, up to approximately 690nm, up to approximately 680nm, up to approximately 670nm, up to approximately 660nm, up to approximately 650nm, up to approximately 640nm, up to approximately 630nm, up to approximately 620nm, up to approximately 610nm, up to approximately 600nm, up to approximately 590nm, up to approximately 580nm, up to approximately 570nm, up to approximately 560nm, up to approximately 550nm, up to approximately 540nm, up to approximately 530nm, up to approximately 520nm,Up to approximately 510nm, up to approximately 500nm, up to approximately 490nm, up to approximately 480nm, up to approximately 470nm, up to approximately 460nm, up to approximately 450nm, up to approximately 440nm, up to approximately 430nm, up to approximately 420nm, up to approximately 410nm, up to approximately 400nm, up to approximately 390nm, up to approximately 380nm, up to approximately 370nm, up to approximately 360nm, maximum At approximately 350nm, at a maximum of approximately 340nm, at a maximum of approximately 330nm, at a maximum of approximately 320nm, at a maximum of approximately 310nm, at a maximum of approximately 300nm, at a maximum of approximately 290nm, at a maximum of approximately 280nm, at a maximum of approximately 270nm, at a maximum of approximately 260nm, at a maximum of approximately 250nm, at a maximum of approximately 240nm, at a maximum of approximately 230nm, at a maximum of approximately 220nm, at a maximum of approximately 210nm, at a maximum of approximately 200nm, at a maximum of approximately 1 90nm, up to approximately 180nm, up to approximately 170nm, up to approximately 160nm, up to approximately 150nm, up to approximately 140nm, up to approximately 130nm, up to approximately 120nm, up to approximately 110nm, up to approximately 100nm, up to approximately 95nm, up to approximately 90nm, up to approximately 85nm, up to approximately 80nm, up to approximately 75nm, up to approximately 70nm, up to approximately 65nm, up to approximately 60nm, up to approximately 55nm, up to approximately 50nm, up to approximately 45nm, up to approximately 40nm, up to approximately 35nm, up to approximately 30nm, up to approximately 25nm, up to approximately 20nm, up to approximately 15nm, up to approximately 10nm, up to approximately 5nm, up to approximately 3nm, up to approximately 2nm, up to approximately 1nm, up to approximately 0.5nm, up to approximately 0.1nm, or longer than 0.1nm.

[0110] In some embodiments, the linear length of the analyte is approximately 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm. Approx. 200nm, approx. 210nm, approx. 220nm, approx. 230nm, approx. 240nm, approx. 250nm, approx. 260nm, approx. 270nm, approx. 280nm , about 290, about 300nm, about 310nm, about 320nm, about 330nm, about 340nm, about 350nm, about 360nm, about 370nm, about 380nm, about 390nm, about 400nm, about 410nm, about 420nm, about 430nm, about 440nm, about 450nm, about 460nm, approx. 470nm, approx. 480nm, approx. 490nm, approx. 500nm, approx. 510nm, approx. 520nm, approx. 530nm, approx. 540nm, approx. 550nm, Approx. 560nm, approx. 570nm, approx. 580nm, approx. 590nm, approx. 600nm, approx. 610nm, approx. 620nm, approx. 630nm, approx. 640nm , about 650nm, about 660nm, about 670nm, about 680nm, about 690nm, about 700nm, about 710nm, about 720nm, about 730nm , about 740nm, about 750nm, about 760nm, about 770nm, about 780nm, about 790nm, about 800nm, about 810nm, about 820n m, approx. 830nm, approx. 840nm, approx. 850nm, approx. 860nm, approx. 870nm, approx. 880nm, approx. 890nm, approx. 900nm, approx. 910n m, approx. 920nm, approx. 930nm, approx. 940nm, approx. 950nm, approx. 960nm, approx. 970nm, approx. 980nm, approx. 990nm, approx. 1,0 00nm, approx. 1,100nm, approx. 1,200nm, approx. 1,300nm, approx. 1,400nm, approx. 1,500nm, approx. 1,600nm, approx. 1,7 00nm, approx. 1,800nm, approx. 1,900nm, approx. 2,000nm, approx. 2,100nm, approx. 2,200nm, approx. 2,300nm, approx. 2, 400nm, approx. 2,500nm, approx. 2,600nm, approx. 2,700nm, approx. 2,800nm, approx. 2,900nm, approx. 3,000nm, approx. 3,100nm, approximately 3,200nm, approximately 3,300nm, approximately 3,400nm, approximately 3,500nm, approximately 3,600nm, approximately 3,700nm, approximately 3,800nm, approximately 3,900nm, approximately 4,000nm, approximately 4,100nm, approximately 4,200nm, approximately 4,300nm, approximately 4,400nm, approximately 4,500nm, approximately 4,600nm, approximately 4,700nm, approximately 4,800nm, approximately 4,900nm, or even longer, approximately 5,000nm.

[0111] In some embodiments, the nanopore includes a lumen. The lumen may be the surface of the nanopore facing the channel. The lumen may include the surface of components of the nanopore facing the channel, including proteins, peptides, or amino acid residues facing the channel. These components may contain an electric charge. The electric charge of these components may provide the net charge of the lumen. These components may have a certain shape. The shape of these components may provide the geometric shape of the lumen. The net charge of the lumen, the geometric shape of the lumen, or a combination thereof may influence the flow of molecules through the lumen (e.g., through the nanopore).

[0112] In some embodiments, the pores include a lumen. In some embodiments, the nanopore lumen includes a net charge of at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, or more than about 200. In some embodiments, the nanopore lumen contains a net charge of up to about 200, up to about 150, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 55, up to about 50, up to about 45, up to about 40, up to about 35, up to about 30, up to about 25, up to about 20, up to about 15, up to about 10, up to about 5, up to about 4, up to about 3, up to about 2, or less than about 2.

[0113] In some embodiments, the nanopore lumen contains a net charge of about 2 to about 200. In some embodiments, the nanopore lumen contains a net charge of up to about 200. In some embodiments, the nanopore lumen contains a net charge of about 2 to about 5, about 2 to about 10, about 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 75, about 2 to about 100, about 2 to about 125, about 2 to about 150, about 2 to about 200, about 5 to about 10, about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 5 to about 75, about 5 Approximately 100, approximately 5-125, approximately 5-150, approximately 5-200, approximately 10-20, approximately 10-30, approximately 10-40, approximately 10-50, approximately 10-75, approximately 10-100, approximately 10-125, approximately 10-150, approximately 10-200, approximately 20-30, approximately 20-40, approximately 20-50, approximately 20-75, approximately 20-100 Approximately 20-125, approximately 20-150, approximately 20-200, approximately 30-40, approximately 30-50, approximately 30-75, approximately 30-100, approximately 30-125, approximately 30-150, approximately 30-200, approximately 40-50, approximately 40-75, approximately 40-100, approximately 40-125, approximately 40-150, approximately 40-200, approximately 50- Includes net charges of 75, approximately 50-100, approximately 50-125, approximately 50-150, approximately 50-200, approximately 75-100, approximately 75-125, approximately 75-150, approximately 75-200, approximately 100-125, approximately 100-150, approximately 100-200, approximately 125-150, approximately 125-200, or approximately 150-200.

[0114] In some embodiments, the nanopore lumen contains a net charge of about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 150, and about 200. In some embodiments, the pore lumen contains a net positive charge. In some embodiments, the pore lumen contains a net negative charge.

[0115] In some embodiments, the analyte may lack a three-dimensional structure. In some cases, an analyte lacking a three-dimensional structure may be a denatured analyte. In some embodiments, one or more parts of the analyte may lack a three-dimensional structure. In some cases, one or more parts may include one or more ends of the analyte. In some cases, one of the ends of the analyte may lack a three-dimensional structure. In some cases, two of the ends of the analyte may lack a three-dimensional structure. In some cases, at least two of the ends of the analyte may lack a three-dimensional structure. In some embodiments, the internal parts (e.g., parts of the analyte that are not ends) may lack a three-dimensional structure. In some embodiments, the analyte may include a three-dimensional structure. In some cases, an analyte having a three-dimensional structure may be a folded analyte.

[0116] The flowing molecules can be analytes, ions, water, or other molecules on the cis or trans side of the nanopore. The flowing molecules can generate an ionic current from the flow of ions. As analytes pass through the pore, they can prevent other molecules, such as ions, from passing through. This allows for a change in the ionic current by altering the ion flow rate. This change can be measured, for example, by a pair of electrodes configured to measure the cis-to-trans current across the nanopore or the membrane in which the nanopore may be located. A narrow lumen geometry can slow the progress of analytes through the pore. Changing the net charge or the lumen geometry can alter the flow of molecules through the pore. For example, altering the lumen to have a more positive net charge can reduce the flow of positively charged molecules (e.g., sodium ions). For example, altering the lumen to have a wider geometry can increase the flow of larger molecules (e.g., glucose molecules or peptide analytes). For example, by altering the lumen to have a more negative net charge and a narrower geometry, the flow of large negatively charged molecules (e.g., glutamate ions) can be reduced. The net charge of the lumen can influence the flow of charged molecules through the nanopores. The net charge can make it easier or more difficult for some charged molecules to pass through.

[0117] A channel may contain constriction zones. A constriction zone may be a portion of a channel that is narrower than the surrounding section. A channel may contain multiple constriction zones. The net charge of the lumen, the geometry of the lumen, or a combination thereof can result in a constriction zone. Modifications to the net charge of the lumen, the geometry of the lumen, or a combination thereof can alter the characteristics of a constriction zone. The characteristics of a constriction zone may be arrangement, position, width, charge, or a combination thereof. For example, a change in the geometry of the lumen can change the width of a constriction zone, or a change in the net charge of the lumen can change the charge of a constriction zone.

[0118] Nanopores may have ion permeability. Ion permeability can be the ability or possibility for ions to flow or diffuse through the channels of the nanopores. Ion permeability (P) may differ for different ions. By comparing different permeability, relative ion selectivity can be generated. Relative ion selectivity is the selectivity (P) for a given cation (e.g., potassium ion). (+) ) the selectivity (P) of a given anion (e.g., chloride ion) (-) Divide by ) and obtain relative ion selectivity (P (+) / P (-) ) can be provided. The net charge and lumen geometry can each affect ion permeability. For example, a positive net charge can reduce cation permeability, or a negative net charge can reduce anion permeability. The shape of the lumen geometry can affect ion permeability based on ion size. For example, a smaller geometry can reduce the permeability of larger ions (e.g., potassium ions) compared to smaller ions (e.g., lithium ions).

[0119] The net charge and lumen geometry can affect the relative ion flux. The relative ion flux may be the net flow of ions through the nanopores. In some embodiments, the nanopores have relative ion selectivity P greater than about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.5, about 3, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. (+) / P (-)may include. In some embodiments, the pore has a relative ion selectivity P of less than about 0.1, less than about 0.2, less than about 0.3, less than about 0.4, less than about 0.5, less than about 0.6, less than about 0.7, less than about 0.8, less than about 0.9, less than about 1.0, less than about 1.2, less than about 1.4, less than about 1.6, less than about 1.8, less than about 2.0, less than about 2.5, less than about 3, less than about 3.2, less than about 3.4, less than about 3.6, less than about 3.8, less than about 4.0, less than about 4.1, less than about 4.2, less than about 4.3, less than about 4.4, less than about 4.5, less than about 4.6, less than about 4.7, less than about 4.8, less than about 4.9, or less than about 5.0 (+) / P (-) may include.

[0120] The nanopores may contain at least one internal pore constriction. In some embodiments, the internal pore constriction may be at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, and at least about 2. 0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1, at least about 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, or at least about 4.0 nm. In some embodiments, the internal pore constriction is approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2. The maximum wavelengths are approximately 0.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or up to approximately 4.0 nm.

[0121] In some embodiments, the nanopore channel includes a lumen. In some embodiments, the nanopore lumen includes a net charge of at least about 2 coulombs, at least about 3 coulombs, at least about 4 coulombs, at least about 5 coulombs, at least about 10 coulombs, at least about 15 coulombs, at least about 20 coulombs, at least about 25 coulombs, at least about 30 coulombs, at least about 35 coulombs, at least about 40 coulombs, at least about 45 coulombs, at least about 50 coulombs, at least about 55 coulombs, at least about 60 coulombs, at least about 70 coulombs, at least about 80 coulombs, at least about 90 coulombs, at least about 100 coulombs, at least about 150 coulombs, at least about 200 coulombs, or more than about 200 coulombs. In some embodiments of the coulombs, the nanopore lumen contains a net charge of up to about 200 coulombs, up to about 150 coulombs, up to about 100 coulombs, up to about 90 coulombs, up to about 80 coulombs, up to about 70 coulombs, up to about 60 coulombs, up to about 55 coulombs, up to about 50 coulombs, up to about 45 coulombs, up to about 40 coulombs, up to about 35 coulombs, up to about 30 coulombs, up to about 25 coulombs, up to about 20 coulombs, up to about 15 coulombs, up to about 10 coulombs, up to about 5 coulombs, up to about 4 coulombs, up to about 3 coulombs, up to about 2 coulombs, or less than about 2 coulombs.

[0122] In some embodiments, the nanopore lumen contains a net charge of about 2 to about 200 coulombs. In some embodiments, the nanopore lumen contains a net charge of up to about 200. In some embodiments, the nanopore lumen contains a net charge of about 2 to about 5, about 2 to about 10, about 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 75, about 2 to about 100, about 2 to about 125, about 2 to about 150, about 2 to about 200, about 5 to about 10, about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 5 to about 75, about 5 to about 100, approximately 5-125, approximately 5-150, approximately 5-200, approximately 10-20, approximately 10-30, approximately 10-40, approximately 10-50, approximately 10-75, approximately 10-100, approximately 10-125, approximately 10-150, approximately 10-200, approximately 20-30, approximately 20-40, approximately 20-50, approximately 20-75, approximately 20-100, approximately 20-approx. 125, approx. 20-approx. 150, approx. 20-approx. 200, approx. 30-approx. 40, approx. 30-approx. 50, approx. 30-approx. 75, approx. 30-approx. 100, approx. 30-approx. 125, approx. 30-approx. 150, approx. 30-approx. 200, approx. 40-approx. 50, approx. 40-approx. 75, approx. 40-approx. 100, approx. 40-approx. 125, approx. 40-approx. 150, approx. 40-approx. 200, approx. 50-approx. 75, Including a net charge of approximately 50-100, 50-125, 50-150, 50-200, 75-100, 75-125, 75-150, 75-200, 100-125, 100-150, 100-200, 125-150, 125-200, or 150-200 coulombs.

[0123] In some embodiments, the nanopore lumen contains a net charge of about 2 coulombs, about 3 coulombs, about 4 coulombs, about 5 coulombs, about 10 coulombs, about 15 coulombs, about 20 coulombs, about 25 coulombs, about 30 coulombs, about 35 coulombs, about 40 coulombs, about 45 coulombs, about 50 coulombs, about 55 coulombs, about 60 coulombs, about 70 coulombs, about 80 coulombs, about 90 coulombs, about 100 coulombs, about 150 coulombs, and about 200 coulombs. In some embodiments, the nanopore lumen contains a net positive charge. In some embodiments, the nanopore lumen contains a net negative charge.

[0124] To some extent, the nanopore lumen may contain a net charge of approximately -20 to approximately +20. In some cases, the nanopore lumen may contain at least approximately -20, at least approximately -19, at least approximately -18, at least approximately -17, at least approximately -16, at least approximately -15, at least approximately -14, at least approximately -13, at least approximately -12, at least approximately -11, at least approximately -10, at least approximately -9, at least approximately -8, at least approximately -7, at least approximately -6, at least approximately -5, at least approximately -4, at least approximately -3, at least approximately -2, at least approximately -1, at least It may include a net charge of approximately 0, at least approximately +1, at least approximately +2, at least approximately +3, at least approximately +4, at least approximately +5, at least approximately +6, at least approximately +7, at least approximately +8, at least approximately +9, at least approximately +10, at least approximately +11, at least approximately +12, at least approximately +13, at least approximately +14, at least approximately +15, at least approximately +16, at least approximately +17, at least approximately +18, at least approximately +19, at least approximately +20, or greater than +20. In some cases, the nanopore lumen is approximately +20, +19, +18, +17, +16, +15, +14, +13, +12, +11, +10, +9, +8, +7, +6, +5, +4, +3, +2, +1, and It may include net charges of approximately 0, up to approximately -1, up to approximately -2, up to approximately -3, up to approximately -4, up to approximately -5, up to approximately -6, up to approximately -7, up to approximately -8, up to approximately -9, up to approximately -10, up to approximately -11, up to approximately -12, up to approximately -13, up to approximately -14, up to approximately -15, up to approximately -16, up to approximately -17, up to approximately -18, up to approximately -19, up to approximately -20, or less than -20.In some cases, the nanopore lumen may contain a net charge of approximately -20, approximately -19, approximately -18, approximately -17, approximately -16, approximately -15, approximately -14, approximately -13, approximately -12, approximately -11, approximately -10, approximately -9, approximately -8, approximately -7, approximately -6, approximately -5, approximately -4, approximately -3, approximately -2, approximately -1, approximately 0, approximately +1, approximately +2, approximately +3, approximately +4, approximately +5, approximately +6, approximately +7, approximately +8, approximately +9, approximately +10, approximately +11, approximately +12, approximately +13, approximately +14, approximately +15, approximately +16, approximately +17, approximately +18, approximately +19, or approximately +20.

[0125] In some embodiments, the nanopores may contain one or more subunits. In some cases, each subunit of the one or more subunits may contain 1 to 20 charged amino acids. In some embodiments, about 1 to 20 charged repeating units can be distributed within the lumen. In some cases, the charged repeating units may be negatively charged. In some cases, the charged repeating units may be positively charged. In some cases, the charged repeating units may be positively charged and negatively charged. In some cases, the lumen may contain at least about 1 charged repeating unit, at least about 2 charged repeating units, at least about 3 charged repeating units, at least about 4 charged repeating units, at least about 5 charged repeating units, at least about 6 charged repeating units, at least about 7 charged repeating units, at least about 8 charged repeating units, at least about 9 charged repeating units, at least about 10 charged repeating units, at least about 11 charged repeating units, at least about 12 charged repeating units, at least about 13 charged repeating units, at least about 14 charged repeating units, at least about 15 charged repeating units, at least about 16 charged repeating units, at least about 17 charged repeating units, at least about 18 charged repeating units, at least about 19 charged repeating units, at least about 20 charged repeating units, or more than 20 charged repeating units.In some cases, the lumen may contain up to approximately 20 charged repeating units, up to approximately 19 charged repeating units, up to approximately 18 charged repeating units, up to approximately 17 charged repeating units, up to approximately 16 charged repeating units, up to approximately 15 charged repeating units, up to approximately 14 charged repeating units, up to approximately 13 charged repeating units, up to approximately 12 charged repeating units, up to approximately 11 charged repeating units, up to approximately 10 charged repeating units, up to approximately 9 charged repeating units, up to approximately 8 charged repeating units, up to approximately 7 charged repeating units, up to approximately 6 charged repeating units, up to approximately 5 charged repeating units, up to approximately 4 charged repeating units, up to approximately 3 charged repeating units, up to approximately 2 charged repeating units, up to approximately 1 charged repeating unit, or less. In some cases, the lumen may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 charged repeating units. In some embodiments, the charged repeating units can be uniformly distributed within the lumen. In some cases, the charged repeating units can be distributed in groups of approximately 2 to 50 repeating units.In some cases, the charged repeating units can be distributed in groups of at least approximately 2 repeating units, at least approximately 5 repeating units, at least approximately 10 repeating units, at least approximately 15 repeating units, at least approximately 20 repeating units, at least approximately 25 repeating units, at least approximately 30 repeating units, at least approximately 40 repeating units, at least approximately 45 repeating units, at least approximately 50 repeating units, or more than 50 repeating units. In some cases, the charged repeating units can be distributed in groups of up to approximately 50 repeating units, up to approximately 45 repeating units, up to approximately 40 repeating units, up to approximately 35 repeating units, up to approximately 30 repeating units, up to approximately 25 repeating units, up to approximately 20 repeating units, up to approximately 15 repeating units, up to approximately 10 repeating units, up to approximately 5 repeating units, up to approximately 2 repeating units, or less than 2 repeating units. In some cases, the charged repeating units can be distributed in groups of approximately 2, 5, 10, 15, 20, 25, 30, 35, 30, 35, 40, 45, or 50 repeating units. In some embodiments, the charged repeating units can be distributed non-uniformly within the lumen.

[0126] The repeating units may be positively or negatively charged. Charged repeating units Asp, Glu, or Asp can be readily introduced by a single amino acid substitution. In some embodiments, the number of negatively charged amino acids that can be uniformly distributed in the lumen may be at least about 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, or more than about 20 charged amino acids. In some embodiments, the number of negatively charged amino acids that can be uniformly distributed within the lumen can be up to about 20 amino acids, up to about 19 amino acids, up to about 18 amino acids, up to about 17 amino acids, up to about 16 amino acids, up to about 15 amino acids, up to about 14 amino acids, up to about 13 amino acids, up to about 12 amino acids, up to about 11 amino acids, up to about 10 amino acids, up to about 9 amino acids, up to about 8 amino acids, up to about 7 amino acids, up to about 6 amino acids, up to about 5 amino acids, up to about 4 amino acids, up to about 3 amino acids, up to about 2 amino acids, up to about 1 amino acid, or less than about 1 charged amino acid.

[0127] In some embodiments, the number of negatively charged amino acids that can be uniformly distributed within the lumen may be about 1 to about 20 charged amino acids. In some embodiments, the number of amino acids that can be uniformly distributed in the lumen is approximately 1 to 2, approximately 1 to 3, approximately 1 to 4, approximately 1 to 5, approximately 1 to 8, approximately 1 to 9, approximately 1 to 10, approximately 1 to 12, approximately 1 to 15, approximately 1 to 18, approximately 1 to 20, approximately 2 to 3, approximately 2 to 4, approximately 2 to 5, approximately 2 to 8, approximately 2 to 9, approximately 2 to 10, approximately 2 to 12, approximately 2 to 15, approximately 2 to 18, approximately 2 to 20, approximately 3 to 4, approximately 3 to 5, approximately 3 to 8, approximately 3 to 9, approximately 3 to 10, approximately 3 to 12, approximately 3 to 15, approximately 3 to 18, approximately 3 to 20, approximately 4 to 5, approximately 4 to 8, approximately 4 to 9, approximately 4 to 1 The number of charged amino acids may be 0, approximately 4-12, approximately 4-15, approximately 4-18, approximately 4-20, approximately 5-8, approximately 5-9, approximately 5-10, approximately 5-12, approximately 5-15, approximately 5-18, approximately 5-20, approximately 8-9, approximately 8-10, approximately 8-12, approximately 8-15, approximately 8-18, approximately 8-20, approximately 9-10, approximately 9-12, approximately 9-15, approximately 9-18, approximately 9-20, approximately 10-12, approximately 10-15, approximately 10-18, approximately 10-20, approximately 12-15, approximately 12-18, approximately 12-20, approximately 15-18, approximately 15-20, or approximately 18-20.

[0128] In some embodiments, the number of negatively charged amino acids that can be uniformly distributed within the lumen may be about 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 charged amino acids. In some cases, the charged repeating units can be distributed in groups of about 2 to 50. In some cases, the charged repeating units can be distributed in groups of at least approximately 2 repeating units, at least approximately 5 repeating units, at least approximately 10 repeating units, at least approximately 15 repeating units, at least approximately 20 repeating units, at least approximately 25 repeating units, at least approximately 30 repeating units, at least approximately 40 repeating units, at least approximately 45 repeating units, at least approximately 50 repeating units, or more than 50 repeating units. In some cases, the charged repeating units can be distributed in groups of up to approximately 50 repeating units, up to approximately 45 repeating units, up to approximately 40 repeating units, up to approximately 35 repeating units, up to approximately 30 repeating units, up to approximately 25 repeating units, up to approximately 20 repeating units, up to approximately 15 repeating units, up to approximately 10 repeating units, up to approximately 5 repeating units, up to approximately 2 repeating units, or less than 2 repeating units. In some cases, the charged repeating units can be distributed in groups of approximately 2, 5, 10, 15, 20, 25, 30, 35, 30, 35, 40, 45, or 50 repeating units.

[0129] In some cases, nanopores may contain one or more subunits. In some cases, each of the one or more subunits may have about 1 to about 20 charged amino acids. In some embodiments, each subunit of one or more subunits is approximately 1 to 2, approximately 1 to 3, approximately 1 to 4, approximately 1 to 5, approximately 1 to 8, approximately 1 to 9, approximately 1 to 10, approximately 1 to 12, approximately 1 to 15, approximately 1 to 18, approximately 1 to 20, approximately 2 to 3, approximately 2 to 4, approximately 2 to 5, approximately 2 to 8, approximately 2 to 9, approximately 2 to 10, approximately 2 to 12, approximately 2 to 15, approximately 2 to 18, approximately 2 to 20, approximately 3 to 4, approximately 3 to 5, approximately 3 to 8, approximately 3 to 9, approximately 3 to 10, approximately 3 to 12, approximately 3 to 15, approximately 3 to 18, approximately 3 to 20, approximately 4 to 5, approximately 4 to 8, approximately 4 to 9, approximately 4 to It may have 10, approximately 4-12, approximately 4-15, approximately 4-18, approximately 4-20, approximately 5-8, approximately 5-9, approximately 5-10, approximately 5-12, approximately 5-15, approximately 5-18, approximately 5-20, approximately 8-9, approximately 8-10, approximately 8-12, approximately 8-15, approximately 8-18, approximately 8-20, approximately 9-10, approximately 9-12, approximately 9-15, approximately 9-18, approximately 9-20, approximately 10-12, approximately 10-15, approximately 10-18, approximately 10-20, approximately 12-15, approximately 12-18, approximately 12-20, approximately 15-18, approximately 15-20, or approximately 18-20 charged amino acids.

[0130] In some embodiments, each subunit of one or more subunits may contain about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, or about 20 charged amino acids. In some cases, the charged repeating units can be distributed in groups of about 2 to about 50 repeating units. In some cases, the charged repeating units can be distributed in groups of at least approximately 2 repeating units, at least approximately 5 repeating units, at least approximately 10 repeating units, at least approximately 15 repeating units, at least approximately 20 repeating units, at least approximately 25 repeating units, at least approximately 30 repeating units, at least approximately 40 repeating units, at least approximately 45 repeating units, at least approximately 50 repeating units, or more than 50 repeating units. In some cases, the charged repeating units can be distributed in groups of up to approximately 50 repeating units, up to approximately 45 repeating units, up to approximately 40 repeating units, up to approximately 35 repeating units, up to approximately 30 repeating units, up to approximately 25 repeating units, up to approximately 20 repeating units, up to approximately 15 repeating units, up to approximately 10 repeating units, up to approximately 5 repeating units, up to approximately 2 repeating units, or less than 2 repeating units. In some cases, the charged repeating units can be distributed in groups of approximately 2, 5, 10, 15, 20, 25, 30, 35, 30, 35, 40, 45, or 50 repeating units.

[0131] At least one negatively charged "adjacent" residue may be located at or introduced to the pore entrance, or at least one positively charged residue may be located at or introduced to the pore exit. In some embodiments, the space between the Cα atom of at least one internally negatively charged amino acid and the Cα atom of a neighboring negatively charged amino acid may be at least about 1 Å, at least about 2 Å, at least about 3 Å, at least about 4 Å, at least about 5 Å, at least about 6 Å, at least about 7 Å, at least about 8 Å, at least about 9 Å, at least about 10 Å, at least about 11 Å, at least about 12 Å, at least about 13 Å, at least about 14 Å, at least about 15 Å, at least about 16 Å, at least about 17 Å, at least about 18 Å, at least about 20 Å, at least about 21 Å, at least about 22 Å, at least about 23 Å, at least about 24 Å, at least about 25 Å, 26 Å, at least about 27 Å, 28 Å, at least about 29 Å, at least about 30 Å, or more than about 30 Å.

[0132] In some embodiments, the space between the Cα atom of at least one internally negatively charged amino acid and the Cα atom of a negatively charged neighboring amino acid can range from about 1 Å to about 30 Å. In some embodiments, the space between the Cα atom of at least one internally negatively charged amino acid and the Cα atom of a negatively charged neighboring amino acid can range from about 1 Å to about 2 Å, about 1 Å to about 3 Å, about 1 Å to about 4 Å, about 1 Å to about 5 Å, about 1 Å to about 6 Å, about 1 Å to about 8 Å, about 1 Å to about 10 Å, about 1 Å to about 15 Å, about 1 Å to about 20 Å, about 1 Å to about 25 Å, about 1 Å to about 30 Å, and about 2 Å to about 3 Å, about 2 Å to about 4 Å, about 2 Å to about 5 Å, about 2 Å to about 6 Å, about 2 Å to about 8 Å, about 2 Å to about 10 Å, about 2 Å to about 15 Å, about 2 Å to about 20 Å, about 2 Å to about 25 Å, about 2 Å to about 30 Å, about 3 Å ~about 4 Å, about 3 Å to about 5 Å, about 3 Å to about 6 Å, about 3 Å to about 8 Å, about 3 Å to about 10 Å, about 3 Å to about 15 Å, about 3 Å to about 20 Å, about 3 Å to about 25 Å, about 3 Å to about 30 Å, about 4 Å to about 5 Å, Approximately 4 Å to 6 Å, approximately 4 Å to 8 Å, approximately 4 Å to 10 Å, approximately 4 Å to 15 Å, approximately 4 Å to 20 Å, approximately 4 Å to 25 Å, approximately 4 Å to 30 Å, approximately 5 Å to 6 Å, approximately 5 Å to 8 Å, approximately 5 Å to 10 Å, approximately 5 Å to 15 Å, approximately 5 Å to 20 Å, approximately 5 Å to 25 Å, approximately 5 Å to 30 Å, approximately 6 Å to 8 Å, approximately 6 Å to 10 Å, approximately 6 Å to 15 Å, approximately 6 Å to 20 Å, approximately 6 Å to 2 It could be 5 Å, approximately 6 Å to approximately 30 Å, approximately 8 Å to approximately 10 Å, approximately 8 Å to approximately 15 Å, approximately 8 Å to approximately 20 Å, approximately 8 Å to approximately 25 Å, approximately 8 Å to approximately 30 Å, approximately 10 Å to approximately 15 Å, approximately 10 Å to approximately 20 Å, approximately 10 Å to approximately 25 Å, approximately 10 Å to approximately 30 Å, approximately 15 Å to approximately 20 Å, approximately 15 Å to approximately 25 Å, approximately 15 Å to approximately 30 Å, approximately 20 Å to approximately 25 Å, approximately 20 Å to approximately 30 Å, or approximately 25 Å to approximately 30 Å.

[0133] In some embodiments, the space between the Cα atom of at least one internally negatively charged amino acid and the Cα atom of the adjacent negatively charged amino acid may be about 1 Å, about 2 Å, about 3 Å, about 4 Å, about 5 Å, about 6 Å, about 7 Å, about 8 Å, about 9 Å, about 10 Å, about 11 Å, about 12 Å, about 13 Å, about 14 Å, about 15 Å, about 16 Å, about 17 Å, about 18 Å, about 20 Å, about 21 Å, about 22 Å, about 23 Å, about 24 Å, about 25 Å, 26 Å, about 27 Å, 28 Å, about 29 Å, or about 30 Å.

[0134] The nanopore lumen may contain distinct sets of charges oriented along the toroidal geometric ring of the nanopore. These sets of charges may be arranged along the longitudinal length of the channel. In some embodiments, the nanopore may contain at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or more than about 20 distinct sets of charges. In some embodiments, the nanopores may contain a set of distinct charges of up to about 20, up to about 19, up to about 18, up to about 17, up to about 16, up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, up to about 1, or less than about 1.

[0135] In some embodiments, the nanopores may contain about 1 to about 20 distinct sets of charges. In some embodiments, the nanopores are approximately 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 15, 1 to 18, 1 to 20, 2 to 3, 2 to 4, 2 to 5, 2 to 8, 2 to 9, 2 to 10, 2 to 12, 2 to 15, 2 to 18, 2 to 20, 2 to 3, 2 to 4, 2 to 5, 2 to 8, 2 to 9, 2 to 10, 2 to 12, 2 to 15, 2 to 18, 2 to 20, 3 to 4, 3 to 5, 3 to 8, 3 to 9, 3 to 10, 3 to 12, 3 to 15, 3 to 18, 3 to 20, 4 to 5, 4 to 8, 4 to 9, 4 to 10, and 4 to 1 2. May include sets of approximately 4-15, 4-18, 4-20, 5-8, 5-9, 5-10, 5-12, 5-15, 5-18, 5-20, 8-9, 8-10, 8-12, 8-15, 8-18, 8-20, 9-10, 9-12, 9-15, 9-18, 8-20, 10-12, 9-15, 9-18, 9-20, 10-12, 10-15, 10-18, 10-20, 12-15, 12-18, 12-20, 15-18, 15-20, or 18-20 distinct charges.

[0136] In some embodiments, the nanopores may contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 distinct sets of charges.

[0137] In some embodiments, each set of charges may be located at least about 0.1 nanometers, at least about 0.2 nanometers, at least about 0.3 nanometers, at least about 0.4 nanometers, at least about 0.5 nanometers, at least about 0.6 nanometers, at least about 0.7 nanometers, at least about 0.8 nanometers, at least about 0.9 nanometers, at least about 1 nanometer, at least about 2 nanometers, at least about 3 nanometers, at least about 4 nanometers, at least about 5 nanometers, at least about 6 nanometers, at least about 7 nanometers, at least about 8 nanometers, at least about 9 nanometers, at least about 10 nanometers, or more than about 10 nanometers apart from each other along the longitudinal length of the channel. In some embodiments, each set of charges may be spaced at a maximum of about 10 nanometers, a maximum of about 9 nanometers, a maximum of about 8 nanometers, a maximum of about 7 nanometers, a maximum of about 6 nanometers, a maximum of about 5 nanometers, a maximum of about 4 nanometers, a maximum of about 3 nanometers, a maximum of about 2 nanometers, a maximum of about 1 nanometer, a maximum of about 0.9 nanometers, a maximum of about 0.8 nanometers, a maximum of about 0.7 nanometers, a maximum of about 0.6 nanometers, a maximum of about 0.5 nanometers, a maximum of about 0.4 nanometers, a maximum of about 0.3 nanometers, a maximum of about 0.2 nanometers, a maximum of about 0.1 nanometers, or less than 0.1 nanometers apart from each other along the longitudinal length of the channel.

[0138] In some embodiments, each set of charges may be spaced about 0.1 nanometers to about 5 nanometers apart from each other along the longitudinal length of the channel. In some embodiments, each set of charges may be spaced about 0.1 nanometers to about 0.2 nanometers, about 0.1 nanometers to about 0.3 nanometers, about 0.1 nanometers to about 0.4 nanometers, about 0.1 nanometers to about 0.5 nanometers, about 0.1 nanometers to about 1 nanometer, about 0.1 nanometers to about 1.5 nanometers, about 0.1 nanometers to about 2 nanometers, about 0.1 nanometers to about 2.5 nanometers, and about 0.1 nanometers to about 3 nanometers apart from each other along the longitudinal length of the channel. Approximately 0.1 nanometers to approximately 4 nanometers, approximately 0.1 nanometers to approximately 5 nanometers, approximately 0.2 nanometers to approximately 0.3 nanometers, approximately 0.2 nanometers to approximately 0.4 nanometers, approximately 0.2 nanometers to approximately 0.5 nanometers, approximately 0.2 nanometers to approximately 1 nanometer, approximately 0.2 nanometers to approximately 1.5 nanometers, approximately 0.2 nanometers to approximately 2 nanometers, approximately 0.2 nanometers to approximately 2.5 nanometers, approximately 0.2 nanometers to approximately 3 nanometers, approximately 0.2 nanometers to approximately 4 nanometers Approximately 0.2 nanometers to approximately 5 nanometers, approximately 0.3 nanometers to approximately 0.4 nanometers, approximately 0.3 nanometers to approximately 0.5 nanometers, approximately 0.3 nanometers to approximately 1 nanometer, approximately 0.3 nanometers to approximately 1.5 nanometers, approximately 0.3 nanometers to approximately 2 nanometers, approximately 0.3 nanometers to approximately 2.5 nanometers, approximately 0.3 nanometers to approximately 3 nanometers, approximately 0.3 nanometers to approximately 4 nanometers, approximately 0.3 nanometers to approximately 5 nanometers, approximately 0.4 nanometers to approximately 0.5 nanometers, Approximately 0.4 nanometers to approximately 1 nanometer, approximately 0.4 nanometers to approximately 1.5 nanometers, approximately 0.4 nanometers to approximately 2 nanometers, approximately 0.4 nanometers to approximately 2.5 nanometers, approximately 0.4 nanometers to approximately 3 nanometers, approximately 0.4 nanometers to approximately 4 nanometers, approximately 0.4 nanometers to approximately 5 nanometers, approximately 0.5 nanometers to approximately 1 nanometer, approximately 0.5 nanometers to approximately 1.5 nanometers, approximately 0.5 nanometers to approximately 2 nanometers, approximately 0.5 nanometers to approximately 2.5 nanometers, approximately 0.5 nanometers to approximately 3 nanometers, approximately 0.5 nanometers to approximately 4 nanometers, approximately 0.5 nanometers to approximately 5 nanometers, approximately 1 nanometer to approximately 1.5 nanometers, approximately 1 nanometer to approximately 2 nanometers, approximately 1 nanometer to approximately 2.5 nanometers, approximately 1 nanometer to approximately 3 nanometers, approximately 1 nanometer to approximately 4 nanometers, approximately 1 nanometer to approximately 5 nanometers, approximately 1.5 nanometers to approximately 2 nanometers, approximately 1.5 nanometers to approximately 2.5 nanometers, approximately 1.5 nanometers to approximately 3 nanometers, approximately 1.5 nanometers to approximately 4 nanometers, approximately 1.5 nanometers to approximately 5 nanometers, approximately 2 nanometers to The distances between the points of contact may be approximately 2.5 nanometers, 2 to 3 nanometers, 2 to 4 nanometers, 2 to 5 nanometers, 2.5 to 3 nanometers, 2.5 to 4 nanometers, 2.5 to 5 nanometers, 3 to 4 nanometers, 3 to 5 nanometers, 4 to 5 nanometers, 5 to 6 nanometers, 6 to 7 nanometers, 7 to 8 nanometers, 8 to 9 nanometers, or 9 to 10 nanometers.

[0139] In some embodiments, each set of charges may be located about 0.1 nanometers, about 0.2 nanometers, about 0.3 nanometers, about 0.4 nanometers, about 0.5 nanometers, about 0.6 nanometers, about 0.7 nanometers, about 0.8 nanometers, about 0.9 nanometers, about 1 nanometer, about 2 nanometers, about 3 nanometers, about 4 nanometers, about 5 nanometers, about 6 nanometers, about 7 nanometers, about 8 nanometers, about 9 nanometers, or about 10 nanometers apart from each other along the longitudinal length of the channel. In some embodiments, one set of charges may be located at the cis inlet of the nanopore. In some embodiments, one set of charges may be located at the transform inlet of the nanopore. In some embodiments, one set of charges may be located at the cis inlet of the nanopore, and one set of charges may be located at the transform inlet of the nanopore.

[0140] In some embodiments, one or more solutions on either the cis or transform side of the fluid chamber may be configured to have a set pH. One or more solutions may have a usable pH of at least about 1, at least about 2, at least about 3, at least about 3.8, at least about 4, at least about 4.5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 10.5, at least about 11, at least about 12, at least about 13, or greater than about 13. One or more solutions may have a usable pH of up to about 13, up to about 12, up to about 11, up to about 10.5, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 4.5, up to about 4, up to about 3.8, up to about 3, up to about 2, up to about 1, or less than about 1.

[0141] One or more solutions may have a usable pH range of approximately 1 to approximately 13. One or more solutions may have usable pH ranges of approximately 1 to approximately 2, approximately 1 to approximately 3, approximately 1 to approximately 4, approximately 1 to approximately 6, approximately 1 to approximately 7, approximately 1 to approximately 8, approximately 1 to approximately 9, approximately 1 to approximately 10, approximately 1 to approximately 11, approximately 1 to approximately 12, approximately 1 to approximately 13, approximately 2 to approximately 3, approximately 2 to approximately 4, approximately 2 to approximately 6, approximately 2 to approximately 7, approximately 2 to approximately 8, approximately 2 to approximately 9, approximately 2 to approximately 10, approximately 2 to approximately 11, approximately 2 to approximately 12, approximately 2 to approximately 13, approximately 3 to approximately 4, approximately 3 to approximately 6, approximately 3 to approximately 7, approximately 3 to approximately 8, approximately 3 to approximately 9, approximately 3 to approximately 10, approximately 3 to approximately 11, approximately 3 to approximately 12, approximately 3 to approximately 13, approximately 4 to approximately 6, approximately 4 to approximately 7, approximately 4 to approximately 8, approximately 4 to approximately 9 It may have a pH of approximately 4-10, approximately 4-11, approximately 4-12, approximately 4-13, approximately 6-7, approximately 6-8, approximately 6-9, approximately 6-10, approximately 6-11, approximately 6-12, approximately 6-13, approximately 7-8, approximately 7-9, approximately 7-10, approximately 7-11, approximately 7-12, approximately 7-13, approximately 8-9, approximately 8-10, approximately 8-11, approximately 8-12, approximately 8-13, approximately 9-10, approximately 9-11, approximately 9-12, approximately 9-13, approximately 10-11, approximately 10-12, approximately 10-13, approximately 11-12, approximately 11-13, or approximately 12-13.

[0142] One or more solutions may have usable pH values ​​of approximately 1, 2, 3, 3.8, 4, 4.5, 6, 7, 8, 9, 10, 10.5, 11, 12, or 13.

[0143] In some embodiments, electroosmotic flow (also called electroosmotic force) acts across the membrane in a cis-to-trans direction or trans-to-cis direction. Electroosmotic flow can be a flow resulting from a net flow of ion migration layers along the surface, as induced by an applied potential. For example, a charged surface may form a static layer of ion migrations charged in opposite directions. Under an applied potential, charged ion migrations can be induced to move in the direction of a higher potential if negative, or in the direction of a lower potential if positive. The flow of charged ions can create a drag force on surrounding solvent (e.g., water) molecules, which can then result in a net flow that exerts a force on surrounding molecules, both charged and neutral. For example, in a negatively charged nanopore lumen, electroosmotic flow may result from a net flow of positive ions in a cis-to-trans direction (due, for example, a lower potential on the trans side), causing surrounding water to flow from cis to trans and exert a force on surrounding molecules. The amount of ion flow and the magnitude of the corresponding electroosmotic flow may be influenced by parameters including differences in ion concentration across the membrane, differences in potential, net charge of the nanopore lumen, geometry of the nanopore lumen, or a combination thereof. In some embodiments, the electroosmotic flow may be a flow arising from one or more constrictions present in the nanopore channel. In some embodiments, the electroosmotic flow may be a flow arising from a net flow of mobile ions along the surface, induced by an applied potential and one or more constrictions present in the nanopore channel.

[0144] In some embodiments, electroosmotic flow can be generated or modified by a difference between the solution on the cis side of the membrane and the solution on the transform side of the membrane. This difference may be a difference in the concentration of ions, electrolytes, or molecules containing osmolite.

[0145] In some embodiments, the difference between solutions may be due to salt asymmetry or ionic asymmetry, where one side of the membrane (e.g., the cis side) contains different concentrations of ions than the other side (e.g., the trans side). Ionic asymmetry can affect the ionic current across the membrane, as described by the Goldman-Hodgkin-Katz equation.

[0146]

number

[0147] A difference in molecular concentration between two sides of a membrane can modify the electroosmotic flux by providing a competing or auxiliary osmotic flux. A concentration difference across the membrane can create an osmotic gradient, and the solvent (e.g., water) can diffuse across the membrane in the direction of higher molecular concentration to minimize the concentration difference between the two sides of the membrane. The osmotic gradient can be oriented to drive the flow of water in the same direction as or different from the electroosmotic force. For example, if the ion concentration on the cis side is higher than on the transform side, the osmotic gradient can drive water from transform to cis, thus creating an osmotic gradient that competes with the electroosmotic force from cis to transform. The ion concentration can also assist the electroosmotic flow from cis to transform, even if it also provides an osmotic gradient.

[0148] In some embodiments, the electroosmotic force may act in the same direction as the electrophoretic force, or in the opposite direction to the electrophoretic force. In some embodiments, the electroosmotic force may be greater than the electrophoretic force. In some embodiments, the electroosmotic force may be less than the electrophoretic force.

[0149] In some embodiments, the cis-to-trans EOF may include a net ionic current flow from the cis side of the membrane to the transform side of the membrane. In some embodiments, the transform-to-cis EOF may include a net ionic current flow from the transform side of the membrane to the cis side of the membrane. In some cases, the nanopore system may include a total ionic current flow. In some cases, the net ionic current flow may include a flow of less than the total number of ions in the nanopore system. In some cases, the net ionic current flow may include a flow of less than the total number of ions in the nanopore system in a particular direction. In some cases, the particular direction may be from the cis side of the membrane to the transform side of the membrane. In some cases, the particular direction may be from the transform side of the membrane to the cis side of the membrane. In some cases, the total ionic current flow may include the total flow of all ions in the nanopore system. In some cases, the total ionic current flow may include the total flow of all ions in the nanopore system in a particular direction. In some cases, the particular direction may be from the cis side of the membrane to the transform side of the membrane. In some cases, the specific direction may be from the trans side of the membrane to the cis side of the membrane.

[0150] In some embodiments, the net ionic current flow may comprise about 0.001% to about 100% of the total ionic current flow. In some cases, the net ionic current flow may comprise about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 0.1% to about 1%, about 1% to about 10%, or about 10% to about 100% of the total ionic current flow. In some cases, the net ionic current flow may include at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 100% of the total ionic current flow. In some cases, the net ionic current flow is approximately 100% of the total ionic current flow, at most about 99.5%, at most about 99%, at most about 98%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most This may include approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, up to approximately 0.05%, up to approximately 0.01%, up to approximately 0.005%, up to approximately 0.001%, or less than 0.001%.In some cases, the net ionic current flow may consist of approximately 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 100% of the total ionic current flow.

[0151] In some embodiments, the rearrangement rate may be about 0.1 amino acids / second (aa / s) to about 1,000 aa / s. In some cases, the rearrangement rate may be at least about 0.1 aa / s, at least about 0.5 aa / s, at least about 1 aa / s, at least about 5 aa / s, at least about 10 aa / s, at least about 50 aa / s, at least about 100 aa / s, at least about 500 aa / s, at least about 1,000 aa / s, or exceed 1,000 aa / s. In some cases, the dislocation rate may be at least approximately 1,000 aa / s, at most approximately 500 aa / s, at most approximately 100 aa / s, at most approximately 50 aa / s, at most approximately 10 aa / s, at most approximately 5 aa / s, at most approximately 1 aa / s, at most approximately 0.5 aa / s, at most approximately 0.1 aa / s, or less than 0.1 aa / s. In some cases, the dislocation rate may be approximately 0.1 aa / s, at most approximately 0.5 aa / s, at most approximately 1 aa / s, at most approximately 5 aa / s, at most approximately 10 aa / s, at most approximately 500 aa / s, or at most approximately 1,000 aa / s.

[0152] In some embodiments, the cis-to-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow of about 0.0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 0.95, or about 0.99, and is also called the relative net current flow from cis to transformer. In some embodiments, the cis-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow, which is less than about 0.0, less than about -0.1, less than about -0.2, less than about -0.3, less than about -0.4, less than about -0.5, less than about -0.6, less than about -0.7, less than about -0.8, less than about -0.9, less than about -0.95, or less than about -0.99, and is also called the relative net current flow from cis to transformer.

[0153] In some embodiments, the cis-to-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow, which is at least about -0.99, at least about -0.95, at least about -0.9, at least about -0.8, at least about -0.7, at least about -0.6, at least about -0.5, at least about -0.4, at least about -0.3, at least about -0.2, at least about -0.1, at least about 0.0, at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 0.95, about 0.99, or about 0.99, and is also called the relative net current flow from cis to transformer. In some embodiments, the cis-to-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow, which is approximately -0.99, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, -0.1, -0.0, -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -0.95, -0.99, or less than approximately -0.99, and is also called the relative net current flow from cis to transformer. In some embodiments, the cis-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow, ranging from approximately -0.99 to approximately 0.99, and is also called the relative net current flow from cis to transformer.In some configurations, the EOF from cis to transform is approximately -0.99 to -0.9, approximately -0.99 to -0.8, approximately -0.99 to -0.6, approximately -0.99 to -0.4, approximately -0.99 to -0.2, approximately -0.99 to 0, approximately -0.99 to 0.2, approximately -0.99 to 0.4, approximately -0.99 to 0.6, approximately -0.99 to 0.8, approximately -0.99 to 0.99, approximately -0.99 to -0.8, approximately -0.9 to -0.6, approximately -0.9 to -0.4, approximately -0.9 Approximately -0.2, approximately -0.9 to approximately 0, approximately -0.9 to approximately 0.2, approximately -0.9 to approximately 0.4, approximately -0.9 to approximately 0.6, approximately -0.9 to approximately 0.8, approximately -0.9 to approximately 0.99, approximately -0.8 to approximately -0.6, approximately -0.8 to approximately -0.4, approximately -0.8 to approximately -0.2, approximately -0.8 to approximately 0, approximately -0.8 to approximately 0.2, approximately -0.8 to approximately 0.4, approximately -0.8 to approximately 0.6, approximately -0.8 to approximately 0.8, approximately -0.8 to approximately 0.99, approximately -0.6 to approximately -0.4, approximately -0.6 to approximately -0.2, approximately -0.6 to approximately 0, approximately -0.6 to approximately 0.2, approximately -0.6 to approximately 0.4, approximately -0.6 to approximately 0.6, approximately -0.6 to approximately 0.8, approximately -0.6 to approximately 0.99, approximately -0.4 to approximately -0.2, approximately -0.4 to approximately 0, approximately -0.4 to approximately 0.2, approximately -0.4 to approximately 0.4, approximately -0.4 to approximately 0.6, approximately -0.4 to approximately 0.8, approximately -0.4 to approximately 0.99, approximately -0.2 to approximately 0, approximately -0.2 to approximately 0.2, approximately -0.2 to approximately 0.4, approximately -0.2 to approximately 0.6, approximately -0.2 to approximately 0.8, approximately -0.2 to approximately 0.99, approximately 0 to approximately 0.2, approximately These arise from net ionic current flows from cis to transformer that exceed the total ionic current flow, ranging from approximately 0 to 0.4, approximately 0 to 0.6, approximately 0 to 0.8, approximately 0 to 0.99, approximately 0.2 to 0.4, approximately 0.2 to 0.6, approximately 0.2 to 0.8, approximately 0.2 to 0.99, approximately 0.4 to 0.6, approximately 0.4 to 0.8, approximately 0.4 to 0.99, approximately 0.6 to 0.8, approximately 0.6 to 0.99, or approximately 0.8 to 0.99, and are also called relative net current flows from cis to transformer.

[0154] In some embodiments, the cis-transformer EOF is approximately -0.99, approximately -0.95, approximately -0.9, approximately -0.8, approximately -0.7, approximately -0.6, approximately -0.5, approximately -0.4, approximately -0.3, approximately -0.2, approximately -0.1, approximately 0.0, approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 0.95, or approximately 0.99, resulting from the net ionic current flow from cis to transformer exceeding the total ionic current flow, and is also called the relative net current flow from cis to transformer.

[0155] In some embodiments, the absolute relative net electroosmotic current (I) with respect to the applied voltage relV ) may be at least about 0.01 pA / mV, at least about 0.02 pA / mV, at least about 0.03 pA / mV, at least about 0.04 pA / mV, at least about 0.05 pA / mV, at least about 0.06 pA / mV, at least about 0.07 pA / mV, at least about 0.08 pA / mV, at least about 0.09 pA / mV, at least about 0.10 pA / mV, at least about 0.15 pA / mV, at least about 0.2 pA / mV, at least about 0.3 pA / mV, at least about 0.4 pA / mV, at least about 0.5 pA / mV, at least about 0.6 pA / mV, at least about 0.7 pA / mV, at least about 0.8 pA / mV, at least about 0.9 pA / mV, at least about 1 pA / mV, or greater than about 1 pA / mV. In some embodiments, the absolute relative net electroosmotic current (I) with respect to the applied voltage relV ) may range from approximately 1 pA / mV to approximately 0.9, approximately 0.8, approximately 0.7, approximately 0.6, approximately 0.5, approximately 0.4, approximately 0.3, approximately 0.2, approximately 0.15, approximately 0.10, approximately 0.09, approximately 0.08, approximately 0.07, approximately 0.06, approximately 0.05, approximately 0.04, approximately 0.03, approximately 0.02, approximately 0.01, or less than approximately 0.1 pA / mV.

[0156] In some embodiments, the absolute relative net electroosmotic current (I) with respect to the applied voltage relVThe osmotic current (I) can be approximately 0.01 pA / mV to approximately 1 pA / mV. In some embodiments, the net electroosmotic current (I) is the absolute relative value of the applied voltage. relV) is approximately 0.01 pA / mV to approximately 0.02 pA / mV, approximately 0.01 pA / mV to approximately 0.04 pA / mV, approximately 0.01 pA / mV to approximately 0.06 pA / mV, approximately 0.01 pA / mV to approximately 0.08 pA / mV, approximately 0.01 pA / mV to approximately 0.1 pA / mV, approximately 0.01 pA / mV to approximately 0.15 pA / mV, approximately 0.01 pA / mV to approximately 0.2 pA / mV, approximately 0.01 pA / mV to approximately 0.4 pA / mV, approximately 0.01 pA / mV to approximately 0.6 pA / mV, approximately 0.01 pA / mV to approximately 0.8 pA / mV, approximately 0.01 pA / mV to approximately 1 pA / mV, approximately 0.02 pA / mV to approximately 0.04 pA / mV, approximately 0.02 pA / mV to approximately 0.06 pA / mV, approximately 0.02 pA / mV to approximately 0.08 pA / mV, approximately 0.02 pA / mV to approximately 0.1 pA / mV, approximately 0.02 pA / mV to approximately 0.15 pA / mV, approximately 0.02 pA / mV to approximately 0.2 pA / mV, approximately 0.02 p A / mV ~ approximately 0.4 pA / mV, approximately 0.02 pA / mV ~ approximately 0.6 pA / mV, approximately 0.02 pA / mV ~ approximately 0.8 pA / mV, approximately 0.02 pA / mV ~ approximately 1 pA / mV, approximately 0.04 pA / mV ~ approximately 0.06 pA / mV, approximately 0.04 pA / mV ~ approximately 0.08 pA / mV, approximately 0.04 pA / mV ~ approximately 0.1 pA / mV, approximately 0.04 pA / mV ~ approximately 0.15 pA / mV, approximately 0.04 pA / mV ~ approximately 0.2 pA / mV, approximately 0.04 pA / mV ~ approximately 0.4 pA / mV, approximately 0.04 pA / mV ~ approximately 0.6 pA / mV, approximately 0.04 pA / mV ~ approximately 0.8 pA / mV, approximately 0 0.04 pA / mV to approximately 1 pA / mV, approximately 0.06 pA / mV to approximately 0.08 pA / mV, approximately 0.06 pA / mV to approximately 0.1 pA / mV, approximately 0.06 pA / mV to approximately 0.15 pA / mV, approximately 0.06 pA / mV to approximately 0.2 pA / mV, approximately 0.06 pA / mV to approximately 0.4 pA / mV, approximately 0.06pA / mV to approx. 0.6pA / mV, approx. 0.06pA / mV to approx. 0.8pA / mV, approx. 0.06pA / mV to approx. 1pA / mV, approx. 0 .08pA / mV~Approx. 0.1pA / mV, Approx. 0.08pA / mV~Approx. 0.15pA / mV, Approx. 0.08pA / mV~Approx. 0.2pA / mV, Approx. 0.08pA / mV to approx. 0.4pA / mV, approx. 0.08pA / mV to approx. 0.6pA / mV, approx. 0.08pA / mV to approx. 0.8pA / mV, Approximately 0.08pA / mV to approximately 1pA / mV, approximately 0.1pA / mV to approximately 0.15pA / mV, approximately 0.1pA / mV to approximately 0.2pA / mV, approximately 0.1pA / mV to approx. 0.4pA / mV, approx. 0.1pA / mV to approx. 0.6pA / mV, approx. 0.1pA / mV to approx. 0.8pA / mV, approx. 0.1pA / mV to approx. 1pA / mV, approx. 0.15pA / mV to approx. 0.2pA / m V, about 0.15pA / mV to about 0.4pA / mV, about 0.15pA / mV to about 0.6pA / mV, about 0.15pA / mV to about 0.8pA / mV, about 0.15pA / mV to about 1pA / mV, about 0.2pA / mV to It could be approximately 0.4 pA / mV, approximately 0.2 pA / mV to approximately 0.6 pA / mV, approximately 0.2 pA / mV to approximately 0.8 pA / mV, approximately 0.2 pA / mV to approximately 1 pA / mV, approximately 0.4 pA / mV to approximately 0.6 pA / mV, approximately 0.4 pA / mV to approximately 0.8 pA / mV, approximately 0.4 pA / mV to approximately 1 pA / mV, approximately 0.6 pA / mV to approximately 0.8 pA / mV, or approximately 0.8 pA / mV to approximately 1 pA / mV.

[0157] In some embodiments, the absolute relative net electroosmotic current (I) with respect to the applied voltage relV ) can be approximately 0.01 pA / mV, approximately 0.02 pA / mV, approximately 0.03 pA / mV, approximately 0.04 pA / mV, approximately 0.05 pA / mV, approximately 0.06 pA / mV, approximately 0.07 pA / mV, approximately 0.08 pA / mV, approximately 0.09 pA / mV, approximately 0.10 pA / mV, approximately 0.15 pA / mV, approximately 0.2 pA / mV, approximately 0.3 pA / mV, approximately 0.4 pA / mV, approximately 0.5 pA / mV, approximately 0.6 pA / mV, approximately 0.7 pA / mV, approximately 0.8 pA / mV, approximately 0.9 pA / mV, or approximately 1 pA / mV. In some cases, the net electroosmotic current relative to the applied voltage may include a value calculated by dividing the net electroosmotic flow by the applied voltage. In some cases, the net electroosmotic flow may include the total flow of a subset of ions or salts within the nanopore system.

[0158] In some embodiments, the pores may contain relative ion selectivity P(+) / P(-) of at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or more than about 5, under the difference of the applied voltage across the membrane. In some embodiments, the pores may contain relative ion selectivity P(+) / P(-) of up to about 5, at least about 4, at least about 3, at least about 2, at least about 1, at least about 0.9, at least about 0.8, at least about 0.7, at least about 0.6, at least about 0.5, at least about 0.4, at least about 0.3, at least about 0.2, at least about 0.1, or less than about 0.1, under the difference of the applied voltage across the membrane.

[0159] In some embodiments, the pores may have relative ion selectivity P(+) / P(-) of about 0.1 to about 5 under the applied voltage difference across the membrane. In some embodiments, the pores may have relative ion selectivity P(+) / P(-) of about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 to about 0.5, about 0.1 to about 1, about 0.1 to about 1.5, about 0.1 to about 2, about 0.1 to about 2.5, about 0.1 to about 3, about 0.1 to about 4, about 0.1 to about 5, about 0.2 to about 0.3, and about 0.2 to about 0. 4, approximately 0.2 to approximately 0.5, approximately 0.2 to approximately 1, approximately 0.2 to approximately 1.5, approximately 0.2 to approximately 2, approximately 0.2 to approximately 2.5, approximately 0.2 to approximately 3, approximately 0.2 to approximately 4, approximately 0.2 to approximately 5, approximately 0.3 to approximately 0.4, approximately 0.3 to approximately 0.5, approximately 0.3 to approximately 1, approximately 0.3 to approximately 1.5, approximately 0.3 to approximately 2, approximately 0.3 to approximately 2.5, approximately 0.3 to approximately 3, approximately 0.3 to approximately 4, approximately 0.3 to approximately 5, Approximately 0.4 to 0.5, approximately 0.4 to 1, approximately 0.4 to 1.5, approximately 0.4 to 2, approximately 0.4 to 2.5, approximately 0.4 to 3, approximately 0.4 to 4, approximately 0.4 to 5, approximately 0.5 to 1, approximately 0.5 to 1.5, approximately 0.5 to 2, approximately 0.5 to 2.5, approximately 0.5 to 3, approximately 0.5 to 4, approximately 0.5 to 5, approximately 1 to 1.5, approximately 1 to 2, approximately 1 to 2.5 It may include relative ion selectivity P(+) / P(-) of approximately 1 to 3, approximately 1 to 4, approximately 1 to 5, approximately 1.5 to 2, approximately 1.5 to 2.5, approximately 1.5 to 3, approximately 1.5 to 4, approximately 1.5 to 5, approximately 2 to 2.5, approximately 2 to 3, approximately 2 to 4, approximately 2 to 5, approximately 2.5 to 3, approximately 2.5 to 4, approximately 2.5 to 5, approximately 3 to 4, approximately 3 to 5, or approximately 4 to 5.

[0160] In some embodiments, the pores may have relative ion selectivity P(+) / P(-) of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5 under the applied voltage difference across the membrane.

[0161] In some embodiments, the cis and transformer solutions in a fluid chamber are configured to generate electroosmosis. Electroosmosis can be generated due to the difference in solute concentration between the cis and transformer solutions. The solute may be one or more ions or one or more osmolites. In some cases, one or more ions are found in chlorides, carbonic acid, chlorous acid, chloric acid, phosphoric acid, bicarbonate, bromide, ammonium sulfate, ammonium, sulfuric acid, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, oxide, glutamic acid, acetic acid, format, acetic acid, butyric acid, benzoic acid, carboxylic acid, alkoxide, penolate, oxalic acid, amlonate, tartaric acid, malic acid, citric acid, gluconic acid, maleic acid, sorbic acid, and stearic acid. It may include lactage, glyceric acid, uric acid, diazonium salt, iminium salt, phosphinic acid, organophosphate, mesylic acid, betigard salt, picolinic acid, cocaine salt, morphine salt, sodium-free glutamate, trolamine salicylic acid, triphenylmethylhexafluorophosphate, choline chloride, copper ibuprofenate, homatropin methyl bromide, beeswax, tetrapropylammonium perruthenate, p-toluenesulfonate collidinium, pyridinium chloride, tetrasodium EDTA, lithium diisopropylamide, lithium bis(trimethylsil)amide, potassium trispirazolylborate, redox salts, ferrocyanides, felicyanides, or any combination thereof. In some embodiments, one or more osmolites may be one or more types of salts.In some cases, one or more types of salts may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide chloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc hydroxide chloride monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof. These ions or osmolites can flow across the membrane through nanopores. These ions may be high-mobility or low-mobility ions.

[0162] In some embodiments, EOF can be generated by an asymmetric salt distribution between the cis and trans sides of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may differ from the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be higher than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be lower than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the trans side of the membrane may be higher than the concentration of one or more salts on the cis side of the membrane. In some cases, the concentration of one or more salts on the trans side of the membrane may be lower than the concentration of one or more salts on the cis side of the membrane.

[0163] In some cases, the concentration of one or more salts on the cis side of the membrane can be about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more salts on the cis side of the membrane can be about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more salts on the cis side of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, and less At least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed 1,000 nM.In some cases, the concentration of one or more salts on the cis side of the membrane can range from approximately 1,000 nM, 950 nM, 900 nM, 850 nM, 800 nM, 750 nM, 700 nM, 650 nM, 600 nM, 550 nM, 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, and more. The maximum value is approximately 150 nM, the maximum is approximately 100 nM, the maximum is approximately 95 nM, the maximum is approximately 90 nM, the maximum is approximately 85 nM, the maximum is approximately 80 nM, the maximum is approximately 75 nM, the maximum is approximately 70 nM, the maximum is approximately 65 nM, the maximum is approximately 60 nM, the maximum is approximately 55 nM, the maximum is approximately 45 nM, the maximum is approximately 40 nM, the maximum is approximately 35 nM, the maximum is approximately 30 nM, the maximum is approximately 25 nM, the maximum is approximately 20 nM, the maximum is approximately 15 nM, the maximum is approximately 10 nM, the maximum is approximately 5 nM, the maximum is approximately 1 nM, or it may be less than 1 nM. In some cases, the concentration of one or more salts on the cis side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM. M may be approximately 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, or 1,000 nM.

[0164] In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte may be at least about 0.01 M, at least about 0.05 M, at least about 0.10 M, at least about 0.20 M, at least about 0.30 M, at least about 0.40 M, at least about 0.50 M, at least about 0.60 M, at least about 0.70 M, at least about 0.80 M, at least about 0.90 M, at least about 1.00 M, at least about 1.10 M, at least about 1.25 M, at least about 1.50 M, at least about 1.75 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 3.5 M, at least about 4 M, at least about 4.5 M, at least about 5 M, or greater than about 5 M. In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte may be up to about 5 M, up to about 4.5 M, up to about 4 M, up to about 3.5 M, up to about 3 M, up to about 2.5 M, up to about 2 M, up to about 1.75 M, up to about 1.50 M, up to about 1.25 M, up to about 1 M, up to about 0.90 M, up to about 0.80 M, up to about 0.70 M, up to about 0.60 M, up to about 0.50 M, up to about 0.40 M, up to about 0.30 M, up to about 0.20 M, up to about 0.10 M, up to about 0.05 M, up to about 0.01 M, or less than about 0.01 M.

[0165] In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte can be about 0.01 M to about 5 M. In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte can be about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to about 0.5 M, Approximately 0.1M to approximately 1M, approximately 0.1M to approximately 1.5M, approximately 0.1M to approximately 2M, approximately 0.1M to approximately 2.5M, approximately 0.1M to approximately 3M, approximately 0.1M to approximately 3.5M, approximately 0.1M to approximately 4M, approximately 0.1M to approximately 4.5M, approximately 0.1 M ~ about 5M, about 0.5M - about 1M, about 0.5M - about 1.5M, about 0.5M - about 2M, about 0.5M - about 2.5M, about 0.5M - about 3M, about 0.5M - about 3.5M, about 0.5M - about 4M, about 0.5M - about 4.5M , about 0.5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2M , about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3.5 M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, It can be about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M, or about 4.5M to about 5M.

[0166] In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte may be about 0.01 M, about 0.05 M, about 0.10 M, about 0.20 M, about 0.30 M, about 0.40 M, about 0.50 M, about 0.60 M, about 0.70 M, about 0.80 M, about 0.90 M, about 1.00 M, about 1.10 M, about 1.25 M, about 1.50 M, about 1.75 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M.

[0167] In some cases, the concentration of one or more salts on the transform side of the membrane can be about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more salts on the transform side of the membrane can be about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more salts on the transform side of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, At least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed about 1,000 nM.In some cases, the concentration of one or more salts on the trans side of the membrane is up to approximately 1,000 nM, up to approximately 950 nM, up to approximately 900 nM, up to approximately 850 nM, up to approximately 800 nM, up to approximately 750 nM, up to approximately 700 nM, up to approximately 650 nM, up to approximately 600 nM, up to approximately 550 nM, up to approximately 500 nM, up to approximately 450 nM, up to approximately 400 nM, up to approximately 350 nM, up to approximately 300 nM, up to approximately 250 nM, up to approximately 200 nM. The maximum nM is approximately 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less than 1 nM. In some cases, the concentration of one or more salts on the trans side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM. It may be nM, approximately 100nM, approximately 150nM, approximately 200nM, approximately 250nM, approximately 300nM, approximately 350nM, approximately 400nM, approximately 450nM, approximately 500nM, approximately 550nM, approximately 600nM, approximately 650nM, approximately 700nM, approximately 750nM, approximately 800nM, approximately 850nM, approximately 900nM, approximately 950nM, or approximately 1,000nM.

[0168] In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be at least about 0.01 M, at least about 0.05 M, at least about 0.10 M, at least about 0.20 M, at least about 0.30 M, at least about 0.40 M, at least about 0.50 M, at least about 0.60 M, at least about 0.70 M, at least about 0.80 M, at least about 0.90 M, at least about 1.00 M, at least about 1.10 M, at least about 1.25 M, at least about 1.50 M, at least about 1.75 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 3.5 M, at least about 4 M, at least about 4.5 M, at least about 5 M, or greater than about 5 M. In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be up to about 5 M, up to about 4.5 M, up to about 4 M, up to about 3.5 M, up to about 3 M, up to about 2.5 M, up to about 2 M, up to about 1.75 M, up to about 1.50 M, up to about 1.25 M, up to about 1 M, up to about 0.90 M, up to about 0.80 M, up to about 0.70 M, up to about 0.60 M, up to about 0.50 M, up to about 0.40 M, up to about 0.30 M, up to about 0.20 M, up to about 0.10 M, up to about 0.05 M, up to about 0.01 M, or less than about 0.01 M.

[0169] In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be about 0.01 M to about 5 M. In some embodiments, the concentration of the cis-side salt, ions, osmolite, or electrolyte may be about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to about 0.5 M. Approximately 0.1M to approximately 1M, approximately 0.1M to approximately 1.5M, approximately 0.1M to approximately 2M, approximately 0.1M to approximately 2.5M, approximately 0.1M to approximately 3M, approximately 0.1M to approximately 3.5M, approximately 0.1M to approximately 4M, approximately 0.1M to approximately 4.5M, approximately 0.1 M ~ about 5M, about 0.5M - about 1M, about 0.5M - about 1.5M, about 0.5M - about 2M, about 0.5M - about 2.5M, about 0.5M - about 3M, about 0.5M - about 3.5M, about 0.5M - about 4M, about 0.5M - about 4.5M , about 0.5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2M , about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3.5 M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, It can be about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M, or about 4.5M to about 5M.

[0170] In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be about 0.01 M, about 0.05 M, about 0.10 M, about 0.20 M, about 0.30 M, about 0.40 M, about 0.50 M, about 0.60 M, about 0.70 M, about 0.80 M, about 0.90 M, about 1.00 M, about 1.10 M, about 1.25 M, about 1.50 M, about 1.75 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M.

[0171] In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis side and the trans side may be at least about 0.01 M, at least about 0.05, at least about 0.10, at least about 0.20, at least about 0.30, at least about 0.40, at least about 0.50, at least about 0.60, at least about 0.70, at least about 0.80, at least about 0.90, at least about 1.00, at least about 1.10, at least about 1.25, at least about 1.50, at least about 1.75, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5 M, or greater than about 5 M. In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis and trans sides may be up to about 5 M, up to about 4.5 M, up to about 4 M, up to about 3.5 M, up to about 3 M, up to about 2.5 M, up to about 2 M, up to about 1.75 M, up to about 1.50 M, up to about 1.25 M, up to about 1 M, up to about 0.90 M, up to about 0.80 M, up to about 0.70 M, up to about 0.60 M, up to about 0.50 M, up to about 0.40 M, up to about 0.30 M, up to about 0.20 M, up to about 0.10 M, up to about 0.05 M, up to about 0.01 M, or less than about 0.01 M.

[0172] In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis and trans sides can be about 0.01 M to about 5 M. In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis and trans sides can be about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, and about 0.1 M to about 0. 5M, about 0.1M to about 1M, about 0.1M to about 1.5M, about 0.1M to about 2M, about 0.1M to about 2.5M, about 0.1M to about 3M, about 0.1M to about 3.5M, about 0.1M to about 4M, about 0.1M to about 4.5M, about 0 .1M to about 5M, about 0.5M to about 1M, about 0.5M to about 1.5M, about 0.5M to about 2M, about 0.5M to about 2.5M, about 0.5M to about 3M, about 0.5M to about 3.5M, about 0.5M to about 4M, about 0.5M to about 4. 5M, about 0.5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2 M, about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3. 5M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, It can be about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M, or about 4.5M to about 5M.

[0173] In some embodiments, the difference in concentration of salt, ion, or electrolyte between the cis side and the trans side may be about 0.01 M, about 0.05 M, about 0.10 M, about 0.20 M, about 0.30 M, about 0.40 M, about 0.50 M, about 0.60 M, about 0.70 M, about 0.80 M, about 0.90 M, about 1.00 M, about 1.10 M, about 1.25 M, about 1.50 M, about 1.75 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M.

[0174] In some embodiments, one or more salts may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide chloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc hydroxide chloride monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, potassium glutamate, sodium ferricyanide, sodium ferrocyanide, potassium ferricyanide, potassium ferrocyanide, or any combination thereof.

[0175] In some embodiments, one or more salts on the cis side of the membrane may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide chloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof. In some embodiments, one or more salts on the trans side of the membrane may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide chloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof.

[0176] In some embodiments, one or more salts on the cis side of the membrane may be the same as one or more salts on the transform side of the membrane. In some cases, one or more salts on the cis side of the membrane may be of the same type as the salts on the transform side of the membrane. In some embodiments, one or more salts on the cis side of the membrane may be different from one or more salts on the transform side of the membrane. In some cases, one or more types of salts on the cis side of the membrane may be different from one or more types of salts on the transform side of the membrane.

[0177] In some embodiments, one or more salts may comprise about one to about ten types of salts. In some cases, one or more salts may comprise at least about one type of salt, at least about two types of salts, at least about three types of salts, at least about four types of salts, at least about five types of salts, at least about six types of salts, at least about seven types of salts, at least about eight types of salts, at least about nine types of salts, at least about ten types of salts, or more than ten types of salts. In some cases, one or more salts may comprise up to about ten types of salts, up to about nine types of salts, up to about eight types of salts, up to about seven types of salts, up to about six types of salts, up to about five types of salts, up to about four types of salts, up to about three types of salts, up to about two types of salts, up to about one type of salt, or less than one type of salt. In some cases, one or more salts may include one type of salt, about two types of salt, about three types of salt, about four types of salt, about five types of salt, about six types of salt, about seven types of salt, about eight types of salt, about nine types of salt, or about ten types of salt.

[0178] In some embodiments, one or more salts on the cis side of the membrane may be the same type of salt as one or more salts on the trans side of the membrane. In some cases, the same type of salt present on the cis and trans sides of the membrane may be present at the same concentration. In some cases, the same type of salt present on the cis and trans sides of the membrane may be present at different concentrations.

[0179] In some embodiments, one or more salts on the cis side of the membrane may be of a different type than one or more salts on the trans side of the membrane. In some embodiments, the different types of salts present on the cis and trans sides of the membrane may be present at the same concentration. In some cases, the different types of salts present on the cis and trans sides of the membrane may be present at different concentrations.

[0180] In some embodiments, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 500% higher than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% Approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% ~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~ It may be approximately 360%, approximately 360% to approximately 370%, approximately 370% to approximately 380%, approximately 380% to approximately 390%, approximately 390% to approximately 400%, approximately 400% to approximately 410%, approximately 410% to approximately 420%, approximately 420% to approximately 430%, approximately 430% to approximately 440%, approximately 440% to approximately 450%, approximately 450% to approximately 460%, approximately 460% to approximately 470%, approximately 470% to approximately 480%, approximately 480% to approximately 490%, or approximately 490% to approximately 500% higher.

[0181] In some cases, the concentration of one or more salts on the cis side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even higher than 500%.

[0182] In some cases, the concentration of one or more salts on the cis side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the trans side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be less than 0.1% higher.

[0183] In some cases, the concentration of one or more salts on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% of the concentration of one or more salts on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

[0184] In some embodiments, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% Approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% ~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~ It may be approximately 360%, approximately 360% to approximately 370%, approximately 370% to approximately 380%, approximately 380% to approximately 390%, approximately 390% to approximately 400%, approximately 400% to approximately 410%, approximately 410% to approximately 420%, approximately 420% to approximately 430%, approximately 430% to approximately 440%, approximately 440% to approximately 450%, approximately 450% to approximately 460%, approximately 460% to approximately 470%, approximately 470% to approximately 480%, approximately 480% to approximately 490%, or even 490% to approximately 500% lower.

[0185] In some cases, the concentration of one or more salts on the cis side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even lower than 500%.

[0186] In some cases, the concentration of one or more salts on the cis side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the trans side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be lower than 0.1%.

[0187] In some cases, the concentration of one or more salts on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% of the concentration of one or more salts on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0188] In some embodiments, the concentration of one or more salts on the transform side of the membrane may be about 0.1% to about 500% higher than the concentration of one or more salts on the cis side of the membrane. In some cases, the concentration of one or more salts on the transform side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% Approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% ~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~ It may be approximately 360%, approximately 360% to approximately 370%, approximately 370% to approximately 380%, approximately 380% to approximately 390%, approximately 390% to approximately 400%, approximately 400% to approximately 410%, approximately 410% to approximately 420%, approximately 420% to approximately 430%, approximately 430% to approximately 440%, approximately 440% to approximately 450%, approximately 450% to approximately 460%, approximately 460% to approximately 470%, approximately 470% to approximately 480%, approximately 480% to approximately 490%, or approximately 490% to approximately 500% higher.

[0189] In some cases, the concentration of one or more salts on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even higher than 500%.

[0190] In some cases, the concentration of one or more salts on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the cis side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be less than 0.1% higher.

[0191] In some cases, the concentration of one or more salts on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more salts on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

[0192] In some embodiments, the concentration of one or more salts on the transform side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more salts on the cis side of the membrane. In some cases, the concentration of one or more salts on the transform side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% Approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% ~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~ It may be approximately 360%, approximately 360% to approximately 370%, approximately 370% to approximately 380%, approximately 380% to approximately 390%, approximately 390% to approximately 400%, approximately 400% to approximately 410%, approximately 410% to approximately 420%, approximately 420% to approximately 430%, approximately 430% to approximately 440%, approximately 440% to approximately 450%, approximately 450% to approximately 460%, approximately 460% to approximately 470%, approximately 470% to approximately 480%, approximately 480% to approximately 490%, or even 490% to approximately 500% lower.

[0193] In some cases, the concentration of one or more salts on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even lower than 500%.

[0194] In some cases, the concentration of one or more salts on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the cis side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be lower than 0.1%.

[0195] In some cases, the concentration of one or more salts on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more salts on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0196] In some embodiments, EOF can be generated by an asymmetric salt distribution. An asymmetric salt distribution may occur when the concentration of one or more salts on the cis side of the membrane is greater than or less than the concentration of one or more salts on the trans side of the membrane.

[0197] Alternatively, EOF can be generated by a symmetric salt distribution between the cis and trans sides of the membrane. A symmetric salt distribution may occur when the concentration of one or more salts on the cis side of the membrane is the same as the concentration of one or more salts on the trans side of the membrane. In some embodiments, the concentration of one or more salts on the cis side of the membrane may be the same as the concentration of one or more salts on the trans side of the membrane.

[0198] In some embodiments, EOF may be generated by an asymmetric ion distribution between the cis and trans sides of the membrane. An asymmetric ion distribution may occur when the concentration of one or more ions on the cis side of the membrane is greater than or less than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be greater than or less than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be higher than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be lower than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the trans side of the membrane may be higher than the concentration of one or more ions on the cis side of the membrane. In some cases, the concentration of one or more ions on the trans side of the membrane may be lower than the concentration of one or more ions on the cis side of the membrane.

[0199] In some cases, the concentration of one or more ions on the cis side of the membrane can be about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more ions on the cis side of the membrane can be about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more ions on the cis side of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, At least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed 1,000 nM.In some cases, the concentration of one or more ions on the cis side of the membrane is up to approximately 1,000 nM, up to approximately 950 nM, up to approximately 900 nM, up to approximately 850 nM, up to approximately 800 nM, up to approximately 750 nM, up to approximately 700 nM, up to approximately 650 nM, up to approximately 600 nM, up to approximately 550 nM, up to approximately 500 nM, up to approximately 450 nM, up to approximately 400 nM, up to approximately 350 nM, up to approximately 300 nM, up to approximately 250 nM, and up to approximately 200 nM. The maximum nM is approximately 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less than 1 nM. In some cases, the salt concentration on the cis side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, and approximately It may be 100 nM, approximately 150 nM, approximately 200 nM, approximately 250 nM, approximately 300 nM, approximately 350 nM, approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, approximately 800 nM, approximately 850 nM, approximately 900 nM, approximately 950 nM, or approximately 1,000 nM.

[0200] In some cases, the concentration of one or more ions on the transform side of the membrane can range from about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more ions on the transform side of the membrane can range from about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more ions on the transform side of the membrane can range from at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, and at least about 95 nM. , at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed 1,000 nM.In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 1,000 nM, 950 nM, 900 nM, 850 nM, 800 nM, 750 nM, 700 nM, 650 nM, 600 nM, 550 nM, 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, and 200 nM. The maximum values ​​may be approximately 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less than 1 nM. In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, and 90 nM. It may be 5 nM, approximately 100 nM, approximately 150 nM, approximately 200 nM, approximately 250 nM, approximately 300 nM, approximately 350 nM, approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, approximately 800 nM, approximately 850 nM, approximately 900 nM, approximately 950 nM, or approximately 1,000 nM.

[0201] In some embodiments, one or more ions may include chlorides, carbonic acid, chlorous acid, chloric acid, phosphoric acid, bicarbonate, bromide, ammonium sulfate, ammonium, sulfuric acid, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, oxide, or any combination thereof.

[0202] In some embodiments, one or more ions on the cis side of the membrane may include chloride, carbonic acid, chlorous acid, chloric acid, phosphoric acid, bicarbonate, bromide, ammonium sulfate, ammonium, sulfuric acid, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, oxide, or any combination thereof.

[0203] In some embodiments, one or more ions on the transformer side of the membrane may include chloride, carbonic acid, chlorous acid, chloric acid, phosphoric acid, bicarbonate, bromide, ammonium sulfate, ammonium, sulfuric acid, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, oxide, or any combination thereof.

[0204] In some embodiments, one or more ions on the cis side of the membrane may be of the same type as one or more ions on the trans side of the membrane. In some embodiments, one or more ions on the cis side of the membrane may be of a different type than one or more ions on the trans side of the membrane.

[0205] In some embodiments, one or more ions may contain about 1 to about 10 ions. In some cases, one or more ions may contain at least about 1 ion, at least about 2 ions, at least about 3 ions, at least about 4 ions, at least about 5 ions, at least about 6 ions, at least about 7 ions, at least about 8 ions, at least about 9 ions, at least about 10 ions, or more than 10 ions. In some cases, one or more ions may contain up to about 10 ions, up to about 9 ions, up to about 8 ions, up to about 7 ions, up to about 6 ions, up to about 5 ions, up to about 4 ions, up to about 3 ions, up to about 2 ions, up to about 1 ion, or less than 1 ion. In some cases, one or more ions may contain about 1 ion, about 2 ions, about 3 ions, about 4 ions, about 5 ions, about 6 ions, about 7 ions, about 8 ions, about 9 ions, or about 10 ions.

[0206] In some embodiments, one or more ions on the cis side of the membrane may be present at the same concentration as one or more ions on the trans side of the membrane. In some cases, one or more ions on the cis side of the membrane may be present at a different concentration than one or more ions on the trans side of the membrane.

[0207] In some embodiments, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 500% higher than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 7 0%~approx. 75%, approx. 75%~approx. 80%, approx. 80%~approx. 85%, approx. 85%~approx. 90%, approx. 90%~approx. 95%, approx. 95%~approx. 100%, approx. 100%~approx. 110%, approx. 110%~approx. 120%, approx. 120%~approx. 130%, approx. 130%~approx. 140%, approx. 140%~approx. 150%, approx. 150%~approx. 160%, approx. 160%~approx. 170%, approx. 170%~approx. 180%, approx. 180%~approx. 190%, approx. 190%~approx. 200%, approx. 20 0% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% It may be approximately 360%, 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or even 490% to 500% higher.

[0208] In some cases, the concentration of one or more ions on the cis side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, and at least about 180%. , at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or may be higher than 500%.

[0209] In some cases, the concentration of one or more ions on the cis side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to At approximately 390%, at a maximum of approximately 380%, at a maximum of approximately 370%, at a maximum of approximately 360%, at a maximum of approximately 350%, at a maximum of approximately 340%, at a maximum of approximately 330%, at a maximum of approximately 320%, at a maximum of approximately 310%, at a maximum of approximately 300%, at a maximum of approximately 290%, at a maximum of approximately 280%, at a maximum of approximately 270%, at a maximum of approximately 260%, at a maximum of approximately 250%, at a maximum of approximately 240%, at a maximum of approximately 230%, and at a maximum At approximately 220%, with a maximum of approximately 210%, with a maximum of approximately 200%, with a maximum of approximately 190%, with a maximum of approximately 180%, with a maximum of approximately 170%, with a maximum of approximately 160%, with a maximum of approximately 150%, with a maximum of approximately 140%, with a maximum of approximately 130%, with a maximum of approximately 120%, with a maximum of approximately 110%, with a maximum of approximately 100%, with a maximum of approximately 95%, with a maximum of approximately 90%, with a maximum of approximately 85%, with a maximum of approximately 80%, with a maximum of approximately 75% %, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be less than 0.1% higher.

[0210] In some cases, the concentration of one or more ions on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

[0211] In some embodiments, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 7 0%~approx. 75%, approx. 75%~approx. 80%, approx. 80%~approx. 85%, approx. 85%~approx. 90%, approx. 90%~approx. 95%, approx. 95%~approx. 100%, approx. 100%~approx. 110%, approx. 110%~approx. 120%, approx. 120%~approx. 130%, approx. 130%~approx. 140%, approx. 140%~approx. 150%, approx. 150%~approx. 160%, approx. 160%~approx. 170%, approx. 170%~approx. 180%, approx. 180%~approx. 190%, approx. 190%~approx. 200%, approx. 20 0% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% It may be approximately 360%, 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or even 490% to 500% lower.

[0212] In some cases, the concentration of one or more ions on the cis side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, and at least about 180%. , at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or lower than 500%.

[0213] In some cases, the concentration of one or more ions on the cis side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to At approximately 390%, at a maximum of approximately 380%, at a maximum of approximately 370%, at a maximum of approximately 360%, at a maximum of approximately 350%, at a maximum of approximately 340%, at a maximum of approximately 330%, at a maximum of approximately 320%, at a maximum of approximately 310%, at a maximum of approximately 300%, at a maximum of approximately 290%, at a maximum of approximately 280%, at a maximum of approximately 270%, at a maximum of approximately 260%, at a maximum of approximately 250%, at a maximum of approximately 240%, at a maximum of approximately 230%, and at a maximum At approximately 220%, with a maximum of approximately 210%, with a maximum of approximately 200%, with a maximum of approximately 190%, with a maximum of approximately 180%, with a maximum of approximately 170%, with a maximum of approximately 160%, with a maximum of approximately 150%, with a maximum of approximately 140%, with a maximum of approximately 130%, with a maximum of approximately 120%, with a maximum of approximately 110%, with a maximum of approximately 100%, with a maximum of approximately 95%, with a maximum of approximately 90%, with a maximum of approximately 85%, with a maximum of approximately 80%, with a maximum of approximately 75% %, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be lower than 0.1%.

[0214] In some cases, the concentration of one or more ions on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0215] In some embodiments, the concentration of one or more ions on the transform side of the membrane may be about 0.1% to about 500% higher than the concentration of salts on the cis side of the membrane. In some cases, the concentration of salts on the transform side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 70% to about 70% 5%, approximately 75% to 80%, approximately 80% to 85%, approximately 85% to 90%, approximately 90% to 95%, approximately 95% to 100%, approximately 100% to 110%, approximately 110% to 120%, approximately 120% to 130%, approximately 130% to 140%, approximately 140% to 150%, approximately 150% to 160%, approximately 160% to 170%, approximately 170% to 180%, approximately 180% to 190%, approximately 190% to 200%, approximately 200% to 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~approximately It may be 360%, approximately 360% to 370%, approximately 370% to 380%, approximately 380% to 390%, approximately 390% to 400%, approximately 400% to 410%, approximately 410% to 420%, approximately 420% to 430%, approximately 430% to 440%, approximately 440% to 450%, approximately 450% to 460%, approximately 460% to 470%, approximately 470% to 480%, approximately 480% to 490%, or even 490% to 500% higher.

[0216] In some cases, the concentration of one or more ions on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, and at least about 180%. , at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or may be higher than 500%.

[0217] In some cases, the concentration of one or more ions on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to At approximately 390%, at a maximum of approximately 380%, at a maximum of approximately 370%, at a maximum of approximately 360%, at a maximum of approximately 350%, at a maximum of approximately 340%, at a maximum of approximately 330%, at a maximum of approximately 320%, at a maximum of approximately 310%, at a maximum of approximately 300%, at a maximum of approximately 290%, at a maximum of approximately 280%, at a maximum of approximately 270%, at a maximum of approximately 260%, at a maximum of approximately 250%, at a maximum of approximately 240%, at a maximum of approximately 230%, and at a maximum At approximately 220%, with a maximum of approximately 210%, with a maximum of approximately 200%, with a maximum of approximately 190%, with a maximum of approximately 180%, with a maximum of approximately 170%, with a maximum of approximately 160%, with a maximum of approximately 150%, with a maximum of approximately 140%, with a maximum of approximately 130%, with a maximum of approximately 120%, with a maximum of approximately 110%, with a maximum of approximately 100%, with a maximum of approximately 95%, with a maximum of approximately 90%, with a maximum of approximately 85%, with a maximum of approximately 80%, with a maximum of approximately 75% %, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be less than 0.1% higher.

[0218] In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

[0219] In some embodiments, the concentration of one or more ions on the transform side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more ions on the cis side of the membrane. In some cases, the concentration of one or more ions on the transform side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 7 0%~approx. 75%, approx. 75%~approx. 80%, approx. 80%~approx. 85%, approx. 85%~approx. 90%, approx. 90%~approx. 95%, approx. 95%~approx. 100%, approx. 100%~approx. 110%, approx. 110%~approx. 120%, approx. 120%~approx. 130%, approx. 130%~approx. 140%, approx. 140%~approx. 150%, approx. 150%~approx. 160%, approx. 160%~approx. 170%, approx. 170%~approx. 180%, approx. 180%~approx. 190%, approx. 190%~approx. 200%, approx. 20 0% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% It may be approximately 360%, 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or even 490% to 500% lower.

[0220] In some cases, the concentration of one or more ions on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, and at least about 180%. , at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or lower than 500%.

[0221] In some cases, the concentration of one or more ions on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to At approximately 390%, at a maximum of approximately 380%, at a maximum of approximately 370%, at a maximum of approximately 360%, at a maximum of approximately 350%, at a maximum of approximately 340%, at a maximum of approximately 330%, at a maximum of approximately 320%, at a maximum of approximately 310%, at a maximum of approximately 300%, at a maximum of approximately 290%, at a maximum of approximately 280%, at a maximum of approximately 270%, at a maximum of approximately 260%, at a maximum of approximately 250%, at a maximum of approximately 240%, at a maximum of approximately 230%, and at a maximum At approximately 220%, with a maximum of approximately 210%, with a maximum of approximately 200%, with a maximum of approximately 190%, with a maximum of approximately 180%, with a maximum of approximately 170%, with a maximum of approximately 160%, with a maximum of approximately 150%, with a maximum of approximately 140%, with a maximum of approximately 130%, with a maximum of approximately 120%, with a maximum of approximately 110%, with a maximum of approximately 100%, with a maximum of approximately 95%, with a maximum of approximately 90%, with a maximum of approximately 85%, with a maximum of approximately 80%, with a maximum of approximately 75% %, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be lower than 0.1%.

[0222] In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0223] Alternatively, EOF can be generated by a symmetric ion distribution between the cis and trans sides of the membrane. A symmetric ion distribution can occur when the concentration of one or more ions on the cis side of the membrane is the same as the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane is the same as the concentration of one or more ions on the trans side of the membrane.

[0224] In some embodiments, the EOF may be generated by asymmetric concentrations of one or more salts and one or more ions between the cis side and the trans side of the membrane.

[0225] In some embodiments, the electroosmotic force may act in the same direction as the electrophoretic force, or in the opposite direction to the electrophoretic force. In some embodiments, the electroosmotic force may be greater than the electrophoretic force. In some embodiments, the electroosmotic force may be less than the electrophoretic force.

[0226] The present invention relates to systems and methods for analyzing target analytes using nanopore-based sensors. More specifically, it relates to methods, nanopore systems, and devices for single-molecule profiling of polymers, such as polypeptides or polysaccharides.

[0227] Various studies have demonstrated both free and motor-controlled migration of polypeptides (proteins that are deployed during or before rearrangement through narrow nanopores, typically less than 2 nm in diameter). However, unlike polynucleotides with a fixed negative charge that can be electrophoretically drawn into nanopores by an electric field from an applied voltage, capturing and controlling the migration of peptides of diverse compositions remains a challenge. This is because diverse compositions result in a range of electrical and / or structural properties (e.g., positive, negative, neutral, hydrophilic, hydrophobic, aromatic mixtures) that prevent simple capture under electrophoretic conditions and rearrangement in the deployed state.

[0228] Due to their complex composition, it has previously been impossible to push / feed analytes (e.g., proteins, peptides, polypeptides) into the pores from the cis side in their native forms (e.g., without attachment to a DNA reader or without adding other (e.g., polyanion) tags to create electrophoretic capture motifs). Because diverse charges can sometimes attract and sometimes repel deployed peptides from nanopores depending on the charge and / or applied voltage, electrophoretic means alone cannot transpose a diverse repertoire of complex peptides through nanopores. Indeed, previous studies have shown the transposition of either very short peptides with contour lengths shorter than the length of the nanopore channels, or very carefully selected (model) protein substrates, whose charge, structure, or added electrophoretic tags favor electrophoretic capture and transposition through nanopores. For example, Cressiot et al., ACS Nano 2015,9(9),9050-9061, Oukhaled et al.,Phys.Rev.Lett.2007,98(15); Merstorf et al., ACS Chem Biol 2012,7(4),652-658, Pastoriza-Gallego et al.,ACS Nano 2014,8(11),11350-11360, Rosen et al.,Nat.Biotechnol.2014,32(2),179-181, Yu et al.,bioRxiv Please refer to 2021,2021.09.28.462155.

[0229] However, this by no means represents the broad amino acid composition of proteins found in nature. For example, see Motone et al. (iScience 24, September 24, 2021), which outlines recent approaches using various techniques aimed at driving protein chains and peptides through nanopores. It states that nanopore protein sequencing remains a challenging and unexplored field.

[0230] Bayat et al. (Nature Comm. 2022 Vol. 13, 5113) reported on the label-free detection and analysis of highly anionic linear polysaccharides using protein nanopores. They found that wild-type erolidine nanopores can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds, and epimers in their uronic acid residues.

[0231] Robertson et al. (BBA - Biomembranes, Vol.1863, Issue 9, 2021) focused on the physicochemistry of nanopore sensing and outlined the types of analytes that can be detected. In particular, they mentioned the size discrimination of PEGs up to approximately 48 repeating units in length using nanopore systems based on modified alpha-hemolysin (aHL) or erolidine.

[0232] This disclosure provides a novel approach that offers a simple and robust means for sequencing or characterizing non-nucleic acid polymers, for example, by supplying long non-nucleic acid polymers through nanopores. In some cases, this disclosure can yield polymers that dislocate against the direction of a dominant EPF acting on them to prevent dislocation, thus eliminating the need for tagging of polymer analytes.

[0233] It has been found that these goals can be achieved by using a large and / or dominant cis-to-trans electroosmotic flow (EOF) generated by a large excess of cis-to-trans ions flowing through nanopores, which can supply and pass a wide variety of elongated and complex polymer substrates through the nanopores from cis to trans, even when contrary to the direction of the electrophoretic force (EPF) acting on the polymer. In some embodiments, the cis-to-trans osmotic flow can be generated by the flow of ions and / or solvents from the cis side of the nanopore system to the trans side of the nanopore system.

[0234] This disclosure provides a system that can capture and feed polymer analytes from the cis side of nanopores using strong electroosmotic force. In some embodiments, the strong electroosmotic force pulls the polymer as it displaces through the pores, allowing for the measurement and / or characterization of changes in current that are structure and / or composition dependent.

[0235] Accordingly, in one embodiment, the present invention relates to a method for dislocating a non-nucleic acid polymer analyte through nanopores, wherein the nanopores are contained in a membrane separating the fluid chamber of the nanopore system into cis and trans sides, and the analyte is added to the cis side, and the dislocation is enabled, wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) resulting from a net ionic current flow from cis to trans. A method is provided for dislocating a non-nucleic acid polymer analyte through nanopores, wherein the nanopores are contained in a membrane separating the fluid chamber of the nanopore system into cis and trans sides, and the polymer analyte is added to the cis side, and the dislocation of the polymer analyte to the trans side of the pores, wherein the length of the elongated polymer analyte is greater than the longitudinal axis of the central channel of the nanopore in a direction perpendicular to the membrane, and the nanopore system has a cis-to-trans EOF resulting from a net ionic current flow from cis to trans, and the cis-to-trans EOF overcomes the trans-to-cis EPF acting on the polymer analyte.

[0236] For example, a nanopore system has a net ion current flow from cis to transformer (as specified herein) that exceeds a total ion current flow of greater than 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, most preferably greater than 0.35 or less than -0.35. rel It has an EOF from cis to trans resulting from (also known as, see below).

[0237] In some embodiments, the nanopore system has an ion selectivity P greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, most preferably greater than 3.0 or less than 0.33. (+) / P (-) The cis-to-trans electrophoretic flow (EOF) is contrary to the trans-to-cis electrophoretic flow (EPF) acting on the analyte. In one embodiment, the nanopore system has an ion selectivity P greater than 3.0 or less than 0.3 under an applied voltage across the membrane. (+) / P (-) It has.

[0238] In some embodiments, ion-selective P (+) / P (-) Under an applied voltage across the film, the magnitude may be at least about 2.0, at least about 2.2, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least about 3.1, at least about 3.2, at least about 3.3, at least about 3.4, at least about 3.5, at least about 3.6, at least about 3.7, at least about 3.8, at least about 3.9, at least about 4.0, at least about 4.1, at least about 4.2, at least about 4.3, at least about 4.4, at least about 4.5, at least about 4.6, at least about 4.8, at least about 5.0, or greater than about 5.0.

[0239] In some embodiments, ion-selective P (+) / P (-) The values ​​may be approximately 0.5, 0.45, 0.42, 0.40 mV, 0.38, 0.36, 0.35, 0.34, 0.33, 0.31, 0.30, 0.29, 0.28, 0.27, 0.26, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, or less than 0.20 under the applied voltage across the film.

[0240] In some embodiments, ion-selective P (+) / P (-) The size can be approximately 2.0 to approximately 5.0. In some embodiments, ion selectivity P (+) / P (-)These are approximately 2.0-2.2, 2.0-2.4, 2.0-2.6, 2.0-2.8, 2.0-3.0, 2.0-3.3, 2.0-3.6, 2.0-3.8, 2.0-4.0, 2.0-4.3, 2.0-4.5, 2.0-4.7, 2.0-5.0, and 2. 0.5~approx. 2.6, approx. 2.5~approx. 2.8, approx. 2.5~approx. 3.0, approx. 2.5~approx. 3.3, approx. 2.5~approx. 3.6, approx. 2.5~approx. 3.8, approx. 2.5~approx. 4.0, approx. 2.5~approx. 4.3, approx. 2.5~approx. 4.5, approx. 2.5~approx. 4.7, approx. 2.5~approx. 5.0, approx. 3.0~approx. 3.2, approx. 3.0~approx. 3.3, approx. 3.0~approx. 3.4, approximately 3.0-3.5, approximately 3.0-3.6, approximately 3.0-3.7, approximately 3.0-3.8, approximately 3.0-4.0, approximately 3.0-4.3, approximately 3.0-4.5, approximately 3.0-4.7, approximately 3.0-5.0, approximately 3.3-3.4, approximately 3.3-3.5, approximately 3.3-3.6, approximately 3.3-3.7, It could be approximately 3.3-3.8, 3.3-4.0, 3.3-4.3, 3.3-4.5, 3.3-4.7, 3.3-5.0, 3.5-3.7, 3.5-3.8, 3.5-4.0, 3.5-4.3, 3.5-4.5, 3.5-4.7, or 3.5-5.0.

[0241] In some embodiments, ion-selective P (+) / P (-) The magnitude can be approximately 0.20 to approximately 0.5. In some embodiments, ion selectivity P (+) / P (-)can be from about 0.2 to about 0.22, from about 0.2 to about 0.24, from about 0.2 to about 0.26, from about 0.20 to about 0.28, from about 0.20 to about 0.3, from about 0.2 to about 0.33, from about 0.2 to about 0.36, from about 0.20 to about 0.38, from about 0.2 to about 0.4, from about 0.2 to about 0.43, from about 0.20 to about 0.45, from about 0.20 to about 0.47, from about 0.2 to about 0.48, from about 0.25 to about 0.27, from about 0.25 to about 0.28, from about 0.25 to about 0.30, from about 0.25 to about 0.33, from about 0.25 to about 0.36, from about 0.25 to about 0.38, from about 0.25 to about 0.40, from about 0.25 to about 0.43, from about 0.25 to about 0.45, from about 0.25 to about 0.47, from about 0.25 to about 5.0, from about 0.30 to about 0.32, from about 0.30 to about 0.33, from about 0.30 to about 0.34, from about 0.30 to about 0.35, from about 0.30 to about 0.36, from about 0.30 to about 0.37, from about 0.30 to about 0.38, from about 0.30 to about 0.40, from about 0.30 to about 0.43, from about 0.30 to about 0.45, from about 0.30 to about 0.47, from about 0.30 to about 0.5.

[0242] In some embodiments, the ion selectivity P (+) / P (-) can be of a magnitude of about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.3, about 3.5, about 3.6, about 3.8, about 4.0, about 4.3, about 4.6, about 4.8, about 5.0, about 0.5, about 0.45, about 0.40, about 0.38, about 0.35, about 0.33, about 0.30, about 0.28, about 0.25, about 0.23 or about 0.20.

[0243] In some embodiments, the applied voltage across the film may be at least about 1 mV, at least about 5 mV, at least about 10 mV, at least about 20 mV, at least about 30 mV, at least about 40 mV, at least about 50 mV, at least about 60 mV, at least about 70 mV, at least about 80 mV, at least about 90 mV, at least about 100 mV, at least about 150 mV, at least about 200 mV, at least about 250 mV, at least about 300 mV, at least about 350 mV, at least about 400 mV, at least about 450 mV, at least about 500 mV, at least about 600 mV, at least about 700 mV, at least about 800 mV, at least about 900 mV, at least about 1000 mV, or greater than about 1000 mV. In some embodiments, the applied voltage across the film may be at least about 1000mV, up to about 900mV, up to about 800mV, up to about 700mV, up to about 600mV, up to about 500mV, up to about 450mV, up to about 400mV, up to about 350mV, up to about 300mV, up to about 250mV, up to about 200mV, up to about 150mV, up to about 100mV, up to about 90mV, up to about 80mV, up to about 70mV, up to about 60mV, up to about 50mV, up to about 40mV, up to about 30mV, up to about 20mV, up to about 10mV, up to about 5mV, up to about 1mV, or less than about 1mV.

[0244] In some embodiments, the applied voltage across the membrane can be on the order of about 1 mV to about 100 mV. In some embodiments, the applied voltage across the membrane can be on the order of about 1 mV to about 5 mV, about 1 mV to about 10 mV, about 1 mV to about 20 mV, about 1 mV to about 30 mV, about 1 mV to about 40 mV, about 1 mV to about 50 mV, about 1 mV to about 60 mV, about 1 mV to about 70 mV, about 1 mV to about 80 mV, about 1 mV to about 90 mV, about 1 mV to about 100 mV, about 5 mV to about 10 mV, about 5 mV to about 20 mV, about 5 mV to about 30 mV, about 5 mV to about 40 mV, about 5 mV to about 50 mV, about 5 mV to about 60 mV, about 5 mV to about 70 mV, about 5 mV to about 80 mV, about 5 mV to about 90 mV, about 5 mV to about 100 mV, about 10 mV to about 20 mV, about 10 mV to about 30 mV, about 10 mV to about 40 mV, about 10 mV to about 50 mV, about 10 mV to about 60 mV, about 10 mV to about 70 mV, about 10 mV to about 80 mV, about 10 mV to about 90 mV, about 10 mV to about 100 mV, about 20 mV to about 30 mV, about 20 mV to about 40 mV, about 20 mV to about 50 mV, about 20 mV to about 60 mV, about 20 mV to about 70 mV, about 20 mV to about 80 mV, about 20 mV to about 90 mV, about 20 mV to about 100 mV, about 30 mV to about 40 mV, about 30 mV to about 50 mV, about 30 mV to about 60 mV, about 30 mV to about 70 mV, about 30 mV to about 80 mV, about 30 mV to about 90 mV, about 30 mV to about 100 mV, about 40 mV to about 50 mV, about 40 mV to about 60 mV, about 40 mV to about 70 mV, about 40 mV to about 80 mV, about 40 mV to about 90 mV, about 40 mV to about 100 mV, about 50 mV to about 60 mV, about 50 mV to about 70 mV, about 50 mV to about 80 mV, about 50 mV to about 90 mV, about 50 mV to about 100 mV, about 60 mV to about 70 mV, about 60 mV to about 80 mV, about 60 mV to about 90 mV, about 60 mV to about 100 mV, about 70 mV to about 80 mV, about 70 mV to about 90 mV, about 70 mV to about 100 mV, about 80 mV to about 90 mV, about 80 mV to about 100 mV, or on the order of about 90 mV to about 100 mV.

[0245] In some embodiments, the applied voltage across the film may be in the range of approximately 100 mV to approximately 1,000 mV.In some embodiments, the applied voltage across the film is approximately 100mV to 150mV, 100mV to 200mV, 100mV to 250mV, 100mV to 300mV, 100mV to 400mV, 100mV to 500mV, 100mV to 600mV, 100mV to 700mV, 100mV to 800mV, 100mV to 900mV, 100mV to 1,000mV, 150mV to 200mV, 150mV to 250mV, 150mV to 300mV, 150mV to 400mV, and 150mV to 200mV. Approx. 500mV, Approx. 150mV ~ Approx. 600mV, Approx. 150mV ~ Approx. 700mV, Approx. 150mV ~ Approx. 800mV, Approx. 150mV ~ Approx. 900m V, approximately 150mV to approximately 1,000mV, approximately 200mV to approximately 250mV, approximately 200mV to approximately 300mV, approximately 200mV to approximately 400mV, approximately 2 00mV~Approx. 500mV, Approx. 200mV~Approx. 600mV, Approx. 200mV~Approx. 700mV, Approx. 200mV~Approx. 800mV, Approx. 200mV~ Approx. 900mV, Approx. 200mV ~ Approx. 1,000mV, Approx. 250mV ~ Approx. 300mV, Approx. 250mV ~ Approx. 400mV, Approx. 250mV ~ Approx. 500 mV, approximately 250mV to approximately 600mV, approximately 250mV to approximately 700mV, approximately 250mV to approximately 800mV, approximately 250mV to approximately 900mV, approximately 2 50mV to about 1,000mV, about 300mV to about 400mV, about 300mV to about 500mV, about 300mV to about 600mV, about 300mV ~700mV, 300mV~800mV, 300mV~900mV, 300mV~1,000mV, 400mV~5 00mV, approximately 400mV to approximately 600mV, approximately 400mV to approximately 700mV, approximately 400mV to approximately 800mV, approximately 400mV to approximately 900mV, approximately The magnitude can be approximately 400mV to 1,000mV, approximately 500mV to 600mV, approximately 500mV to 700mV, approximately 500mV to 800mV, approximately 500mV to 900mV, approximately 500mV to 1,000mV, approximately 600mV to 700mV, approximately 600mV to 800mV, approximately 600mV to 900mV, approximately 600mV to 1,000mV, approximately 700mV to 800mV, approximately 700mV to 900mV, approximately 700mV to 1,000mV, approximately 800mV to 900mV, approximately 800mV to 1,000mV, or approximately 900mV to 1,000mV.

[0246] In some embodiments, the applied voltage across the film may be of a magnitude of approximately 1mV, 5mV, 10mV, 20mV, 30mV, 40mV, 50mV, 60mV, 70mV, 80mV, 90mV, 100mV, 150mV, 200mV, 250mV, 300mV, 350mV, 400mV, 450mV, 500mV, 600mV, 700mV, 800mV, 900mV, or 1000mV. In some embodiments, the voltage is negative from cis to transformer. In some embodiments, the voltage is positive from cis to transformer.

[0247] In some embodiments, a signal is measured. The signal may include an electrical signal. The signal may be related to or caused by dislocations of the analyte. The signal may include an ionic current or a change in ionic current. The signal may include a voltage or a change in voltage across the membrane and / or nanopores. The signal may include a measurement of the change in current between nanopore states. The nanopore states may include an open channel, trapping of the analyte by the nanopore, or passage of the polymer from the trapped state through the nanopore. In some embodiments, measuring the signal may include comparing the signals between different states of the nanopore.

[0248] In some embodiments, a pair of electrodes are configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the net electroosmotic current relative to the applied voltage is greater than about 0.10 pA / mV. In some embodiments, the nanopores include internal pore constriction of about 0.5 nanometers to about 2 nanometers (nm).

[0249] Furthermore, a nanopore system is provided for dislocating a non-nucleic acid polymer analyte (e.g., analyte) through nanopores, wherein the system includes nanopores contained in a membrane that separates the fluid chamber of the nanopore system into a cis side and a trans side, the analyte is added to the cis side, and the nanopore system has an electroosmotic flow (EOF) from cis to trans resulting from a net ionic current flow from cis to trans, as a result of which the target polymer is trapped in the nanopores. The dominant cis-to-trans EOF results from a net ionic current flow from cis to trans exceeding a total ionic current flow of, for example, greater than 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, most preferably greater than 0.35 or less than -0.35.

[0250] In certain embodiments, the nanopore system of the present invention has an ion selectivity P greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, most preferably greater than 3.0 or less than 0.33, for example, greater than 3.5 or less than 0.28. (+) / P (-) It has.

[0251] The present invention, which relies on a dominant cis-to-trans EOF resulting from a net cis-to-trans ionic current flow, is neither taught nor suggested in the art.

[0252] In some cases, EPFs can be the dominant process driving trapping and / or dislocation in nanopore systems. Therefore, all previous demonstrations have used selected model polymers (which have a net charge that aids EPFs) or modified polymers with highly charged tags (e.g., by adding polyanion tags) so that the resulting EPF forces acting on the polymer are cis-to-trans to drive dislocations. If EOFs have been used in nanopore systems previously, they are most often trans-to-cis acting against cis-to-trans EPFs (slowing EPF-driven dislocations or trapping molecules in nanopores), or cis-to-trans combined with cis-to-trans EPFs to aid dislocations. While some previous studies have shown the capture of neutral or weakly charged small molecules or polymers within nanopores via weak electroosmosis (https: / / doi.org / 10.1073 / pnas.2531778100, https: / / pubs.acs.org / doi / full / 10.1021 / ja4026193, https: / / doi.org / 10.1063 / 1.2723088), there is no feasible disclosure that long and / or complex polymers (with contour lengths greater than the pore length) can be captured and / or displaced using cis-to-trans electroosmosis (EOF), which can overcome trans-to-cis EPF acting in the opposite direction.

[0253] See also US2022 / 0283140 A1, which discloses a method and system for performing single-molecule proteomics using a nanopore sensor, measuring the electronic signature of a protein or peptide transported through a nanopore using a drug such as guanidinium chloride, and binding to the inside of the nanopore and / or providing electroosmotic force into the nanopore. In this system, the EOF is used to assist in the rearrangement, but the EPF is set to be in the same direction. This disclosure provides a method and system in which a cis-to-trans EOF can overcome a reverse EPF.

[0254] While some previous studies have shown the capture of neutral or weakly charged small molecules or polymers within nanopores via weak electroosmosis, there is no feasible disclosure that long and / or complex polymers (with contour lengths greater than the pore length) can be captured and / or displaced using cis-to-trans electroosmosis (EOF), which can overcome trans-to-cis EPF acting in the opposite direction.

[0255] In some embodiments, the novel system relies on positioning a specific strong electroosmotic means in the direction of dislocation. This was unpredictable because the EOF acting on the polymer analyte could repel the analyte and thus hinder its capture and / or dislocation. Furthermore, long polymer analytes could become clogged within the nanopores. In fact, this is how EOF is most commonly used in conventional nanopore systems, acting to create traps to keep the analyte within the pores. In some cases, the nanopores capture the free ends of the polymer because there are no tags at the ends to create a strong EPF.

[0256] The polymer analyte may be synthetic, semi-synthetic, or of biological origin. For example, it may be a biopolymer other than DNA. It may contain, or consist of, peptide units, sugar units, or water-soluble plastic monomers, and any combination thereof. Preferably, the polymer analyte is a polypeptide, polysaccharide, or water-soluble plastic such as PEG, or a PEGylated polypeptide.

[0257] In one embodiment, the polymer analyte is an unmodified (label-free) analyte. Preferably, in the method of the present invention, the polymer ends are unstructured, and preferably, the polymer is modified or partially modified. In one embodiment, the length of the elongated polymer is greater than the longitudinal axis of the central channel of the nanopore perpendicular to the membrane, and preferably, the length of the polymer is more than 50 monomer units, for example, more than 50 peptide units.

[0258] In preferred embodiments, the present invention provides a method for rearranging a non-nucleoside polymer analyte (e.g., analyte, polypeptide) comprising at least 30 peptide units and / or positively and / or negatively charged residues. The polypeptide may be in a denatured / expanded state, and preferably, the polypeptide is added in a pre-denatured state.

[0259] The method may further include (c) measuring the change in ionic current caused by dislocation of the target polymer through the nanopores, preferably, operation (c) includes measuring the change in current for the states of (i) open channels, (ii) polymer capture by the nanopores, and / or (iii) polymer passing through the nanopores from (ii), more preferably, measuring includes detecting the difference between states (i), (ii), and / or (iii). In one embodiment, measuring includes measuring the difference between states (iii) caused by the composition and / or structure of the polymer passing through the nanopores. cis-to-trans EOF can be achieved by various means. For example, cis-to-trans EOF can be controlled by modifying the charge of the nanopores (e.g., genetic engineering) or any combination thereof, by adjusting the pH, type and / or concentration of salts and / or osmotic pressure across the membrane of the nanopore system. Preferably, dominant EOF is achieved by modifying the nanopores and / or by an asymmetric salt distribution between the cis and trans sides of the chamber.

[0260] In certain embodiments, the system has an ion selectivity P greater than 2.0, preferably greater than 2.5, and most preferably greater than 3.0. (+) / P (-) The system preferably includes cation-selective (mutant) nanopores.

[0261] In one embodiment, the nanopores are solid-state nanopores or biological nanopores, and preferably have internal pore constriction with a diameter in the range of 0.5 to 2 nm.

[0262] In some embodiments, the nanopores may be biological nanopores, more preferably alpha-helical or beta-barrel oligomeric pore-forming toxins or porins. The nanopores are preferably selected from the group consisting of erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, phage-derived portal proteins (e.g., Phi29, G20c), or their variants. In certain embodiments, the nanopores are selected from the variant CytK nanopores listed in Table 1. Those skilled in the art will understand that the nanopores may also be constructed from elements of existing nanopores (see, e.g., WO2021 / 101378) or newly developed using predictive protein manipulation software (see, e.g., Shimizu et al. 2022, Nature Nanotechnology volume 17, pg. 67-75).

[0263] The method according to the present invention may further include operation (c) measuring a change in ionic current caused by dislocation of a polymer analyte through nanopores. Operation (c) preferably includes measuring a change in current for the states of (i) open channels, (ii) capture of the polymer by nanopores, and / or (iii) the passage of the polymer from (ii) through the nanopores. For example, this includes detecting the difference between states (i), (ii), and / or (iii). In certain embodiments, the measurement includes measuring the difference between states (iii) caused by the composition and / or structure of a non-nucleic acid polymer analyte (e.g., analyte, protein) passing through the nanopores. The method preferably includes obtaining one or more measurements characteristic of the target polymer. The one or more measurements may be one, two, three, four, or five or more characteristics characteristic of the polymer analyte. One or more features are preferably selected from (i) polymer length, (ii) polymer identity, (iii) polymer arrangement, (iv) polymer secondary or tertiary structure, and / or (v) whether the polymer has been modified (post-translation). Any combination of (i) to (v) may be measured according to the present invention.

[0264] A further embodiment of the present invention is a nanopore system for dislocating polymer analytes through nanopores, The present invention relates to a nanopore system comprising: (a) a membrane having nanopores therein, the membrane separating a chamber into a cis side and a trans side, to which a polymer analyte is added on the cis side and displaced through the nanopores to the trans side; (b) a polymer analyte captured on the cis side of the chamber by a protein translocase that can bind to and displace the polymer analyte through the nanopores in a continuous sequence; and (c) means for providing a voltage difference between the cis side and the trans side of the membrane.

[0265] In some embodiments, the nanopore system is further characterized by an electroosmotic force (EOF) from cis to trans resulting from the net ionic current flow from cis to trans, such that the polymeric analyte is captured in the nanopore. Preferably, the nanopore system has a net ionic current flow from cis to trans that exceeds the flow of the total ionic current of greater than 0.2 or less than -0.2, more preferably greater than 0.3 or less than -0.3, and most preferably greater than 0.35 or less than -0.35 (I rel ), resulting in an EOF from cis to trans.

[0266] In certain aspects, the nanopore system has an ion selectivity P (+) / P (-) greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, more preferably greater than 3.0 or less than 0.33, or even greater than 3.5 or less than 0.2.

[0267] The voltage difference can be provided in various ways. For example, in one circuit, a voltage can be applied and / or a current can be measured, or the system can include a first circuit for applying a voltage and / or a second circuit for measuring a current. It is also possible to create a voltage difference with an asymmetric salt across the membrane. For example, the device includes a circuit for providing a voltage between the cis side and the trans side and measuring the ionic current flowing through the nanopore. See FIG. 1. Preferably, a negative voltage is applied to the trans side.

[0268] The system can further include means for measuring a signal based on the ionic current flowing through the nanopore during translocation. These measuring means are set to detect changes in the signal that reflect the characteristics of the analyte (e.g., protein) when it translocates.

[0269] The system may use alternative means of measuring the voltage-current characteristics of the nanopore system, such as using ion flux fluorescence probes or field-effect transistor systems, in addition to measuring voltage changes. However, other suitable detection methods also exist, such as tunneling, surface-enhanced Raman, plasmonics, and / or other spectroscopic methods that do not measure ion current but instead directly measure the properties of the target analyte in the nanopores.

[0270] Furthermore, analytical devices are provided that include one or more nanopore systems disclosed herein, for example, in the form of an array.

[0271] Further embodiments relate to the use of methods, nanopore systems, or devices according to the present invention for the detection and / or analysis of one or more polymer analytes, preferably at the single-molecule level, for characterizing at least one feature of a polymer analyte. Since the method is independent of the charge of the polymer analyte being analyzed, it can, in principle, analyze any type of polymer. The method and corresponding systems provide a highly desired single-molecule polymer sequencing approach. Embodiments of the present invention may be applied to protein or glycan sequencing, single-molecule protein or glycan sequencing, proteomics, detection of post-translational modifications in single cells, glycopeptide analysis, detection of protein or glycan biomarkers, and / or their post-translational modifications, and / or detection of disease biomarkers. One preferred application of this method is single-molecule protein sequencing, and / or discovery and / or quantification of post-translational modifications in proteins.

[0272] Definition of Terms electroosmosis According to the present invention, the nanopore system has a cis-to-trans electroosmotic flow, or vice versa, which creates a drag force (often called electroosmotic force (EOF)) on particles dispersed in a solution (independent of their charge). The EOF arises from a net ion flow (e.g., cis-to-trans) that creates a force strong enough to move the fluid against the solvent itself (water) (Chinappi et al., 2020, ACS Nano, 14, 11, pg. 15816-15828), whic...

Claims

1. It is a method, (a) (i) A nanopore system comprising (1) a fluid chamber and (2) a membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side and a transform side. (ii) To provide a non-nucleic acid polymer analyte, wherein the non-nucleic acid polymer analyte includes a linear length greater than the channel length of the nanopore, (b) A method for dislocating the non-nucleic acid polymer analyte from the cis side to the trans side of the fluid chamber, wherein the non-nucleic acid polymer analyte comprises an elongated structure, the nanopore system has a cis-to-trans electroosmotic force resulting from a net ionic current flow from cis to trans, and the cis-to-trans electroosmotic force dislocates the non-nucleic acid polymer analyte through the nanopores against an electrophoretic force acting in the opposite direction to the cis-to-trans electroosmotic force.

2. The method according to claim 1, wherein the electroosmotic force is at least 10% greater than the electrophoretic force.

3. The method according to claim 1 or 2, wherein the electroosmotic force is at least 50% greater than the electrophoretic force.

4. The method according to any one of claims 1 to 3, wherein the electroosmotic force is at least 100% greater than the electrophoretic force.

5. The method according to any one of claims 1 to 4, wherein the cis side of the fluid chamber contains a first solution, and the transformer side of the fluid chamber contains a second solution.

6. The method according to claim 5, wherein the first solution contains a solute at a first concentration, and the second solution contains a solute at a second concentration.

7. The method according to claim 6, wherein the solute comprises ions or osmolite.

8. The method according to claim 6, wherein the difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force from the cis to the transformer in the presence of an applied potential.

9. The method according to any one of claims 1 to 8, wherein the non-nucleic acid polymer analyte is an unmodified (label-free) non-nucleic acid polymer analyte.

10. The method according to any one of claims 1 to 9, wherein the ends of the non-nucleic acid polymer analyte lack a three-dimensional structure.

11. The method according to any one of claims 1 to 10, wherein at least a portion of the non-nucleic acid polymer analyte is modified.

12. The method according to any one of claims 1 to 11, wherein the linear length of the non-nucleic acid polymer analyte is greater than the channel length of the nanopores traversing the membrane when the non-nucleic acid polymer analyte is elongated.

13. The method according to claim 12, wherein the non-nucleic acid polymer analyte comprises at least about 25 repeating units.

14. The method according to claim 12 or 13, wherein the non-nucleic acid polymer analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof.

15. The method according to any one of claims 12 to 14, wherein the non-nucleic acid polymer analyte comprises a polypeptide, a polysaccharide, or a water-soluble plastic.

16. The method according to any one of claims 1 to 15, wherein the non-nucleic acid polymer analyte comprises a polypeptide of at least 30 peptide units.

17. The method according to claim 16, wherein the at least 30 peptide units include positively or negatively charged peptide units.

18. The method according to claim 16 or 17, wherein the polypeptide is in a denatured state.

19. The method according to any one of claims 16 to 18, wherein the polypeptide is provided in a folded state.

20. The method according to any one of claims 1 to 19, further comprising measuring a signal generated by dislocating the non-nucleic acid polymer analyte through the nanopores.

21. The method according to claim 20, wherein the measurement includes measuring a signal about (a) the open channels of the nanopores, (b) the capture of the non-nucleoside polymer analyte by the nanopores, or (c) the state of passage of the non-nucleoside polymer analyte through the nanopores.

22. The method according to claim 21, wherein the measurement includes detecting the difference between states (a), (b), and (c).

23. The method according to claim 20, wherein the signal includes an ion current, a change in ion current, or a derivative thereof.

24. The method according to any one of claims 1 to 23, wherein the linear length of the non-nucleic acid polymer analyte is at least 1 kDa.

25. The method according to any one of claims 1 to 24, wherein the linear length of the non-nucleic acid polymer analyte is a maximum of 4,000 kDa.

26. The method according to any one of claims 1 to 25, wherein the linear length of the non-nucleic acid polymer analyte is at least twice as large as the channel length of the nanopore.

27. The method according to any one of claims 1 to 25, wherein the linear length of the non-nucleic acid polymer analyte is up to twice as large as the channel length of the nanopore.

28. The method according to any one of claims 1 to 27, wherein the linear length of the non-nucleic acid polymer analyte is at least 3 nanometers.

29. The method according to any one of claims 1 to 28, wherein the electroosmotic force from cis to transformer includes a net ionic current flow from cis to transformer.

30. The method according to any one of claims 1 to 29, wherein the electroosmotic force from cis to transform is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the system, modification of the nanopores, or any combination thereof.

31. The method according to any one of claims 1 to 30, wherein the electroosmotic force from cis to transform is adjusted by modifying the charge of the nanopores.

32. The method according to any one of claims 1 to 31, wherein the electroosmotic force from cis to transform is regulated by an asymmetric salt distribution between the cis side and the transform side of the membrane.

33. The method according to any one of claims 1 to 32, wherein the nanopores have an ion selectivity P(+) / P(-) greater than 2.

0.

34. The method according to any one of claims 1 to 32, wherein the nanopores have an ion selectivity P(+) / P(-) of less than 0.

50.

35. The method according to any one of claims 1 to 34, wherein the nanopore system further comprises a pair of electrodes.

36. The method according to claim 35, wherein the pair of electrodes are configured to provide a voltage applied to generate the electrophoretic force.

37. The method according to claim 36, wherein the applied voltage is a negative voltage relative to the transformer side.

38. The method according to claim 36, wherein the applied voltage is a positive voltage relative to the transformer side.

39. The method according to any one of claims 36 to 38, wherein the magnitude of the applied voltage is less than 300 mV.

40. The method according to any one of claims 36 to 39, wherein the magnitude of the applied voltage exceeds 20 mV.

41. The method according to any one of claims 36 to 40, wherein the absolute relative net electroosmotic current with respect to the applied voltage is greater than about 0.10 pA / mV.

42. The method according to any one of claims 1 to 41, wherein the nanopores include internal pore constriction of about 0.5 nanometers to about 2 nanometers (nm).

43. The method according to any one of claims 1 to 42, wherein the nanopores include an alpha-helical oligomer pore structure.

44. The method according to any one of claims 1 to 43, wherein the nanopores include a beta-barrel oligomer pore structure.

45. The method according to any one of claims 1 to 44, wherein the nanopores include recombinant nanopores.

46. The method according to any one of claims 1 to 45, wherein the nanopores contain proteins of erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, modified variants thereof, or ion-selective variants thereof.

47. The method according to any one of claims 1 to 46, wherein the nanopores include biological nanopores.

48. The method according to claim 47, wherein the biological nanopore is modified to restrict the passage of one or more ions through the channels of the nanopore.

49. The method according to claim 48, wherein the biological nanopore restricts the passage of one or more ions through the channels of the nanopore by altering the charge of the channels of the nanopore.

50. The method according to claim 47 or 48, wherein the net charge of the channel is negative.

51. The method according to claim 47 or 48, wherein the net charge of the channel is positive.

52. The method according to any one of claims 1 to 51, wherein the nanopores include mutant CytK nanopores.

53. The method according to claim 52, wherein the mutant CytK nanopore comprises one or more amino acid substitutions.

54. The method according to claim 53, wherein the one or more amino acid substitutions include K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof.

55. The method according to claim 53, wherein the one or more amino acid substitutions include K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof.

56. The mutant CytK nanopores (i) K128D and K155D, (ii) K128D, K155D, and T116D, (iii) T147D or S151D, (iv) K128D, K155D, and S120D, (v) Q122D, T147D, or S155D, and (vi) The method according to claim 52, comprising one of the amino acid substitution combinations of K128D, K155D, Q145D, and S151D.

57. The mutant CytK nanopores (i) S120D, G122D, or K155D, (ii) S120D combined with K128F / K128D, (iii) Q122D or S151D, (iv) K128D or K128F, (v) S120D, K115D, and Q122D, (vi) K128F, S120D, and G122D, and (vii) The method according to claim 52, comprising one or more combinations of amino acid substitutions of K128F, S120D, G122D, and K155D.

58. It is a system, (a) Fluid chamber, (b) A membrane comprising nanopores, wherein the membrane separates the fluid chamber into a cis side (i) containing a first solution and a trans side (ii) containing a second solution, and the first and second solutions are configured to displace a non-nucleic acid polymer analyte across the nanopores using electroosmotic flow, wherein the non-nucleic acid polymer analyte comprises elongated structures, and the non-nucleic acid polymer analyte comprises a linear length greater than the channel length of the nanopores, (c) A system comprising a pair of electrodes, the first electrode and the second electrode, wherein the first electrode is positioned on the cis side of the fluid chamber and the second electrode is positioned on the transformer side of the fluid chamber, and the pair of electrodes is configured to generate electrophoretic force acting in the opposite direction to the electroosmotic flow.

59. It is a system, Fluid chamber and A membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, and the first and second solutions are configured to displace a non-nucleic acid polymer analyte using electroosmotic flow, A pair of electrodes including a first electrode and a second electrode, The fluid chamber, the nanopores, and a controller operably connected to the pair of electrodes, wherein the controller (a) Using the pair of electrodes, generate an electrophoretic force acting in the opposite direction to the electroosmotic flow that displaces the non-nucleic acid polymer analyte through the nanopores, (b) A system for detecting one or more signals related to at least one feature of the non-nucleic acid polymer analyte during or after the dislocation of the non-nucleic acid polymer analyte passing through the nanopores, wherein the non-nucleic acid polymer analyte has a linear length greater than the channel length of the nanopores.

60. The system according to claim 59, wherein the controller uses the pair of electrodes to detect one or more signals related to at least one feature of the non-nucleic acid polymer analyte.

61. The system according to claim 58 or 59, wherein the electroosmotic flow is greater than the electrophoretic force.

62. The system according to claim 61, wherein the electroosmotic flow is at least 10% greater than the electrophoretic force.

63. The system according to claim 61, wherein the electroosmotic flow is at least 50% greater than the electrophoretic force.

64. The system according to claim 61, wherein the electroosmotic flow is at least 100% greater than the electrophoretic force.

65. The system according to any one of claims 58 to 64, wherein the first solution contains a solute at a first concentration, and the second solution contains a solute at a second concentration.

66. The system according to claim 65, wherein the solute comprises ions or osmolite.

67. The system according to claim 65 or 66, wherein the difference between the first concentration of the solute and the second concentration of the solute is configured to generate the electroosmotic flow in the presence of an applied potential.

68. The system according to any one of claims 58 to 67, wherein the electroosmotic flow includes a net ionic current flow from cis to transformer.

69. The system according to any one of claims 58 to 68, wherein the electroosmotic flow is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the system, modification of the nanopores, or any combination thereof.

70. The system according to any one of claims 58 to 69, wherein the electroosmotic flow is regulated by modifying the charge of the nanopores.

71. The system according to any one of claims 58 to 70, wherein the electroosmotic flow is regulated by an asymmetric salt distribution between the cis side and the transform side of the membrane.

72. The system according to any one of claims 58 to 71, wherein the nanopores have an ion selectivity P(+) / P(-) greater than 2.

0.

73. The system according to any one of claims 58 to 72, wherein the nanopores have an ion selectivity P(+) / P(-) of less than 0.

50.

74. The system according to any one of claims 58 to 73, wherein the pair of electrodes are configured to provide the voltage applied to generate the electrophoretic force.

75. The system according to claim 74, wherein the applied voltage is a negative voltage relative to the transformer.

76. The system according to claim 74, wherein the applied voltage is a positive voltage relative to the transformer.

77. The system according to any one of claims 74 to 76, wherein the magnitude of the applied voltage is less than 300 mV.

78. The system according to any one of claims 74 to 77, wherein the magnitude of the applied voltage exceeds 20 mV.

79. The system according to any one of claims 74 to 78, wherein the absolute relative net electroosmotic current with respect to the applied voltage is greater than about 0.10 pA / mV.

80. The system according to any one of claims 58 to 79, wherein the nanopores include internal pore constriction of about 0.5 nanometers to about 2 nanometers (nm).

81. The system according to any one of claims 58 to 80, wherein the nanopores include an alpha-helical oligomer pore structure.

82. The system according to any one of claims 58 to 81, wherein the nanopores include a beta-barrel oligomer pore structure.

83. The system according to any one of claims 58 to 82, wherein the nanopores include recombinant nanopores.

84. The system according to any one of claims 58 to 83, wherein the nanopores contain proteins of erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, modified variants thereof, or ion-selective variants thereof.

85. The system according to any one of claims 58 to 84, wherein the nanopores include biological nanopores.

86. The system according to claim 85, wherein the biological nanopores are modified to restrict the passage of one or more ions through the channels of the nanopores.

87. The system according to claim 86, wherein the biological nanopore restricts the passage of one or more ions through the channels of the nanopore by altering the charge of the channels of the nanopore.

88. The system according to claim 86, wherein the net charge of the channel is negative.

89. The system according to claim 86, wherein the net charge of the channel is positive.

90. The system according to any one of claims 58 to 89, wherein the nanopores include mutant CytK nanopores.

91. The system according to claim 90, wherein the mutant CytK nanopore comprises one or more amino acid substitutions.

92. The system according to claim 91, wherein the one or more amino acid substitutions include K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof.

93. The system according to claim 91, wherein the one or more amino acid substitutions include K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof.

94. The mutant CytK nanopores (i) K128D and K155D, (ii) K128D, K155D, and T116D, (iii) T147D or S151D, (iv) K128D, K155D, and S120D, (v) Q122D, T147D, or S155D, and (vi) The system according to claim 90, comprising one of the amino acid substitution combinations of K128D, K155D, Q145D, and S151D.

95. The mutant CytK nanopores (i) S120D, G122D, or K155D, (ii) S120D combined with K128F / K128D, (iii) Q122D or S151D, (iv) K128D or K128F, (v) S120D, K115D, and Q122D, (vi) K128F, S120D, and G122D, and (vii) The system according to claim 90, comprising one or more combinations of amino acid substitutions of K128F, S120D, G122D, and K155D.

96. The system according to any one of claims 58 to 95, wherein the non-nucleic acid polymer analyte is an unmodified (label-free) non-nucleic acid polymer analyte.

97. The system according to any one of claims 58 to 96, wherein the ends of the non-nucleic acid polymer analyte lack a three-dimensional structure.

98. The system according to any one of claims 58 to 97, wherein at least a portion of the non-nucleic acid polymer analyte is modified.

99. The system according to any one of claims 58 to 98, wherein the linear length of the non-nucleic acid polymer analyte is greater than the channel length of the nanopores traversing the membrane when the non-nucleic acid polymer analyte is elongated.

100. The system according to any one of claims 58 to 99, wherein the non-nucleic acid polymer analyte comprises at least about 25 repeating units.

101. The system according to any one of claims 58 to 100, wherein the non-nucleic acid polymer analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof.

102. The system according to claim 101, wherein the non-nucleic acid polymer analyte comprises a polypeptide, a polysaccharide, or a water-soluble plastic.

103. The system according to claim 102, wherein the non-nucleic acid polymer analyte comprises a polypeptide of at least 30 peptide units.

104. The system according to claim 103, wherein the at least 30 peptide units include positively or negatively charged peptide units.

105. The system according to any one of claims 102 to 104, wherein the polypeptide is in a denatured state.

106. The system according to any one of claims 102 to 104, wherein the polypeptide is provided in a folded state.

107. The system according to any one of claims 58 to 106, wherein the linear length of the non-nucleic acid polymer analyte is at least 1 kDa.

108. The system according to any one of claims 58 to 107, wherein the linear length of the non-nucleic acid polymer analyte is a maximum of 4,000 kDa.

109. The system according to any one of claims 58 to 108, wherein the linear length of the non-nucleic acid polymer analyte is at least twice as large as the channel length of the nanopore.

110. The system according to any one of claims 58 to 108, wherein the linear length of the non-nucleic acid polymer analyte is up to twice as large as the channel length of the nanopore.

111. A device including an array of systems, comprising a system according to any one of claims 58 to 110.

112. Use of the method according to any one of claims 1 to 57 for characterizing at least one feature of the non-nucleic acid polymer analyte.

113. Use of the system according to any one of claims 58 to 110 for characterizing at least one feature of the non-nucleic acid polymer analyte.

114. Use of the method according to any one of claims 1 to 57 for the detection and analysis of one or more non-nucleic acid polymer analytes at the single-molecule level.

115. Use of the system according to any one of claims 58 to 110 for the detection and analysis of one or more non-nucleic acid polymer analytes at the single-molecule level.

116. Use of the method according to any one of claims 1 to 57 for the detection and analysis of one or more polypeptides.

117. Use of the system according to any one of claims 58 to 110 for the detection and analysis of one or more polypeptides.

118. A method for dislocating a non-nucleic acid polymer analyte through nanopores, wherein the nanopores are contained within a membrane separating the fluid chamber of the nanopore system into cis and trans sides, and the method comprises adding the polymer analyte to the cis side and enabling the dislocation of the polymer analyte, wherein the length of the elongated polymer analyte is greater than the longitudinal axis of the central channel of the nanopore in a direction perpendicular to the membrane. A method wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) arising from a net ionic current flow from cis to trans, and the cis-to-trans EOF overcomes the trans-to-cis electrophoretic force (EPF) acting on the polymer analyte.

119. The method according to claim 118, wherein the polymer analyte is an unmodified (labeled) analyte.

120. The method according to claim 118 or 119, wherein the ends of the polymer are not structured, and preferably the polymer is modified or partially modified.

121. The method according to any one of claims 118 to 120, wherein the polymer analyte comprises at least 25 repeating units, preferably at least 35 repeating units, and more preferably at least 45 repeating units.

122. The method according to any one of claims 118 to 121, wherein the polymer analyte is synthetic, semi-synthetic, or of biological origin, for example, a biopolymer, and preferably comprises or consists of peptide units, sugar units, and water-soluble plastic monomers, and any combination thereof.

123. The method according to claim 122, wherein the polymer analyte is a polypeptide, a polysaccharide, a water-soluble plastic such as PEG, or a PEG-modified polypeptide.

124. The method according to claim 123, wherein the polymer analyte is a polypeptide comprising at least 30 peptide units and includes positively and negatively charged residues.

125. The method according to claim 123 or 124, wherein the polypeptide is in a denatured / expanded state, and preferably the polypeptide is added in a pre-denatured state.

126. The method according to any one of claims 1 to 125, further comprising (c) measuring a change in ionic current caused by dislocation of a target polymer through the nanopores, preferably (c) measuring a change in current for states of (i) open channels, (ii) trapping of the polymer by the nanopores, and (iii) passing of the polymer from (ii) through the nanopores, more preferably the measurement comprising detecting a difference between states (i), (ii), and (iii).

127. A nanopore system for dislocating a polymer analyte through nanopores, wherein the system comprises nanopores contained in a membrane separating the fluid chamber of the nanopore system into a cis side and a trans side, the analyte is added to the cis side, and the nanopore system has an electroosmotic force (EOF) from cis to trans resulting from a net ionic current flow from cis to trans, the EOF from cis to trans overcomes the electrophoretic force (EPF) from trans to cis acting on the polymer analyte.

128. The method according to any one of claims 118 to 126 or the nanopore system according to claim 127, wherein the nanopore system has an EOF from cis to transform resulting from a net ionic current flow from cis to transform exceeding a total ionic current flow of 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, more preferably greater than 0.35 or less than -0.

35.

129. The method or nanopore system according to any one of claims 118 to 128, wherein the cis-to-trans EOF is controlled by modifying the charge of the nanopores (e.g., genetically engineered) or any combination thereof, by adjusting the pH, type and / or concentration of salts and / or osmotic pressure across the membrane of the nanopore system.

130. The method or nanopore system according to any one of claims 118 to 129, wherein the cis-to-trans EOF is regulated by modification of the nanopores and / or by an asymmetric salt distribution between the cis side and the trans side of the chamber.

131. The nanopore system has an ion selectivity P greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, most preferably greater than 3.0 or less than 0.

33. （＋） / P （－） A method or nanopore system according to any one of claims 118 to 130, comprising:

132. The method or nanopore system according to any one of claims 118 to 131, wherein the system has an ion selectivity P(+) / P(-) greater than 2.0, preferably greater than 2.5, and more preferably greater than 3.0, and a negative voltage is applied to the transformer side, and preferably the system comprises cation-selective (mutant) nanopores.

133. The method or nanopore system according to any one of claims 118 to 132, wherein the nanopores are biological nanopores, preferably biological nanopores having internal pore constriction in the range of 0.5 to 2 nm.

134. The method or nanopore system according to any one of claims 118 to 133, wherein the nanopores are alpha-helical or beta-barrel oligomer pore-forming toxins or porins, and preferably the nanopores are selected from the group consisting of erolysine (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, phage-derived portal proteins, and modified variants thereof, or ion-selective variants thereof.

135. The method or system according to any one of claims 118 to 134, wherein the nanopores include biological nanopores that have been modified, for example, by genetic engineering, to provide desired ion selectivity, and preferably the ion-selective nanopores have been modified to have a net charge in the region facing the lumen greater than 21, preferably greater than 28, more preferably greater than 35, and most preferably the net charge is negative.

136. The nanopore is a mutant CytK nanopore containing one or more amino acid substitutions selected from the group consisting of K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, and S151D, and the numbering corresponds to a CytK amino acid available in UniPro under accession number A0A2S1A9G3_9BACI, preferably the CytK mutant nanopore is K128D and K155D; K128D, K155D, and T116D (optionally further comprising T147D and / or S151D); K128D, K155D, and S120D (optionally further comprising Q122D, T147D, and / or S155D); the method or system according to any one of claims 118 to 135, comprising one of the amino acid substitution combinations of K128D, K155D, Q145D, and S151D.

137. An analytical device comprising an array of nanopore systems according to any one of claims 127 to 136.

138. Use of the method, nanopore system, or device according to any one of claims 118 to 137, preferably at the single-molecule level, for the detection and analysis of one or more target polymers, more preferably for the detection and analysis of one or more target polypeptides, for characterizing at least one feature of a target polymer.

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