Nanopore systems and methods for single molecule polymer analysis

Abstract

The present invention relates to devices and methods for analyzing target analytes using nanopore-based sensors, and more particularly, to methods, nanopore systems, and devices for single molecule analysis of polymers, such as polypeptides or polysaccharides. The present invention provides a method of translocating a non-nucleic acid-based polymeric analyte through a nanopore contained in a membrane separating a fluid chamber of a nanopore system into a cis-side and a trans-side, the method comprising adding the analyte to the cis-side and allowing it to translocate, wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) resulting from a cis-to-trans net ionic current, preferably wherein the cis-to-trans EOF originates from a ratio of cis-to-trans net ionic current to total ionic current greater than 0.2 or less than-0.2.

Description

[0001] Cross-reference

[0002] This application claims the benefit of European Application No. EP22204589.0, filed on October 28, 2022, the entire content of which is incorporated herein by reference.

[0003] Background

[0004] Characterizing and identifying analytes is an important aspect of scientific research. These scientific research may have important impacts on clinical and research work.

[0005] Overview

[0006] In one aspect, the present disclosure provides a method comprising: providing: a nanopore system, wherein the nanopore system comprises a fluidic chamber; and a membrane comprising a nanopore, wherein the membrane separates the fluidic chamber into a cis side and a trans side; and a non-nucleic acid-based polymeric analyte, wherein the non-nucleic acid-based polymeric analyte has a linear length greater than the channel length of the nanopore; translocating the non-nucleic acid-based polymeric analyte from the cis side of the fluidic chamber to the trans side, wherein the non-nucleic acid-based polymeric analyte comprises an elongated structure, wherein the nanopore system has a cis-to-trans electroosmotic force generated by a net cis-to-trans ionic current, wherein the cis-to-trans electroosmotic force resists the electrophoretic force acting in the opposite direction to the cis-to-trans electroosmotic force to translocate the non-nucleic acid-based polymeric analyte through the nanopore.

[0007] 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 fluidic chamber comprises a first solution and the trans side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of solute and the second solution comprises a second concentration of solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, the difference between the first concentration of the solute and the second concentration of the solute is configured to generate a cis-to-trans electroosmotic force upon application of an electric potential.

[0008] In some embodiments, the non-nucleic acid-based polymeric analyte is an unmodified (unlabeled) non-nucleic acid-based polymeric analyte. In some embodiments, the analyte is an unmodified analyte. In some cases, the unmodified analyte can be the wild-type version of the analyte. In some cases, the unmodified analyte may not contain any additional molecules conjugated to the unmodified analyte. In some embodiments, the analyte is a label-free or tag-free analyte. In some cases, the label-free analyte can include an analyte not conjugated to any peptide label, any protein label, any nucleic acid label, any carbohydrate label, any lipid label, or any combination thereof. In some cases, the tag-free analyte can include an analyte not conjugated to any peptide tag, any protein tag, any nucleic acid tag, any carbohydrate tag, any tag label, or any combination thereof. In some embodiments, the termini of the non-nucleic acid-based polymeric analyte lack three-dimensional structure. In some embodiments, at least a portion of the non-nucleic acid-based polymeric analyte is denatured. In some embodiments, when the non-nucleic acid-based polymeric analyte is stretched, the linear length of the non-nucleic acid-based polymeric analyte is greater than the channel length of the nanopore through the membrane. In some embodiments, the non-nucleic acid-based polymeric analyte contains at least about 25 repeating units. In some embodiments, the non-nucleic acid-based polymeric analyte contains peptide units, sugar units, water-soluble plastic monomers, or any combination thereof. In some embodiments, the non-nucleic acid-based polymeric analyte contains polypeptides, polysaccharides, or water-soluble plastics.

[0009] In some embodiments, the non-nucleic acid-based polymeric analyte contains a polypeptide having at least 30 peptide units. In some embodiments, the at least 30 peptide units include positively charged 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.

[0010] In some embodiments, the method further includes measuring a signal generated by translocation of the non-nucleic acid-based polymeric analyte through the nanopore.

[0011] In some embodiments, the measuring includes: measuring the signal in the following states: (a) the open channel of the nanopore; (b) the capture of the non-nucleic acid-based polymeric analyte by the nanopore; or (c) the non-nucleic acid-based polymeric analyte passing through the nanopore. In some embodiments, the measuring includes detecting the differences between states (a), (b), and (c). In some embodiments, the signal includes an ion current, a change in ion current, or a deviation thereof.

[0012] In some embodiments, the linear length of the non-nucleic acid-based polymer analyte is at least 1 kDa. In some embodiments, the linear length of the non-nucleic acid-based polymer analyte is at most 4,000 kDa. In some embodiments, the linear length of the non-nucleic acid-based polymer analyte is at least twice the length of the nanopore channel. In some embodiments, the linear length of the non-nucleic acid-based polymer analyte is at most twice the length of the nanopore channel. In some embodiments, the linear length of the non-nucleic acid-based polymer analyte is at least 3 nanometers.

[0013] In some embodiments, the cis-to-trans electroosmotic force includes a cis-to-trans net ionic current. In some embodiments, the cis-to-trans electroosmotic force is regulated by the pH value, type of salt, concentration of salt, osmotic pressure, modification of the nanopore, or any combination thereof on the membrane of the system. In some embodiments, the cis-to-trans electroosmotic force is regulated by a change in the charge of the nanopore. In some embodiments, the cis-to-trans electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane. In some embodiments, the ionic selectivity P(+) / P(-) of the nanopore is greater than 2.0. In some embodiments, the ionic selectivity P(+) / P(-) of the nanopore is less than 0.50.

[0014] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the trans side. In some embodiments, the applied voltage is a positive voltage on the trans side. In some embodiments, the amplitude of the applied voltage is less than 300 mV. In some embodiments, the amplitude of the applied voltage is greater than 20 mV. In some embodiments, the absolute relative net electroosmotic current under the applied voltage is greater than about 0.10 pA / mV. In some embodiments, the nanopore comprises an inner pore constriction of about 0.5 nanometers to about 2 nanometers (nm).

[0015] In some embodiments, the nanopore comprises an α-helical oligomeric pore structure. In some embodiments, the nanopore comprises a β-barrel oligomeric pore structure. In some embodiments, the nanopore comprises a recombinant nanopore. In some embodiments, the nanopore comprises a protein selected from Aerolysin (Aer), Cytolysin K (CytK), MspA, alpha-hemolysin (aHL), Curli subunit G (CsgG), Fragaceatoxin C (FraC), Lysenin, Outer membrane protein F (OmpF), Outer membrane protein G (OmpG), Ferric hydroxamate uptake protein (FhuA), phage derived portal protein, a modified variant thereof, or an ion-selective mutant thereof.

[0016] In some embodiments, the nanopore comprises a biological nanopore. In some embodiments, the biological nanopore is modified to restrict the passage of one or more ions through the nanopore channel. In some embodiments, the biological nanopore restricts the passage of one or more ions through the nanopore channel by altering the charge of the nanopore channel. In some embodiments, the net charge of the channel is negative. In some embodiments, the net charge of the channel is positive.

[0017] In some embodiments, the nanopore comprises a mutant CytK nanopore. In some embodiments, the mutant CytK nanopore comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions include K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof. In some embodiments, the 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 nanopore comprises one of the following combinations of amino acid substitutions: (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) combinations thereof. In some embodiments, the mutant CytK nanopore comprises one or more of the following combinations of amino acid substitutions: (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 (h) combinations thereof.

[0018] On the other hand, the present disclosure provides a system comprising: a fluid chamber; a membrane comprising a nanopore, wherein the membrane divides the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte through the nanopore using electroosmotic flow, wherein the non-nucleic acid-based polymer analyte comprises an elongated structure, and wherein the linear length of the non-nucleic acid-based polymer analyte is greater than the channel length of the nanopore; a pair of electrodes comprising a first electrode and a second electrode, wherein the first electrode is disposed on the cis side of the fluid chamber and the second electrode is disposed on the trans side of the fluid chamber, and wherein the pair of electrodes is configured to generate an electrophoretic force opposite to the direction of electroosmotic flow.

[0019] On the other hand, the present disclosure provides a system comprising: a fluid chamber; a membrane comprising nanopores, wherein the membrane separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based 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: generates an electrophoretic force acting in a direction opposite to the electroosmotic flow using the pair of electrodes, translocates the non-nucleic acid-based polymer analyte through the nanopores, and detects one or more signals related to at least one characteristic of the non-nucleic acid-based polymer analyte during or after the non-nucleic acid-based polymer analyte translocates through the nanopores, wherein the linear length of the non-nucleic acid-based polymer analyte is greater than the channel length of the nanopores.

[0020] In some embodiments, the controller uses the pair of electrodes to detect one or more signals related to at least one characteristic of the non-nucleic acid-based polymer analyte. In some embodiments, the electroosmotic flow is greater than the electrophoretic force.

[0021] 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 first concentration of solute, and the second solution contains a second concentration of solute.

[0022] In some embodiments, the solute includes an ion or an osmolyte. In some embodiments, the difference between the first concentration of solute and the second concentration of solute is configured to generate electroosmotic flow upon application of an electric potential.

[0023] In some embodiments, the electroosmotic flow includes a net ion current from cis to trans. In some embodiments, the electroosmotic flow is regulated by the pH value on the system membrane, the type of salt, the concentration of salt, the osmotic pressure, the modification of the nanopores, or any combination thereof. In some embodiments, the electroosmotic flow is regulated by changing the charge of the nanopores. In some embodiments, the electroosmotic flow is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane. In some embodiments, the ion selectivity P(+) / P(-) of the nanopores is greater than 2.0. In some embodiments, the ion selectivity P(+) / P(-) of the nanopores is less than 0.50.

[0024] In some embodiments, a pair of electrodes can be configured to provide an applied voltage. The applied voltage can be across the membrane. The applied voltage can generate an electrophoretic force. In some embodiments, a pair of electrodes can be configured to provide an electrophoretic force across the membrane. The pair of electrodes can be configured for measuring signals.

[0025] In some embodiments, the nanopore comprises an α-helical oligomeric pore structure. In some embodiments, the nanopore comprises a β-barrel oligomeric pore structure. In some embodiments, the nanopore comprises a recombinant nanopore. In some embodiments, the nanopore comprises a protein such as aerolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, fragaceatoxin C (FraC), lumbrokinase, OmpF, OmpG, FhuA, a phage-derived portal protein, a modified variant thereof, or an ion-selective mutant thereof.

[0026] In some embodiments, the nanopore comprises a biological nanopore. In some embodiments, the biological nanopore is modified to restrict the passage of one or more ions through the nanopore channel. In some embodiments, the biological nanopore restricts the passage of one or more ions through the nanopore channel by altering the charge of the nanopore channel. In some embodiments, the net charge of the channel is negative. In some embodiments, the net charge of the channel is positive.

[0027] In some embodiments, the nanopore comprises a mutant CytK nanopore. In some embodiments, the mutant CytK nanopore comprises one or more amino acid substitution ions. 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 nanopore comprises one of the following combinations of amino acid substitutions: (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) combinations thereof. In some embodiments, the mutant CytK nanopore comprises one or more of the following combinations of amino acid substitutions: (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 (h) combinations thereof. In some embodiments, the non-nucleic acid-based polymeric analyte is an unmodified (unlabeled) non-nucleic acid-based polymeric analyte. In some embodiments, the termini of the non-nucleic acid-based polymeric analyte lack three-dimensional structure. In some embodiments, at least a portion of the non-nucleic acid-based polymeric analyte is denatured. In some embodiments, when the non-nucleic acid-based polymeric analyte is stretched, the linear length of the non-nucleic acid-based polymeric analyte is greater than the channel length of the nanopore through the membrane. In some embodiments, the non-nucleic acid-based polymeric analyte comprises at least about 25 repeating units.

[0028] In some embodiments, the non-nucleic acid-based polymeric analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof. In some embodiments, the non-nucleic acid-based polymeric analyte comprises polypeptides, polysaccharides, or water-soluble plastics. In some embodiments, the non-nucleic acid-based polymeric analyte comprises a polypeptide having at least 30 peptide units. In some embodiments, the at least 30 peptide units comprise positively charged 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 non-nucleic acid-based polymeric analyte has a linear length of at least 1 kDa. In some embodiments, the non-nucleic acid-based polymeric analyte has a linear length of at most 4,000 kDa. In some embodiments, the non-nucleic acid-based polymeric analyte has a linear length that is at least 2 times the channel length of the nanopore. In some embodiments, the non-nucleic acid-based polymeric analyte has a linear length that is at most 2 times the channel length of the nanopore.

[0029] In another aspect, the present disclosure provides an apparatus comprising a system array, the system comprising any of the systems disclosed herein.

[0030] In another aspect, the present disclosure provides the use of any of the methods, kits, or apparatuses disclosed herein for characterizing at least one feature of a non-nucleic acid-based polymeric analyte.

[0031] In another aspect, the present disclosure provides the use of any of the systems disclosed herein for characterizing at least one feature of a non-nucleic acid-based polymeric analyte.

[0032] In another aspect, the present disclosure provides the use of any of the methods, kits, or apparatuses disclosed herein for detecting and analyzing one or more non-nucleic acid-based polymeric analytes at the single molecule level.

[0033] In another aspect, the present disclosure provides the use of any of the systems disclosed herein for detecting and analyzing one or more non-nucleic acid-based polymeric analytes at the single molecule level.

[0034] In another aspect, the present disclosure provides the use of any of the methods, kits, or apparatuses disclosed herein for detecting and analyzing one or more polypeptides.

[0035] In another aspect, the present disclosure provides the use of any of the systems disclosed herein for detecting and analyzing one or more polypeptides.

[0036] Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0037] Another aspect of the present disclosure provides a system that includes one or more computer processors and a computer memory coupled thereto. The computer memory includes machine-executable code that, when executed by the one or more computer processors, implements any of the methods described above or elsewhere in this document.

[0038] Another aspect of the present disclosure provides a method for translocating a non-nucleic acid-based polymeric analyte through a nanopore, the nanopore being included in a membrane that separates the fluidic chamber of the nanopore system into a cis side and a trans side, the method including adding the polymeric analyte to the cis side of the nanopore system and allowing the polymeric analyte to translocate, wherein the length of the elongated polymeric analyte is greater than the length of the central channel of the nanopore in a direction perpendicular to the membrane.

[0039] And wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) generated by a net cis-to-trans ionic current, and wherein the cis-to-trans EOF overcomes the trans-to-cis electrophoretic force (EPF) acting on the polymeric analyte.

[0040] In some embodiments, the polymeric analyte is an unmodified (unlabeled) analyte.

[0041] In some embodiments of any of the foregoing embodiments, the ends of the polymer are unstructured, preferably, wherein the polymer is denatured or partially denatured.

[0042] In some embodiments of any of the foregoing embodiments, the polymeric analyte includes at least 25 repeating units, preferably at least 35 repeating units, more preferably at least 45 repeating units.

[0043] In some embodiments of any of the foregoing embodiments, the polymeric analyte is synthetic, semi-synthetic, or of biological origin, such as a biopolymer, preferably comprising or consisting of peptide units, sugar units, and water-soluble plastic monomers and any combination thereof. In some embodiments, the polymeric analyte is a polypeptide, a polysaccharide, or a water-soluble plastic, such as polyethylene glycol (PEG) or a PEGylated polypeptide. In some embodiments, the polymeric analyte is a polypeptide of at least 30 peptide units, comprising positively and negatively charged residues. In some embodiments, the polypeptide is in a denatured / unfolded state, preferably the polypeptide is added in a pre-denatured state.

[0044] In some embodiments of any of the foregoing embodiments, it further includes (c) measuring the change in ionic current caused by the translocation of the target polymer through the nanopore. Preferably, wherein operation (c) includes measuring the change in current in the following states: (i) open channel, (ii) nanopore capturing the polymer, and (iii) the polymer from (ii) passing through the nanopore. More preferably, wherein the measurement includes detecting the differences between states (i), (ii), and (iii).

[0045] On the other hand, the present disclosure provides a nanopore system for translocating a polymer analyte through a nanopore. The system includes a nanopore contained in a membrane that separates the fluid chambers of the nanopore system into a cis side and a trans side, where the analyte will be added to the cis side. The nanopore system has a cis-to-trans electroosmotic force (EOF) generated by the net ion current flow from cis to trans, and wherein the cis-to-trans EOF overcomes the trans-to-cis electrophoretic force (EPF) acting on the polymer analyte.

[0046] In some embodiments of any of the foregoing embodiments, the nanopore system has a cis-to-trans EOF generated by a ratio of the cis-to-trans net ion current to the total ion current 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.

[0047] In some embodiments of any of the foregoing embodiments, the cis-to-trans EOF is set by adjusting the pH, type and / or concentration of salt, and / or osmotic pressure on the membrane of the nanopore system, by changing (e.g., through genetic engineering) the charge of the nanopore, or any combination thereof.

[0048] In some embodiments of any of the foregoing embodiments, the cis-to-trans EOF is set by changing the asymmetric salt distribution between the cis side and the trans side of the nanopore and / or the chamber.

[0049] In some embodiments of any of the foregoing embodiments, the ion selectivity P(+) / P(-) of the nanopore system is greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, and most preferably greater than 3.0 or less than 0.33.

[0050] In some embodiments of any of the foregoing embodiments, the ion selectivity P(+) / P(-) of the system is greater than 2.0, preferably greater than 2.5, more preferably greater than 3.0, and wherein a negative applied voltage is present on the trans side. Preferably, wherein the system includes a cation-selective (mutated) nanopore.

[0051] In some embodiments of any of the foregoing embodiments, the nanopore is a biological nanopore, preferably having an inner pore constriction structure in the range of 0.5 - 2 nanometers.

[0052] In some embodiments of any of the foregoing embodiments, the nanopore is an α-helix or β-barrel oligomeric pore-forming toxin or porin. Preferably, the nanopore is selected from aerolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, fragaceatoxin C (FraC), lumbrokinase, phage-derived portal protein and its modified variants, or its ion-selective mutants.

[0053] In some embodiments of any of the foregoing embodiments, the nanopore includes, for example, a biological nanopore modified by genetic engineering to provide the desired ion selectivity. Preferably, the ion-selective nanopore is modified such that the net charge of the region facing the cavity > 21, preferably > 28, more preferably > 35, and most preferably where the net charge is negative.

[0054] In some embodiments of any of the foregoing embodiments, the nanopore is a mutant CytK nanopore, which contains one or more amino acid substitutions selected from the group consisting of K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, and S151D, where the numbering corresponds to the CytK amino acids with the accession number A0A2S1A9G3_9BACI in UniProt. Preferably, the CytK mutant nanopore contains one of the following combinations of amino acid substitutions: K128D and K155D; K128D, K155D, and T116D, optionally further containing T147D and / or S151D; K128D, K155D, and S120D, optionally further containing Q122D, T147D, and / or S155D; K128D, K155D, Q145D, and S151D.

[0055] Another aspect of the present disclosure provides an analysis device that includes an array of nanopore systems according to any of the foregoing embodiments.

[0056] Another aspect of the present disclosure provides the use of a method, nanopore system, or device according to any of the foregoing embodiments for characterizing at least one feature of a target polymer, preferably for detecting and analyzing one or more target polymers at the single-molecule level, and more preferably for detecting and analyzing one or more target polypeptides.

[0057] Those skilled in the art will readily understand other aspects and advantages of the present disclosure from the following detailed description, in which only exemplary embodiments of the present disclosure are shown and described. It should be understood that the present disclosure can adopt other different embodiments, and several details can be modified in various obvious aspects, all of which do not depart from the present disclosure. Therefore, the drawings and descriptions should be regarded as exemplary rather than restrictive.

[0058] Incorporation by reference

[0059] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If a publication, patent, or patent application incorporated by reference conflicts with the disclosure in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material.

[0060] Detailed description

[0061] Although various embodiments of the present invention have been shown and described herein, those skilled in the art will understand that these embodiments are provided by way of example only. Those skilled in the art may make various changes, alterations, and substitutions without departing from the spirit of the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0062] When a numerical value is described in a range format, it should be understood that such disclosure includes the disclosure of all possible sub-ranges within such range, as well as the specific numerical values that fall within such range, whether or not the specific numerical values or specific sub-ranges are explicitly stated.

[0063] As used herein, the terms "a", "an", and "the" generally refer to both the singular and the plural, unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or", unless otherwise stated.

[0064] When the terms "at least", "greater than", or "greater than or equal to" appear before the first value in a series of two or more numerical values, these terms apply to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0065] When the terms "not greater than", "less than", or "less than or equal to" appear before the first value in a series of two or more numerical values, these terms apply to each value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0066] In some embodiments, methods related to analyte analysis are provided.

[0067] One aspect of the present disclosure provides a method that includes providing a nanopore system. The nanopore system can include a fluid chamber. The fluid chamber can be divided into a cis side and a trans side. The method can further include providing a non-nucleic acid-based polymer analyte. In some cases, the linear length of the non-nucleic acid-based polymer analyte can be greater than the channel length of the nanopore. The method can further include translocating the non-nucleic acid-based polymer analyte. In some cases, the non-nucleic acid-based polymer analyte can translocate from the cis side to the trans side of the fluid chamber. The non-nucleic acid-based polymer analyte can include an elongated structure. In some cases, the nanopore system can have a cis-to-trans electroosmotic force. The electroosmotic force can include a cis-to-trans net ionic current. In some cases, the cis-to-trans electroosmotic force can resist the electrophoretic force to translocate the non-nucleic acid-based polymer analyte through the nanopore. In some cases, the electrophoretic force can act in a direction opposite to the cis-to-trans electroosmotic force.

[0068] One aspect of the present disclosure provides a system that includes a nanopore system. The nanopore system can include a fluid chamber. The system can further include a membrane. In some cases, the membrane can include a nanopore. In some cases, the membrane can divide the fluid chamber into a cis side and a trans side. The fluid chamber can be divided into a cis side and a trans side. In some cases, the cis side can contain a first solution. In some cases, the trans side can contain a second solution. In some cases, the cis side can contain a first solution and the trans side can contain a second solution. The system can further include a non-nucleic acid-based polymer analyte. The first solution and the second solution can be configured to translocate the non-nucleic acid-based polymer analyte through the nanopore using electroosmotic flow. In some cases, the non-nucleic acid-based polymer analyte can have an elongated structure. In some cases, the linear length of the non-nucleic acid-based polymer analyte can be greater than the channel length of the nanopore. The system can further include a pair of electrodes. In some cases, the pair of electrodes can include a first electrode and a second electrode. The first electrode can be disposed on the cis side of the fluid chamber. The second electrode can be disposed on the trans side of the fluid chamber. The first electrode can be disposed on the cis side of the fluid chamber and the second electrode can be disposed on the trans side of the fluid chamber. In some cases, the pair of electrodes can be configured to generate an electrophoretic force. In some cases, the electrophoretic force can act in a direction opposite to the electroosmotic flow.

[0069] In some embodiments, the change in ionic current can be measured when the analyte translocates through the nanopore. In some cases, the change in ionic current can be measured by a voltage-based chip. In some cases, the voltage-based chip can measure the voltage and / or current change across the nanopore. In some cases, the voltage-based chip can be a trans electrode.

[0070] Characterization methods may involve measuring the ionic current through the pore, typically by measuring the current. Alternatively, the ionic flow through the pore can be measured optically, such as the method disclosed by Heron et al. in J. Am. Chem. Soc. 9 Vol. 131, No. 5, 2009. Thus, the device may also include a circuit that is capable of applying an electric potential and measuring the electrical signals across the membrane and the pore. The characterization method can 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 can be attached to the analyte.

[0071] In some embodiments, the analyte includes a polymeric analyte. The analyte can include a nucleic acid-based polymeric analyte or a non-nucleic acid-based polymeric analyte. The analyte can be synthetic, semi-synthetic, or of biological origin. For example, a synthetic analyte can include an analyte constructed by a non-biological chemical process, such as polyethylene glycol (PEG), a synthetically constructed protein peptide, or a synthetically constructed DNA molecule. A biological analyte can include an analyte produced by a biological process, such as a protein produced by a cell or a system that employs cell (or cell-derived) components (e.g., an in vitro enzymatic translation system). A semi-synthetic analyte can include portions produced from biological and non-biological sources, such as a biologically produced protein conjugated to a PEG molecule. Possible electrical measurements can include current measurements, impedance measurements, tunneling, electron tunneling measurements (Ivanov AP et al., Nano Lett. January 12, 2011; 11(1):279 - 85), field effect transistor (FET) measurements (International Application WO 2005 / 124888), voltage field effect transistor (FET) measurements, or any combination thereof. In some embodiments, the signal can be an electron tunneling through a solid-state nanopore or a voltage field effect transistor (FET) measurement through a solid-state nanopore.

[0072] Characterization methods can include measuring the ionic current through the pore by measuring the current. Alternatively, the ionic flow through the pore can be measured optically, such as that disclosed by Heron et al. in J. Am. Chem. Soc. 9 Vol. 131, No. 5, 2009. Thus, the device may also include a circuit that is capable of applying an electric potential and measuring the electrical signals across the membrane and the pore. The characterization method can be performed using a patch clamp or a voltage clamp. The characterization method preferably uses a voltage clamp.

[0073] The characterization method can be performed on a well or an array of nanopore channels, where each array contains 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more pores or nanopore channels.

[0074] The characterization method can involve measuring the current flowing through the pore. The method is typically carried out with a voltage applied across the membrane and the pore. The voltage used is typically from +2V to -2V, and typically from -400mV to +400mV. The lower limit of the voltage used is preferably -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and the upper limit is independently selected from +10mV, 20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. The voltage used is more preferably in the range of -120mV to 240mV, and most preferably in the range of 120mV to 220mV. By using an increased applied potential, the discrimination of the pore for different nucleotides can be enhanced.

[0075] In some embodiments, the analyte comprises a protein or a peptide. The protein or peptide can comprise a folded state, an unfolded state, or an intermediate state thereof. The folded state includes a state of the protein or peptide in which the polymer is in a low energy state such that the protein or peptide maintains a two-dimensional or three-dimensional structure. This low energy state can be based on the interactions between the amino acids of the peptide or protein. The unfolded state can include a state of the protein or peptide in which the polymer is in a high energy state such that the protein or peptide does not maintain a two-dimensional or three-dimensional structure. The intermediate state between the folded state and the unfolded state can be an energy state in which some part or parts of the peptide or protein maintain a two-dimensional or three-dimensional structure while other parts of the peptide or protein do not maintain a two-dimensional or three-dimensional structure.

[0076] In some embodiments, the analyte can include a non-nucleic acid-based polymeric analyte. In some embodiments, a portion of the non-nucleic acid-based polymeric analyte can include a nucleic acid molecule. In certain cases, the portion of the non-nucleic acid polymeric analyte can be from 0% to about 100% of the non-nucleic acid polymeric analyte. In certain cases, the portion of the non-nucleic acid polymeric analyte can 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 polymeric analyte. In some cases, the portion of the non-nucleic acid polymeric analyte can be at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5% or at most about 0% of the non-nucleic acid polymeric analyte. In certain cases, the portion of the non-nucleic acid polymeric analyte can be about 0%, 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 75%, about 80%, about 85%, about 90%, about 95% or about 100% of the non-nucleic acid polymeric analyte.

[0077] In some embodiments, a portion of the non-nucleic acid-based polymer analyte can comprise an oligosaccharide molecule. In certain cases, the portion of the non-nucleic acid polymer analyte can be from 0% to about 100% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte can 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 can be at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or at most about 0% of the non-nucleic acid polymer analyte. In certain cases, the portion of the non-nucleic acid polymer analyte can be about 0%, 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 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the non-nucleic acid polymer analyte.

[0078] An analyte can have a contour length. In some embodiments, the contour length can include the length of the analyte when it is not fully unfolded. In certain cases, the analyte can be between 1% and about 100% unfolded. In some cases, the analyte can 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 analyte can be at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than 1% unfolded. In certain cases, the analyte can be 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% unfolded. In certain cases, the linear length of the analyte can be the length of the analyte when it is 100% unfolded. The analyte can include a linear length. In some embodiments, the contour length of the analyte can be the linear length of the analyte. The linear length can be the length of the analyte in an unfolded state. In some embodiments, the linear length of the analyte can be from about 3 nanometers (nm) to about 5,000 nm. In some embodiments, the linear length of the analyte can be from 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, about 70 nm to about 75 nm, about 75 nm to about 80 nm, about 80 nm to about 85 nm, about 85 nm to about 90 nm, about 90 nm to about 95 nm, about 95 nm to about 100 nm, about 100 nm to about 150 nm, about 150 nm to about 200 nm, about 200 nm to about 250 nm, about 250 nm to about 300 nm, about 300 nm to about 350 nm, about 350 nm to about 400 nm, about 400 nm to about 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650 nm, about 650 nm to about 700 nm, about 700 nm to about 750 nm, about 750 nm to about 800 nm, about 800 nm to about 850 nm, about 850 nm to about 900 nm, about 900 nm to about 950 nm, about 950 nm to about 1,000 nm, about 1,000 nm to about 1,100 nm, about 1,100 nm to about 1,200 nm, about 1,200 nm to about 1,300 nm, about 1,300 nm to about 1,400 nm, about 1,400 nm to about 1,500 nm, about 1,500 nm to about 1,600 nm, about 1,600 nm to about 1,700 nm, about 1,700 nm to about 1,800 nm, about 1,800 nm to about 1,900 nm, about 1,900 nm to about 2,000 nm, about 2,000 nm to about 2,100 nm, about 2,100 nm to about 2,200 nm, about 2,200 nm to about 2,300 nm, about 2,300 nm to about 2,400 nm, about 2,400 to about 2,500 nm, about 2,500 nm to about 2,600 nm, about 2,600 nm to about 2,700 nm, about 2,700 nm to about 2,800 nm, about 2,800 nm to about 2,900 nm, about 2,900 nm to about 3,000 nm, about 3,000 nm to about 3,100 nm, about 3,200 nm to about 3,300 nm, about 3,300 nm to about 3,400 nm, about 3,400 nm to about 3,500 nm, about 3,500 nm to about 3,600 nm, about 3,600 nm to about 3,700 nm, about 3,700 nm to about 3,800 nm, from about 3,800 nm to about 3,900 nm, from about 3,900 nm to about 4,000 nm, from about 4,000 nm to about 4,100 nm, from about 4,100 nm to about 4,200 nm, from about 4,200 nm to about 4,300 nm, from about 4,300 nm to about 4,400 nm, from about 4,400 nm to about 4,500 nm, from about 4,500 nm to about 4,600 nm, from about 4,600 nm to about 4,700 nm, from about 4,700 nm to about 4,800 nm, from about 4,800 nm to about 4,900 nm or from about 4,900 nm to about 5,000 nm.,

[0079] In some embodiments, the linear length of the analyte can be 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, 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 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 nm, at least about 160 nm, at least about 170 nm, at least about 180 nm, at least about 190 nm, at least about 200 nm, at least about 210 nm, at least about 220 nm, at least about 230 nm, at least about 240 nm, at least about 250 nm, at least about 260 nm, at least about 270 nm, at least about 280 nm, at least about 290, at least about 300 nm, at least about 310 nm, at least about 320 nm, at least about 330 nm, at least about 340 nm, at least about 350 nm, at least about 360 nm, at least about 370 nm, at least about 380 nm, at least about 390 nm, at least about 400 nm, at least about 410 nm, at least about 420 nm, at least about 430 nm, at least about 440 nm, at least about 450 nm, at least about 460 nm, at least about 470 nm, at least about 480 nm, at least about 490 nm, at least about 500 nm, at least about 510 nm, at least about 520 nm, at least about 530 nm, at least about 540 nm, at least about 550 nm, at least about 560 nm, at least about 570 nm, at least about 580 nm, at least about 590 nm, at least about 600 nm, at least about 610 nm, at least about 620 nm, at least about 630 nm, at least about 640 nm, at least about 650 nm, at least about 660 nm, at least about 670 nm, at least about 680 nm, at least about 690 nm, at least about 700 nm, at least about 710 nm, at least about 720 nm, at least about 730 nm, at least about 740 nm, at least about 750 nm, at least about 760 nm, at least about 770 nm, at least about 780 nm, at least about 790 nm, at least about 800 nm, at least about 810 nm, at least about 820 nm, at least about 830 nm, at least about 840 nm, at least about 850 nm, at least about 860 nm, at least about 870 nm, at least about 880 nm, at least about 890 nm, at least about 900 nm, at least about 910 nm, at least about 920 nm, at least about 930 nm, at least about 940 nm, at least about 950 nm, at least about 960 nm, at least about 970 nm, at least about 980 nm, at least about 990 nm, at least about 1,000 nm, at least about 1,100 nm, at least about 1,200 nm, at least about 1,300 nm, at least about 1,400 nm, at least about 1,500 nm, at least about 1,600 nm, at least about 1,700 nm, at least about 1,800 nm, at least about 1,900 nm, at least about 2,000 nm, at least about 2,100 nm, at least about 2,200 nm, at least about 2,300 nm, at least about 2,400 nm, at least about 2,500 nm, at least about 2,600 nm, at least about 2,700 nm, at least about 2,800 nm, at least about 2,900 nm, at least about 3,000 nm, at least about 3,100 nm, at least about 3,200 nm, at least about 3,300 nm, at least about 3,400 nm, 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 greater than about 5,000 nm.,

[0080] In some embodiments, the linear length of the analyte can be at most about 5,000 nm, at most about 4,900 nm, at most about 4,800 nm, at most about 4,700 nm, at most about 4,600 nm, at most about 4,500 nm, at most about 4,400 nm, at most about 4,300 nm, at most about 4,200 nm, at most about 4,100 nm, at most about 4,000 nm, at most about 3,900 nm, at most about 3,800 nm, at most about 3,700 nm, at most about 3,600 nm, at most about 3,500 nm, at most about 3,400 nm, at most about 3,300 nm, at most about 3,200 nm, at most about 3,100 nm, at most about 3,000 nm, at most about 2,900 nm, at most about 2,800 nm, at most about 2,700 nm, at most about 2,600 nm, at most about 2,500 nm, at most about 2,400 nm, at most about 2,300 nm, at most about 2,200 nm, at most about 2,100 nm, at most about 2,000 nm, at most about 1,900 nm, at most about 1,800 nm, at most about 1,700 nm, at most about 1,600 nm, at most about 1,500 nm, at most about 1,400 nm, at most about 1,300 nm, at most about 1,200 nm, at most about 1,100 nm, at most about 1,000 nm, up to about 990 nm, up to about 980 nm, up to about 970 nm, up to about 960 nm, up to about 950 nm, up to about 940 nm, up to about 930 nm, up to about 920 nm, up to about 910 nm, up to about 900 nm, up to about 890 nm, up to about 880 nm, up to about 870 nm, up to about 860 nm, up to about 850 nm, up to about 840 nm, up to about 830 nm, up to about 820 nm, up to about 810 nm, up to about 800 nm, up to about 790 nm, 780 nm, up to about 770 nm, up to about 760 nm, up to about 750 nm, up to about 740 nm, up to about 730 nm, up to about 720 nm, up to about 710 nm, up to about 700 nm, up to about 690 nm, up to about 680 nm, up to about 670 nm, up to about 660 nm, up to about 650 nm, up to about 640 nm, up to about 630 nm, up to about 620 nm, up to about 610 nm, up to about 600 nm, up to about 590 nm, up to about 580 nm, up to about 570 nm, up to about 560 nm, up to about 550 nm, up to about 540 nm, up to about 530 nm, up to about 520 nm, up to about 510 nm, up to about 500 nm, up to about 490 nm, up to about 480 nm, up to about 470 nm, up to about 460 nm, up to about 450 nm, up to about 440 nm, up to about 430 nm, up to about 420 nm, up to about 410 nm, up to about 400 nm, up to about 390 nm, up to about 380 nm, up to about 370 nm, up to about 360 nm, up to about 350 nm, up to about 340 nm, up to about 330 nm, up to about 320 nm, up to about 310 nm, up to about 300 nm, up to about 290 nm, up to about 280 nm, up to about 270 nm, up to about 260 nm, up to about 250 nm, up to about 240 nm, up to about 230 nm, up to about 220 nm, up to about 210 nm, up to about 200 nm, up to about 190 nm, up to about 180 nm, up to about 170 nm, up to about 160 nm, up to about 150 nm, up to about 140 nm, up to about 130 nm, up to about 120 nm, up to about 110 nm, up to about 100 nm, up to about 95 nm, up to about 90 nm, up to about 85 nm, up to about 80 nm, up to about 75 nm, up to about 70 nm, up to about 65 nm, up to about 60 nm, up to about 55 nm, up to about 50 nm, up to about 45 nm, up to about 40 nm, up to about 35 nm, up to about 30 nm, about 25 nanometers, up to about 20 nanometers, up to about 15 nanometers, up to about 10 nanometers, up to about 5 nanometers, up to about 3 nanometers or less than about 3 nanometers.,

[0081] In some embodiments, the linear length of the analyte can be about 3 nm, about 4 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, about 700 nm, about 710 nm, about 720 nm, about 730 nm, about 740 nm, about 750 nm, about 760 nm, about 770 nm, about 780 nm, about 790 nm, about 800 nm, about 810 nm, about 820 nm, about 830 nm, about 840 nm, about 850 nm, about 860 nm, about 870 nm, about 880 nm, about 890 nm, about 900 nm, about 910 nm, about 920 nm, about 930 nm, about 940 nm 950 nm, about 960 nm, about 970 nm, about 980 nm, about 990 nm, about 1,000 nm, about 1,100 nm, about 1,200 nm, about 1,300 nm, about 1,400 nm, about 1,500 nm, about 1,600 nm, about 1,700 nm, about 1,800 nm, about 1,900 nm, about 2,000 nm, about 2,100 nm, about 2,200 nm, about 2,300 nm, about 2,400 nm, about 2,500 nm, about 2,600 nm, about 2,700 nm, about 2,800 nm, about 2,900 nm, about 3,000 nm, about 3,100 nm, about 3,200 nm 3,300 nm, about 3,400 nm, about 3,500 nm, about 3,600 nm, about 3,700 nm, about 3,800 nm, about 3,900 nm, about 4,000 nm, about 4,100 nm, about 4,200 nm, about 4,300 nm, about 4,400 nm, about 4,500 nm, about 4,600 nm, about 4,700 nm, about 4,800 nm, about 4,900 nm, or about 5,000 nm.,

[0082] An analyte in an unfolded state may or may not contain secondary structure elements. Secondary structure elements can include α-helices, β-helices, turns, or β-sheets. The helix can be left-handed or right-handed. An analyte in an unfolded state can be fully or partially unfolded. The contour length can be the length when the two ends of the polymeric analyte fully extend to each other. Alternatively, in some cases, the contour length of the analyte can be the length when the two ends of the analyte do not fully extend to each other. In some embodiments, the analyte can include a structured portion, an unstructured portion, a denatured portion, a partially denatured portion, or a combination thereof. In some embodiments, the analyte includes 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 can include a structured portion, an unstructured portion, a denatured portion, a partially denatured portion, or a combination thereof.

[0083] The analyte can include repeating units. In some embodiments, the analyte can include from about 2 to about 100 repeating units. In some cases, the analyte can include from about 2 to about 5 repeating units, from about 5 to about 10 repeating units, from about 10 to about 15 repeating units, from about 15 to about 20 repeating units, from about 20 to about 25 repeating units, from about 25 to about 30 repeating units, from about 30 to about 35 repeating units, from about 35 to about 40 repeating units, from about 40 to about 45 repeating units, from about 45 to about 50 repeating units, from about 50 to about 55 repeating units, from about 55 to about 60 repeating units, from about 60 to about 65 repeating units, from about 65 to about 70 repeating units, from about 70 to about 75 repeating units, from about 75 to about 80 repeating units, from about 80 to about 85 repeating units, from about 85 to about 90 repeating units, from about 90 to about 95 repeating units, or from about 95 to about 100 repeating units.

[0084] In some embodiments, the analyte may comprise at least about 2 repeat units, at least about 3 repeat units, at least about 4 repeat units, at least about 5 repeat units, at least about 10 repeat units, at least about 15 repeat units, at least about 20 repeat units, at least about 25 repeat units, at least about 30 repeat units, at least about 35 repeat units, at least about 40 repeat units, at least about 45 repeat units, at least about 50 repeat units, at least about 55 repeat units, at least about 60 repeat units, at least about 65 repeat units, at least about 70 repeat units, at least about 75 repeat units, at least about 80 repeat units, at least about 85 repeat units, at least about 90 repeat units, at least about 95 repeat units, at least about 100 repeat units, or more than 100 repeat units. In some embodiments, the analyte may comprise at most about 100 repeat units, at most about 95 repeat units, at most about 90 repeat units, at most about 85 repeat units, at most about 80 repeat units, at most about 75 repeat units, at most about 70 repeat units, at most about 65 repeat units, at most about 60 repeat units, at most about 55 repeat units, at most about 50 repeat units, at most about 45 repeat units, at most about 40 repeat units, at most about 35 repeat units, at most about 30 repeat units, at most about 25 repeat units, at most about 20 repeat units, at most about 15 repeat units, at most about 10 repeat units, at most about 5 repeat units, at most about 4 repeat units, at most about 3 repeat units, at most about 2 repeat units, or fewer than 2 repeat units. In some embodiments, the analyte may comprise about 2 repeat units, about 3 repeat units, about 4 repeat units, about 5 repeat units, about 10 repeat units, about 15 repeat units, about 20 repeat units, about 25 repeat units, about 30 repeat units, about 35 repeat units, about 40 repeat units, about 45 repeat units, about 50 repeat units, about 55 repeat units, about 60 repeat units, about 65 repeat units, about 70 repeat units, at least about 75 repeat units, at least about 80 repeat units, about 85 repeat units, about 90 repeat units, about 95 repeat units, or about 100 repeat units.

[0085] The unit can include a peptide unit, a sugar unit, a lipid unit, a nucleotide, a water-soluble plastic monomer, or a combination thereof. The analyte can include a polypeptide, a polysaccharide, a lipid, a nucleic acid, a water-soluble plastic, or a combination thereof. In some embodiments, the analyte can be charged. The charge can be a positive charge or a negative charge. The charge can be uniformly distributed or non-uniformly distributed on the analyte. In some embodiments, the charge can be caused by an amino acid residue. The amino acid residue can be a natural or mutant residue. In some cases, the mutant residue can be a point mutation in the analyte. In some cases, the mutant residue can be a residue different from the wild-type sequence of the analyte. In some embodiments, the analyte can contain a peptide. The peptide can include a polypeptide or a protein. The protein can be a full-length protein or a truncated protein. The truncated protein (e.g., a peptide) can be a protein shorter in length than when it was initially prepared. For example, the protein may be shortened due to cleavage (e.g., by a peptidase) or degradation (e.g., due to acidic or basic conditions). The protein can contain a natural protein sequence or a sequence that modifies the protein sequence. The sequence can be modified by mutation, deletion, or insertion of the sequence. The sequence can be a combination of sequences. For example, a first natural sequence can be appended or inserted into a second natural sequence to form a third sequence composed of the combination of the first sequence and the second sequence.

[0086] In another aspect, the present disclosure provides a system for determining one or more characteristics 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 trans side. The cis side can contain a fluid solution. The trans side can contain a fluid solution. The fluid solution can be configured to provide electroosmotic flow, also known as electroosmotic force. The electroosmotic force can act across the entire membrane. In some embodiments, the membrane includes nanopores. In some embodiments, a pair of electrodes is provided. The pair of electrodes can be arranged such that one electrode is on the cis side of the fluid chamber and the other electrode is on the trans side of the fluid chamber. In some embodiments, an electrophoretic force is provided.

[0087] In another aspect, the present disclosure provides methods for determining one or more characteristics of an analyte. In some embodiments, the method includes translocating the analyte through a nanopore. The translocation can be assisted by electroosmotic forces, electrophoretic forces, or a combination thereof. The electroosmotic forces, electrophoretic forces, or a combination thereof can impede such translocation. In some embodiments, the analyte is in a pre-denatured state prior to translocation. In some embodiments, the analyte can translocate through the nanopore in an elongated form. In some cases, the elongated form of the analyte may not contain a three-dimensional structure. In some cases, the elongated form of the analyte can be a completely linear structure. In some cases, the elongated form of the analyte can be a linear structure. In some cases, the analyte can translocate through the nanopore in a folded structure. In some cases, the analyte in the folded structure can include a non-elongated analyte. In some cases, in the analyte, the non-elongated structure may not contain any three-dimensional structure. In some cases, the analyte having a folded structure can include a three-dimensional structure. In some embodiments, the method includes measuring a signal. The signal can be caused or affected by the translocation of the analyte. In some embodiments, one or more analytes are translocated. The signal of one or more translocated analytes can be measured.

[0088] In some embodiments, the analyte translocation through the nanopore occurs in the cis-to-trans direction. In some embodiments, the analyte translocation through the nanopore occurs in the trans-to-cis direction. In some embodiments, the analyte translocation through the nanopore occurs in the direction of the electroosmotic force (EOF). In some embodiments, the analyte translocation through the nanopore occurs in a direction opposite to the direction of the electrophoretic force (EPF). In some embodiments, the analyte translocation through the nanopore occurs in the same direction as the EOF direction or in a direction opposite to the EPF direction.

[0089] In some embodiments, the EOF can be greater than the EPF. In certain cases, the EOF is about 0.1% to about 500% greater than the EPF. In some cases, the 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%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490% or about 490% to about 500% greater than the EPF.

[0090] In some cases, EOF can be 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 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 more than 500% larger than EPF.

[0091] In some cases, the EOF can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1% larger than the EPF.

[0092] In some cases, the EOF can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% larger than the EPF.

[0093] In some embodiments, analyte translocation occurs through the nanopore in the EOF direction. In some embodiments, analyte translocation occurs through the nanopore in the EPF direction. In some embodiments, analyte translocation occurs through the nanopore in the EOF direction or the EPF direction.

[0094] Alternatively, in some embodiments, analyte translocation occurs through the nanopore in the EPF direction. In some embodiments, analyte translocation occurs through the nanopore in a direction opposite to the EOF. In some embodiments, analyte translocation occurs through the nanopore in the direction of the EPF or in a direction opposite to the EOF.

[0095] Alternatively, in some embodiments, the EPF can be greater than the EOF. In some embodiments, the EPF can be greater than the EOF. In certain cases, the EPF is approximately 0.1% to 500% greater than the EOF. In some cases, the EPF is approximately 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%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490% or about 490% to about 500% greater than the EOF.

[0096] In some cases, the EPF can be 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 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 more than 500% greater than the EOF.

[0097] In some cases, the EPF can be up to about 500%, up to about 490%, up to about 480%, up to about 470%, up to about 460%, up to about 450%, up to about 440%, up to about 430%, up to about 420%, up to about 410%, up to about 400%, up to about 390%, up to about 380%, up to about 370%, up to about 360%, up to about 350%, up to about 340%, up to about 330%, up to about 320%, up to about 310%, up to about 300%, up to about 290%, up to about 280%, up to about 270%, up to about 260%, up to about 250%, up to about 240%, up to about 230%, up to about 220%, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, 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 1%, up to about 0.5%, up to about 0.1%, or less than 0.1% larger than the EOF.

[0098] In certain cases, the EPF can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500% larger than the EOF.

[0099] In some embodiments, analyte translocation through the nanopore can occur in the absence of one or more auxiliary proteins. In some embodiments, analyte translocation through the nanopore can occur in the presence of EOF. In certain cases, analyte translocation can occur in the presence of EOF and the absence of one or more auxiliary proteins. In certain cases, one or more auxiliary proteins may be able to move the analyte through the nanopore. In some cases, one or more auxiliary proteins can include translocases, helicases, unfoldases, DNA polymerases, RNA polymerases, topoisomerases, or any combination thereof.

[0100] In some embodiments, the nanopore includes a biological nanopore or a solid-state nanopore. The biological nanopore can contain a mutation to a portion of the biological nanopore. The mutation can include an insertion, a substitution, a deletion, or a combination thereof. In some embodiments, the nanopore can comprise a recombinant nanopore. In some cases, the recombinant nanopore can contain components from one or more different types of nanopores. In some embodiments, the nanopore can be modified to restrict the passage of one or more ions through the nanopore channel. In some cases, the nanopore can restrict the passage of one or more ions through the nanopore channel by changing the charge of the nanopore channel. In some cases, the nanopore can be modified to have a channel with a net negative charge. In certain cases, the channel with a net negative charge can restrict the passage of one or more anions through the nanopore. In certain cases, the nanopore can be modified to have a channel with a net positive charge. In certain cases, the channel with a net positive charge can restrict the passage of one or more cations through the nanopore. In certain cases, the charge of the nanopore can be modified at the cis entrance of the channel. In certain cases, the charge of the nanopore can be modified at the trans entrance of the channel. In certain cases, the charge of the nanopore can be modified at the central channel of the nanopore.

[0101] The nanopore can have a geometry. The geometry can be annular, including a ring or a channel. The annular shape can be an annular polyhedron shape, including a ring or a channel. The ring can include one or more proteins that form the nanopore. The ring can have a cross-sectional geometry similar to that of one or more proteins that form the nanopore. The cis side of the ring can be wider than the trans side, or the trans side can be wider than the cis side. The ring can include portions having a conical geometry, a cylindrical geometry, an amorphous geometry, or a combination thereof. The channel can include a central portion of the nanopore geometry that does not contain the proteins or peptides of the nanopore. The channel can allow molecules to pass through the nanopore (e.g., through the channel). The channel can restrict the passage of molecules through the nanopore. Such restriction can be based on the width of the channel or the charge of the channel. The channel can include a channel length. The channel length can be the length of the channel measured along the longitudinal axis of the channel, which is perpendicular to the annular ring of the nanopore geometry. The channel length can be measured as the distance along the longitudinal axis of the channel between the farthest points of the nanopore along the longitudinal axis of the channel. In some embodiments, the starting point of the channel can be on the cis side of the nanopore and the ending point on the trans side of the nanopore, or the starting point can be on the trans side of the nanopore and the ending point on the cis side of the nanopore. In some embodiments, the channel length is less than the linear length or the contour length of the analyte. In some embodiments, the channel length is greater than the linear length or the contour length of the analyte. In some embodiments, the channel length of the channel is about 2 - 40 nm. In some embodiments, the channel length of the channel is about 2 - 5 nm, about 5 - 10 nm, about 10 - 15 nm, about 15 - 20 nm, about 20 - 25 nm, about 25 - 30 nm, about 30 - 35 nm, or about 35 - 40 nm. In some cases, the channel includes a channel length of at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 21 nm, at least about 22 nm, at least about 23 nm, at least about 24 nm, at least about 25 nm, at least about 26 nm, at least about 27 nm, at least about 28 nm, at least about 29 nm, at least about 30 nm, at least about 31 nm, at least about 32 nm, at least about 33 nm, at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 nm, at least about 38 nm, at least about 39 nm, at least about 40 nm or greater than 40 nm.In some cases, the channel includes a channel length of at most about 40 nm, at most about 39 nm, at most about 38 nm, at most about 37 nm, at most about 36 nm, at most about 35 nm, at most about 34 nm, at most about 33 nm, at most about 32 nm, at most about 31 nm, at most about 30 nm, at most about 29 nm, at most about 28 nm, at most about 27 nm, at most about 26 nm, at most about 25 nm, at most about 24 nm, at most about 23 nm, at most about 22 nm, at most about 21 nm, at most about 20 nm, at most about 19 nm, at most about 18 nm, at most about 17 nm, at most about 16 nm, at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4 nm, at most about 3 nm, at most about 2 nm, or less than 2 nm. In some cases, the channel includes a channel length of about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 39 nm, or about 40 nm.

[0102] In some embodiments, the internal nanopore channel can include a lumen. In some cases, the lumen of the nanopore channel can be from about 0.5 nanometers to about 10 nanometers. In some cases, the lumen of the nanopore channel can 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 can be at most about 10.0 nm, at most about 9.5 nm, at most about 9.0 nm, at most about 8.5 nm, at most about 8.0 nm, at most about 7.5 nm, at most about 7.0 nm, at most about 6.5 nm, at most about 6.0 nm, at most about 5.5 nm, at most about 5.0 nm, at most about 4.5 nm, at most about 4.0 nm, at most about 3.5 nm, at most about 3.0 nm, at most about 2.5 nm, at most about 2.0 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.5 nm, or less than 0.5 nm. In some cases, the lumen of the nanopore channel can be about 0.5 nm, about 1.0 nm, about 1.5 nm, about 2.0 nm, about 2.5 nm, about 3.0 nm, about 3.5 nm, about 4.0 nm, about 4.5 nm, about 5.0 nm, about 5.5 nm, about 6.0 nm, about 6.5 nm, about 7.0 nm, about 7.5 nm, about 8.0 nm, about 8.5 nm, about 9.0 nm, about 9.5 nm, or about 10.0 nm.

[0103] In some embodiments, the internal nanopore channel may include one or more constriction structures. In some cases, the internal nanopore channel may include from about 1 to about 50 constriction structures. In some cases, the internal nanopore channel may include at least about 1 constriction structure, at least about 5 constriction structures, at least about 10 constriction structures, at least about 15 constriction structures, at least about 20 constriction structures, at least about 25 constriction structures, at least about 30 constriction structures, at least about 25 constriction structures, at least about 30 constriction structures, at least about 35 constriction structures, at least about 40 constriction structures, at least about 45 constriction structures, at least about 50 constriction structures, or more than 50 constriction structures. In some cases, the internal nanopore channel may include at most about 50 constriction structures, at most about 45 constriction structures, at most about 40 constriction structures, at most about 35 constriction structures, at most about 30 constriction structures, at most about 25 constriction structures, at most about 20 constriction structures, at most about 15 constriction structures, at most about 10 constriction structures, at most about 5 constriction structures, at most about 1 constriction structure, or less than 1 constriction structure. In some cases, the internal nanopore channel may include about 1 constriction structure, about 5 constriction structures, about 10 constriction structures, about 15 constriction structures, about 20 constriction structures, about 25 constriction structures, about 30 constriction structures, about 35 constriction structures, about 40 constriction structures, about 45 constriction structures, or about 50 constriction structures.

[0104] In some embodiments, the dimensions of one or more constriction structures can be from about 0.2 nm to about 2 nm. In some cases, the dimensions of one or more constriction structures can be 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, the dimensions of one or more constriction structures can be at most about 2.0 nm, at most about 1.9 nm, at most about 1.8 nm, at most about 1.7 nm, at most about 1.6 nm, at most about 1.5 nm, at most about 1.4 nm, at most about 1.3 nm, at most about 1.2 nm, at most about 1.1 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, or less than 0.2 nm. In some cases, the dimensions of one or more constriction structures can be about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1.0 nm, about 1.1 nm, about 1.2 nm, about 1.3 nm, about 1.4 nm, about 1.5 nm, about 1.6 nm, about 1.7 nm, about 1.8 nm, about 1.9 nm, or about 2.0 nm.

[0105] In some embodiments, the analyte is longer than the length of the nanopore channel. In some embodiments, the analyte can 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 the length of the nanopore channel, at least about 90 times, at least about 95 times, at least about 100 times, or greater than about 100 times. In some embodiments, the analyte can be at most about 100 times, at most about 95 times, at most about 90 times, at most about 80 times, at most about 75 times, at most about 70 times, at most about 65 times, at most about 60 times, at most about 55 times, at most about 50 times, at most about 45 times, at most about 40 times, at most about 35 times, at most about 30 times, at most about 25 times, at most about 20 times, at most about 19 times, at most about 18 times, at most about 17 times, at most about 16 times, at most about 15 times, at most about 14 times, at most about 13 times, at most about 12 times, at most about 11 times, at most about 10 times, at most about 9 times, at most about 8 times, at most about 7 times, at most about 6 times, at most about 5 times, at most about 4 times, at most about 3 times, at most about 2 times, or less than about 2 times the length of the nanopore channel.

[0106] In some embodiments, the analyte can be from about 2 to about 100 times the length of the nanopore channel. In some embodiments, the analyte can be from about 2 to about 5 times, about 2 to about 10 times, about 2 to about 20 times, about 2 to about 30 times, about 2 to about 40 times, about 2 to about 50 times, about 2 to about 60 times, about 2 to about 70 times, about 2 to about 80 times, about 2 to about 90 times, about 2 to about 100 times, about 5 to about 10 times, about 5 to about 20 times, about 5 to about 30 times, about 5 to about 40 times, about 5 to about 50 times, about 5 to about 60 times, about 5 to about 70 times, about 5 to about 80 times, about 5 to about 90 times, about 5 to about 100 times, about 10 to about 20 times, about 10 to about 30 times, about 10 to about 40 times, about 10 to about 50 times, about 10 to about 60 times, about 10 to about 70 times, about 10 to about 80 times, about 10 to about 90 times, about 10 to about 100 times, about 20 to about 30 times, about 20 to about 40 times, about 20 to about 50 times, about 20 to about 60 times, about 20 to about 70 times, about 20 to about 80 times, about 20 to about 90 times, about 20 to about 100 times, about 30 to about 40 times, about 30 to about 50 times, about 30 to about 60 times, about 30 to about 70 times, about 30 to about 80 times, about 30 to about 90 times, about 30 to about 100 times, about 40 to about 50 times, about 40 to about 60 times, about 40 to about 70 times, about 40 to about 80 times, about 40 to about 90 times, about 40 to about 100 times, about 50 to about 60 times, about 50 to about 70 times, about 50 to about 80 times, about 50 to about 90 times, about 50 to about 100 times, about 60 to about 70 times, about 60 to about 80 times, about 60 to about 90 times, about 60 to about 100 times, about 70 to about 80 times, about 70 to about 90 times, about 70 to about 100 times, about 80 to about 90 times, about 80 to about 100 times, or about 90 to about 100 times the length of the nanopore channel.

[0107] In some embodiments, the analyte can be at least about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times or about 100 times the length of the nanopore channel.

[0108] 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%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490%, or about 490% to about 500% longer than the channel length of the nanopore.

[0109] 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 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 greater than 500% longer than the channel length of the nanopore.

[0110] In some cases, the linear length of the analyte is up to about 500%, up to about 490%, up to about 480%, up to about 470%, up to about 460%, up to about 450%, up to about 440%, up to about 430%, up to about 420%, up to about 410%, up to about 400%, up to about 390%, up to about 380%, up to about 370%, up to about 360%, up to about 350%, up to about 340%, up to about 330%, up to about 320%, up to about 310%, up to about 300%, up to about 290%, up to about 280%, up to about 270%, up to about 260%, up to about 250%, up to about 240%, up to about 230%, up to about 220%, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, 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 1%, up to about 0.5%, up to about 0.1%, or less than 0.1% longer than the channel length of the nanopore.

[0111] In some cases, the linear length of the analyte is about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500% longer than the channel length of the nanopore.

[0112] 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 can be 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 nm 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, about 70 nm to about 75 nm, about 75 nm to about 80 nm, about 80 nm to about 85 nm, about 85 nm to about 90 nm, about 90 nm to about 95 nm, about 95 nm to about 100 nm, about 100 nm to about 150 nm, about 150 nm to about 200 nm, about 200 nm to about 250 nm, about 250 nm to about 300 nm, about 300 nm to about 350 nm, about 350 nm to about 400 nm, about 400 nm to about 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650 nm, about 650 nm to about 700 nm, about 700 nm to about 750 nm, about 750 nm to about 800 nm, about 800 nm to about 850 nm, about 850 nm to about 900 nm, about 900 nm to about 950 nm, about 950 nm to about 1,000 nm, about 1,000 nm to about 1,100 nm, about 1,100 nm to about 1,200 nm, about 1,200 nm to about 1,300 nm, about 1,300 nm to about 1,400 nm, about 1,400 nm to about 1,500 nm, about 1,500 nm to about 1,600 nm, about 1,600 nm to about 1,700 nm, about 1,700 nm to about 1,800 nm, about 1,800 nm to about 1,900 nm, about 1,900 nm to about 2,000 nm, about 2,000 nm to about 2,100 nm, about 2,100 nm to about 2,200 nm, about 2,200 nm to about 2,300 nm, about 2,300 nm to about 2,400 nm, about 2,400 nm to about 2,500 nm, about 2,500 nm to about 2,600 nm, about 2,600 nm to about 2,700 nm, about 2,700 nm to about 2,800 nm, about 2,800 nm to about 2,900 nm, about 2,900 nm to about 3,000 nm, about 3,000 nm to about 3,100 nm, about 3,200 nm to about 3,300 nm, about 3,from about 300 nm to about 3,400 nm, from about 3,400 nm to about 3,500 nm, from about 3,500 nm to about 3,600 nm, from about 3,600 nm to about 3,700 nm, from about 3,700 nm to about 3,800 nm, from about 3,800 nm to about 3,900 nm, from about 3,900 nm to about 4,000 nm, from about 4,000 nm to about 4,100 nm, from about 4,100 to about 4,200 nm, from about 4,200 nm to about 4,300 nm, from about 4,300 nm to about 4,400 nm, from about 4,400 nm to about 4,500 nm, from about 4,500 nm to about 4,600 nm, from about 4,600 nm to about 4,700 nm, from about 4,700 nm to about 4,800 nm, from about 4,800 nm to about 4,900 nm, or from about 4,900 nm to about 5,000 nm.,

[0113] In some embodiments, the analyte can be present on the cis side of the nanopore system. In some embodiments, the analyte can be present on the trans side of the nanopore system. In some embodiments, the analyte can be present in the channel of the nanopore. In some embodiments, the analyte can be present simultaneously on the cis side of the nanopore system and in the channel of the nanopore. In some embodiments, the analyte can be present simultaneously on the trans side of the nanopore system and in the channel of the nanopore. In some embodiments, the analyte can be present simultaneously on the cis side of the nanopore system, in the channel of the nanopore, and on the trans side of the nanopore system.

[0114] In some embodiments, the linear length of the analyte can be 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 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 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 nm, at least about 160 nm, at least about 170 nm, at least about 180 nm, at least about 190 nm, at least about 200 nm, at least about 210 nm, at least about 220 nm, at least about 230 nm, at least about 240 nm, at least about 250 nm, at least about 260 nm, at least about 270 nm, at least about 280 nm, at least about 290 nm, at least about 300 nm, at least about 310 nm, at least about 320 nm, at least about 330 nm, at least about 340 nm, at least about 350 nm, at least about 360 nm, at least about 370 nm, at least about 380 nm, at least about 390 nm, at least about 400 nm, at least about 410 nm, at least about 420 nm, at least about 430 nm, at least about 440 nm, at least about 450 nm, at least about 460 nm, at least about 470 nm, at least about 480 nm, at least about 490 nm, at least about 500 nm, at least about 510 nm, at least about 520 nm, at least about 530 nm, at least about 540 nm, at least about 550 nm, at least about 560 nm, at least about 570 nm, at least about 580 nm, at least about 590 nm, at least about 600 nm, at least about 610 nm, at least about 620 nm, at least about 630 nm, at least about 640 nm, at least about 650 nm, at least about 660 nm, at least about 670 nm, at least about 680 nm, at least about 690 nm, at least about 700 nm, at least about 710 nm, at least about 720 nm, at least about 730 nm, at least about 740 nm, at least about 750 nm, at least about 760 nm, at least about 770 nm, at least about 780 nm, at least about 790 nm, at least about 800 nm, at least about 810 nm, at least about 820 nm, at least about 830 nm, at least about 840 nm, at least about 850 nm, at least about 860 nm, at least about 870 nm, at least about 880 nm, at least about 890 nm, at least about 900 nm, at least about 910 nm, at least about 920 nm, at least about 930 nm, at least about 940 nm, at least about 950 nm longer than the channel length of the nanopore.At least about 960 nm, at least about 970 nm, at least about 980 nm, at least about 990 nm, at least about 1,000 nm, at least about 1,100 nm, at least about 1,200 nm, at least about 1,300 nm, at least about 1,400 nm, at least about 1,500 nm, at least about 1,600 nm, at least about 1,700 nm, at least about 1,800 nm, at least about 1,900 nm, at least about 2,000 nm, at least about 2,100 nm, at least about 2,200 nm, at least about 2,300 nm, at least about 2,400 nm, at least about 2,500 nm, at least about 2,600 nm, at least about 2,700 nm, at least about 2,800 nm, at least about 2,900 nm, at least about 3,000 nm, at least about 3,100 nm, at least about 3,200 nm, at least about 3,300 nm, at least about 3,400 nm, 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 more than 5,000 nm.

[0115] In some embodiments, the linear length of the analyte can be up to about 5,000 nm, up to about 4,900 nm, up to about 4,800 nm, up to about 4,700 nm, up to about 4,600 nm, up to about 4,500 nm, up to about 4,400 nm, up to about 4,300 nm, up to about 4,200 nm, up to about 4,100 nm, up to about 4,000 nm, up to about 3,900 nm, up to about 3,800 nm, up to about 3,700 nm, up to about 3,600 nm, up to about 3,500 nm, up to about 3,400 nm, up to about 3,300 nm, up to about 3,200 nm, up to about 3,100 nm, up to about 3,000 nm, up to about 2,900 nm, up to about 2,800 nm, up to about 2,700 nm, up to about 2,600 nm, up to about 2,500 nm, up to about 2,400 nm, up to about 2,300 nm, up to about 2,200 nm, up to about 2,100 nm, up to about 2,000 nm, up to about 1,900 nm, up to about 1,800 nm, up to about 1,700 nm, up to about 1,600 nm, up to about 1,500 nm, up to about 1,400 nm, up to about 1,300 nm, up to about 1,200, up to about 1,100, up to about 1,000 nm, up to about 990, up to about 980 nm, up to about 970 nm, about 960 nm, up to about 950 nm, up to about 940 nm, up to about 930 nm, up to about 920 nm, up to about 910 nm, up to about 900 nm, up to about 890 nm, up to about 880 nm, up to about 870 nm, up to about 860 nm, up to about 850 nm, up to about 840 nm, up to about 830 nm, up to about 820 nm, up to about 810 nm, up to about 800 nm, up to about 790 nm, up to about 780 nm, up to about 770 nm, up to about 760 nm, up to about 750 nm, up to about 740 nm, up to about 730 nm, up to about 720 nm, up to about 710 nm, up to about 700 nm, 690 nm, up to about 680 nm, up to about 670 nm, up to about 660 nm, up to about 650 nm, up to about 640 nm, up to about 630 nm, up to about 620 nm, up to about 610 nm, up to about 600 nm, up to about 590 nm, up to about 580 nm, up to about 570 nm, up to about 560 nm, up to about 550 nm, up to about 540 nm, up to about 530 nm, up to about 520 nm, up to about 510 nm, up to about 500 nm, up to about 490 nm, up to about 480 nm, up to about 470 nm, up to about 460 nm, up to about 450 nm, up to about 440 nm, up to about 430 nm, up to about 420 nm, up to about 410 nm longer than the channel length of the nanopore.At most about 400 nm, at most about 390 nm, at most about 380 nm, at most about 370 nm, at most about 360 nm, at most about 350 nm, at most about 340 nm, at most about 330 nm, at most about 320 nm, at most about 310 nm, at most about 300 nm, at most about 290 nm, at most about 280 nm, at most about 270 nm, at most about 260 nm, at most about 250 nm, at most about 240 nm, at most about 230 nm, at most about 220 nm, at most about 210 nm, at most about 200 nm, at most about 190 nm, at most about 180 nm, at most about 170 nm, at most about 160 nm, at most about 150 nm, at most about 140 nm, at most about 130 nm, at most about 120 nm, at most about 110 nm, at most about 100 nm, at most about 95 nm, at most about 90 nm, at most about 85 nm, at most about 80 nm, at most about 75 nm, at most about 70 nm, at most about 65 nm, at most about 60 nm, at most about 55 nm, at most about 50 nm, at most about 45 nm, at most about 40 nm, at most about 35 nm, at most about 30 nm, at most about 25 nm, at most about 20 nm, at most about 15 nm, at most about 10 nm, at most about 5 nm, at most about 3 nm, at most about 2 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.1 nm or less than 0.1 nanometer.

[0116] In some embodiments, the linear length of the analyte can be longer than the channel length of the nanopore by about 0.1 nm, about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, about 700 nm, about 710 nm, about 720 nm, about 730 nm, about 740 nm, about 750 nm, about 760 nm, about 770 nm, about 780 nm, about 790 nm, about 800 nm, about 810 nm, about 820 nm, about 830 nm, about 840 nm, about 850 nm, about 860 nm, about 870 nm, about 880 nm, about 890 nm, about 900 nm, about 910 nm, about 920 nm, about 930 nm, about 940 nm, about 950 nm, about 960 nm, about 970 nm, about 980 nm, about 990 nm, about 1,000 nm, about 1,100 nm, about 1,200 nm, about 1,300 nm, about 1,400 nm, about 1,500 nm, about 1,600 nm, about 1,700 nm, about 1,800 nm, about 1,900 nm, about 2,000 nm, about 2,100 nm, about 2,200 nm, about 2,300 nm, about 2,400 nm, about 2,500 nm, about 2,600 nm, about 2,700 nm, about 2,800 nm, about 2,900 nm, about 3,000 nm, about 3,100 nm, about 3,200 nm, about 3,300 nm, about 3,400 nm, about 3,500 nm, about 3,600 nm, about 3,700 nm, about 3,800 nm, about 3,900 nm, about 4,000 nm, about 4,100 nm, about 4,200 nm, about 4,300 nm, about 4,400 nm, about 4,500 nm, about 4,600 nm, about 4,700 nm, about 4,800 nm, about 4,900 nm, or about 5,000 nm.,

[0117] In some embodiments, the nanopore comprises a cavity. The cavity can be the channel-facing surface of the nanopore. The cavity can include the channel-facing surface of the nanopore components, including proteins, peptides, or amino acid residues facing the channel. These components can be charged. The charge of these components can determine the net charge of the cavity. These components can have a shape. The shape of these components can determine the geometry of the cavity. The net charge of the cavity, the geometry of the cavity, or a combination thereof can affect the flow of molecules through the cavity (e.g., through the nanopore).

[0118] In some embodiments, the pore comprises a cavity. In some embodiments, the cavity of the nanopore has 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 greater than about 200. In some embodiments, the cavity of the nanopore has a net charge of at most about 200, at most about 150, at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 55, at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at most about 20, at most about 15, at most about 10, at most about 5, at most about 4, at most about 3, at most about 2, or less than about 2.

[0119] In some embodiments, the cavity of the nanopore contains a net charge of from about 2 to about 200. In some embodiments, the cavity of the nanopore contains a net charge of at most about 200. In some embodiments, the cavity of the nanopore contains a net charge of from about 2 to about 5, from about 2 to about 10, from about 2 to about 20, from about 2 to about 30, from about 2 to about 40, from about 2 to about 50, from about 2 to about 75, from about 2 to about 100, from about 2 to about 125, from about 2 to about 150, from about 2 to about 200, from about 5 to about 10, from about 5 to about 20, from about 5 to about 30, from about 5 to about 40, from about 5 to about 50, from about 5 to about 75, from about 5 to about 100, from about 5 to about 125, from about 5 to about 150, from about 5 to about 200, from about 10 to about 20, from about 10 to about 30, from about 10 to about 40, from about 10 to about 50, from about 10 to about 75, from about 10 to about 100, from about 10 to about 125, from about 10 to about 150, from about 10 to about 200, from about 20 to about 30, from about 20 to about 40, from about 20 to about 50, from about 20 to about 75, from about 20 to about 100, from about 20 to about 125, from about 20 to about 150, from about 20 to about 200, from about 30 to about 40, from about 30 to about 50, from about 30 to about 75, from about 30 to about 100, from about 30 to about 125, from about 30 to about 150, from about 30 to about 200, from about 40 to about 50, from about 40 to about 75, from about 40 to about 100, from about 40 to about 125, from about 40 to about 150, from about 40 to about 200, from about 50 to about 75, from about 50 to about 100, from about 50 to about 125, from about 50 to about 150, from about 50 to about 200, from about 75 to about 100, from about 75 to about 125, from about 75 to about 150, from about 75 to about 200, from about 100 to about 125, from about 100 to about 150, from about 100 to about 200, from about 125 to about 150, from about 125 to about 200, or from about 150 to about 200.

[0120] In some embodiments, the cavity of the nanopore 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, or about 200. In some embodiments, the cavity of the pore contains a net positive charge. In some embodiments, the cavity of the pore contains a net negative charge.

[0121] In some embodiments, the analyte may lack a three-dimensional structure. In some cases, the analyte lacking a three-dimensional structure can be a denatured analyte. In some embodiments, one or more portions of the analyte may lack a three-dimensional structure. In some cases, the one or more portions can include one or more termini of the analyte. In some cases, one terminus of the analyte may lack a three-dimensional structure. In some cases, two termini of the analyte may lack a three-dimensional structure. In some cases, at least two termini of the analyte may lack a three-dimensional structure. In some embodiments, internal portions (e.g., non-terminal portions of the analyte) may lack a three-dimensional structure. In some embodiments, the analyte can include a three-dimensional structure. In some cases, the analyte having a three-dimensional structure can be a folded analyte. The flowing molecules can be analytes, ions, water, or other molecules on the cis side or the trans side of the nanopore. The flowing molecules can generate an ionic current through an ion flow. When the analyte passes through the nanopore, other molecules (e.g., ions) may be hindered from passing through the nanopore. This can change the ionic current by changing the ion flow rate. Such a change can be measured by setting, for example, a pair of electrodes for measuring the current passing through the nanopore (or the membrane where the nanopore is located) from cis to trans. The narrow geometry of the cavity can slow down the speed of the analyte passing through the nanopore. Changes in the net charge or the cavity geometry can change the flow rate of molecules passing through the nanopore. For example, changing the cavity to have a more positive net charge can reduce the flow rate of positively charged molecules (e.g., sodium ions). For example, changing the cavity to a wider geometry can increase the flow rate of larger molecules (e.g., glucose molecules or peptide analytes). For example, changing the cavity to have a more negative net charge and a narrower geometry can reduce the flow rate of larger negatively charged molecules (e.g., glutamate ions). The net charge of the cavity affects the flow of charged molecules through the nanopore. The net charge can make it easier or more difficult for certain charged molecules to pass through.

[0122] The channel can include a constriction region. The constriction region can be a portion of the channel that is narrower than the surrounding portions. The channel can include multiple constriction regions. The net charge of the cavity, the geometry of the cavity, or a combination thereof can form the constriction region. Changing the net charge of the cavity, the geometry of the cavity, or a combination thereof can change the characteristics of the constriction region. The characteristics of the constriction region can be placement, location, width, charge, or a combination thereof. For example, changing the geometry of the cavity can change the width of the constriction region, or changing the net charge of the cavity can change the charge of the constriction region.

[0123] The nanopore can have ion permeability. Ion permeability can be the ability or likelihood of ions to flow through or diffuse through the nanopore channel. The ion permeability (P) of different ions may be different. By comparing different permeabilities, the relative ion selectivity can be calculated. The relative ion selectivity can be a given cation (P(+) the permeability of (e.g., potassium ions) divided by the permeability of a given anion (P (-) ), e.g., chloride ions), to yield a relative ion selectivity (P (+) / P (-) ). Both the net charge and geometry of the cavity can affect ion permeability. For example, a positive net charge can reduce the permeability of cations, or a negative net charge can reduce the permeability of anions. The geometry of the cavity can affect ion permeability based on the size of the ion. For example, a smaller geometry will reduce the permeability of larger ions (e.g., potassium ions) relative to smaller ions (e.g., lithium ions).

[0124] The net charge and geometry of the cavity can affect the relative ion flux. The relative ion flux can be the net flow of ions through the nanopore. In some embodiments, the nanopore can have a relative ion selectivity (P (+) / P (-) ) greater than about 0.1, greater than about 0.2, greater than about 0.3, greater than about 0.4, greater than about 0.5, greater than about 0.6, greater than about 0.7, greater than about 0.8, greater than about 0.9, greater than about 1.0, greater than about 1.2, greater than about 1.4, greater than about 1.6, greater than about 1.8, greater than about 2.0, greater than about 2.5, greater than about 3, greater than about 3.2, greater than about 3.4, greater than about 3.6, greater than about 3.8, greater than about 4.0, greater than about 4.1, greater than about 4.2, greater than about 4.3, greater than about 4.4, greater than about 4.5, greater than about 4.6, greater than about 4.7, greater than about 4.8, greater than about 4.9, or greater than about 5.0. In some embodiments, the pore can have a relative ion selectivity (P (+) / P (-) ) 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.

[0125] The nanopore may include at least one inner pore constriction structure. In some embodiments, the inner pore constriction structure is 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, 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 inner pore constriction structure is at most about 0.2, at most about 0.3, at most about 0.4, at most about 0.5, at most about 0.6, at most about 0.7, at most about 0.8, at most about 0.9, at most about 1.0, at most about 1.1, at most about 1.2, at most about 1.3, at most about 1.4, at most about 1.5, at most about 1.6, at most about 1.7, at most about 1.8, at most about 1.9, at most about 2.0, at most about 2.1, at most about 2.2, at most about 2.3, at most about 2.4, at most about 2.5, at most about 2.6, at most about 2.7, at most about 2.8, at most about 2.9, at most about 3.0, at most about 3.1, at most about 3.2, at most about 3.3, at most about 3.4, at most about 3.5, at most about 3.6, at most about 3.7, at most about 3.8, at most about 3.9, or at most about 4.0 nm.

[0126] In some embodiments, the nanopore channel includes a cavity. In some embodiments, the nanopore cavity 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 greater than about 200 coulombs. In some embodiments, the nanopore cavity includes a net charge of at most about 200 coulombs, at most about 150 coulombs, at most about 100 coulombs, at most about 90 coulombs, at most about 80 coulombs, at most about 70 coulombs, at most about 60 coulombs, at most about 55 coulombs, at most about 50 coulombs, at most about 45 coulombs, at most about 40 coulombs, at most about 35 coulombs, at most about 30 coulombs, at most about 25 coulombs, at most about 20 coulombs, at most about 15 coulombs, at most about 10 coulombs, at most about 5 coulombs, at most about 4 coulombs, at most about 3 coulombs, at most about 2 coulombs, or less than about 2 coulombs.

[0127] In some embodiments, the nanopore cavity contains a net charge of from about 2 to about 200 Coulombs. In some embodiments, the nanopore cavity contains a net charge of at most about 200. In some embodiments, the nanopore cavity contains a net charge of from about 2 to about 5, from about 2 to about 10, from about 2 to about 20, from about 2 to about 30, from about 2 to about 40, from about 2 to about 50, from about 2 to about 75, from about 2 to about 100, from about 2 to about 125, from about 2 to about 150, from about 2 to about 200, from about 5 to about 10, from about 5 to about 20, from about 5 to about 30, from about 5 to about 40, from about 5 to about 50, from about 5 to about 75, from about 5 to about 100, from about 5 to about 125, from about 5 to about 150, from about 5 to about 200, from about 10 to about 20, from about 10 to about 30, from about 10 to about 40, from about 10 to about 50, from about 10 to about 75, from about 10 to about 100, from about 10 to about 125, from about 10 to about 150, from about 10 to about 200, from about 20 to about 30, from about 20 to about 40, from about 20 to about 50, from about 20 to about 75, from about 20 to about 100, from about 20 to about 125, from about 20 to about 150, from about 20 to about 200, from about 30 to about 40, from about 30 to about 50, from about 30 to about 75, from about 30 to about 100, from about 30 to about 125, from about 30 to about 150, from about 30 to about 200, from about 40 to about 50, from about 40 to about 75, from about 40 to about 100, from about 40 to about 125, from about 40 to about 150, from about 40 to about 200, from about 50 to about 75, from about 50 to about 100, from about 50 to about 125, from about 50 to about 150, from about 50 to about 200, from about 75 to about 100, from about 75 to about 125, from about 75 to about 150, from about 75 to about 200, from about 100 to about 125, from about 100 to about 150, from about 100 to about 200, from about 125 to about 150, from about 125 to about 200, or from about 150 to about 200 Coulombs.

[0128] In some embodiments, the nanopore cavity 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, about 200 Coulombs. In some embodiments, the nanopore cavity contains a net positive charge. In some embodiments, the nanopore cavity contains a net negative charge.

[0129] In some embodiments, the nanopore cavity can have a net charge of from about -20 to about +20. In some cases, the nanopore cavity can have a net charge of at least about -20, at least about -19, at least about -18, at least about -17, at least about -16, at least about -15, at least about -14, at least about -13, at least about -12, at least about -11, at least about -10, at least about -9, at least about -8, at least about -7, at least about -6, at least about -5, at least about -4, at least about -3, at least about -2, at least about -1, at least about 0, 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 +9, 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 greater than +20. In some cases, the nanopore cavity can have a net charge of at most about +20, at most about +19, at most about +18, at most about +17, at most about +16, at most about +15, at most about +14, at most about +13, at most about +12, at most about +11, at most about +10, at most about +9, at most about +8, at most about +7, at most about +6, at most about +5, at most about +4, at most about +3, at most about +2, at most about +1, at most about 0, at most about -1, at most about -2, at most about -3, at most about -4, at most about -5, at most about -6, at most about -7, at most about -8, at most about -9, at most about -10, at most about -11, at most about -12, at most about -13, at most about -14, at most about -15, at most about -16, at most about -17, at most about -18, at most about -19, at most about -20, or less than -20. In some cases, the nanopore cavity can have a net charge of about -20, about -19, about -18, about -17, about -16, about -15, about -14, about -13, about -12, about -11, about -10, about -9, about -8, about -7, about -6, about -5, about -4, about -3, about -2, about -1, about 0, about +1, about +2, about +3, about +4, about +5, about +6, about +7, about +8, about +9, about +10, about +11, about +12, about +13, about +14, about +15, about +16, about +17, about +18, about +19, or about +20.

[0130] In some embodiments, the nanopore can comprise one or more subunits. In some cases, each of the one or more subunits can comprise from 1 to 20 charged amino acids. In some embodiments, from about 1 to 20 charged repeating units can be distributed within the cavity. In some cases, the charged repeating units can be negatively charged. In some cases, the charged repeating units can be positively charged. In some cases, the charged repeating units can be both positively and negatively charged. In some cases, the cavity can comprise 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 cavity can comprise at most about 20 charged repeating units, at most about 19 charged repeating units, at most about 18 charged repeating units, at most about 17 charged repeating units, at most about 16 charged repeating units, at most about 15 charged repeating units, at most about 14 charged repeating units, at most about 13 charged repeating units, at most about 12 charged repeating units, at most about 11 charged repeating units, at most about 10 charged repeating units, at most about 9 charged repeating units, at most about 8 charged repeating units, at most about 7 charged repeating units, at most about 6 charged repeating units, at most about 5 charged repeating units, at most about 4 charged repeating units, at most about 3 charged repeating units, at most about 2 charged repeating units, at most about 1 charged repeating unit or less.In some cases, the cavity may contain about 1 charged repeating unit, about 2 charged repeating units, about 3 charged repeating units, about 4 charged repeating units, about 5 charged repeating units, about 6 charged repeating units, about 7 charged repeating units, about 8 charged repeating units, about 9 charged repeating units, about 10 charged repeating units, about 11 charged repeating units, about 12 charged repeating units, about 13 charged repeating units, about 14 charged repeating units, about 15 charged repeating units, about 16 charged repeating units, about 17 charged repeating units, about 18 charged repeating units, about 19 charged repeating units, or about 20 charged repeating units. In some embodiments, the charged repeating units may be evenly distributed in the cavity. In some cases, the charged repeating units may be distributed every about 2 to about 50 repeating units. In some cases, the charged repeating units may be distributed at least about every 2 repeating units, at least about every 5 repeating units, at least about every 10 repeating units, at least about every 15 repeating units, at least about every 20 repeating units, at least about every 25 repeating units, at least about every 30 repeating units, at least about every 40 repeating units, at least about every 45 repeating units, at least about every 50 repeating units, or more than every 50 repeating units. In some cases, the charged repeating units may be distributed at most about every 50 repeating units, at most about every 45 repeating units, at most about every 40 repeating units, at most about every 35 repeating units, at most about every 30 repeating units, at most about every 25 repeating units, at most about every 20 repeating units, at most about every 15 repeating units, at most about every 10 repeating units, at most about every 5 repeating units, at most about every 2 repeating units, or less than every 2 repeating units. In some cases, the charged repeating units may be distributed about every two repeating units, about every 5 repeating units, about every 10 repeating units, about every 15 repeating units, about every 20 repeating units, about every 25 repeating units, about every 30 repeating units, about every 35 repeating units, about every 30 repeating units, about every 35 repeating units, about every 40 repeating units, about every 45 repeating units, or about every 50 repeating units. In some embodiments, the charged repeating units may be unevenly distributed within the cavity.

[0131] The repeating unit can be positively charged or negatively charged. The charged repeating units Asp, Glu or Asp can be conveniently introduced by a single amino acid substitution. In some embodiments, the number of negatively charged amino acids that can be uniformly distributed within the cavity can 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 greater than about 20 negatively charged amino acids. In some embodiments, the number of negatively charged amino acids that can be uniformly distributed within the cavity can be at most about 20 amino acids, at most about 19 amino acids, at most about 18 amino acids, at most about 17 amino acids, at most about 16 amino acids, at most about 15 amino acids, at most about 14 amino acids, at most about 13 amino acids, at most about 12 amino acids, at most about 11 amino acids, at most about 10 amino acids, at most about 9 amino acids, at most about 8 amino acids, at most about 7 amino acids, at most about 6 amino acids, at most about 5 amino acids, at most about 4 amino acids, at most about 3 amino acids, at most about 2 amino acids, at most about 1 amino acid or less than about 1 charged amino acid.

[0132] In some embodiments, the number of negatively charged amino acids that can be evenly distributed within the cavity can be from about 1 to about 20 charged amino acids. In some embodiments, the number of amino acids that can be evenly distributed within the cavity can be about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 12, about 1 to about 15, about 1 to about 18, about 1 to about 20, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 2 to about 12, about 2 to about 15, about 2 to about 18, about 2 to about 20, about 3 to about 4, about 3 to about 5, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 3 to about 12, about 3 to about 15, about 3 to about 18, about 3 to about 20, about 4 to about 5, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 4 to about 12, about 4 to about 15, about 4 to about 18, about 4 to about 20, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5 to about 12, about 5 to about 15, about 5 to about 18, about 5 to about 20, about 8 to about 9, about 8 to about 10, about 8 to about 12, about 8 to about 15, about 8 to about 18, about 8 to about 20, about 9 to about 10, about 9 to about 12, about 9 to about 15, about 9 to about 18, about 9 to about 20, about 10 to about 12, about 10 to about 15, about 10 to about 18, about 10 to about 20, about 12 to about 15, about 12 to about 18, about 12 to about 20, about 15 to about 18, about 15 to about 20, or about 18 to about 20 charged amino acids.

[0133] In some embodiments, the number of negatively charged amino acids that may be evenly distributed within the cavity may be 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 negatively charged repeating units may be distributed between about every 2 repeating units to about every 50 repeating units. In some cases, the charged repeating units may be distributed at least about every 2 repeating units, at least about every 5 repeating units, at least about every 10 repeating units, at least about every 15 repeating units, at least about every 20 repeating units, at least about every 25 repeating units, at least about every 30 repeating units, at least about every 40 repeating units, at least about every 45 repeating units, at least about every 50 repeating units, or more than every 50 repeating units. In some cases, the charged repeating units may be distributed at most about every 50 repeating units, at most about every 45 repeating units, at most about every 40 repeating units, at most about every 35 repeating units, at most about every 30 repeating units, at most about every 25 repeating units, at most about every 20 repeating units, at most about every 15 repeating units, at most about every 10 repeating units, at most about every 5 repeating units, at most about every 2 repeating units, or less than every 2 repeating units. In some cases, the charged repeating units may be distributed about every two repeating units, about every 5 repeating units, about every 10 repeating units, about every 15 repeating units, about every 20 repeating units, about every 25 repeating units, about every 30 repeating units, about every 35 repeating units, about every 30 repeating units, about every 35 repeating units, about every 40 repeating units, about every 45 repeating units, or about every 50 repeating units.

[0134] In some cases, the nanopore can comprise one or more subunits. In some cases, each of the one or more subunits can have from about 1 to about 20 charged amino acids. In some embodiments, each of the one or more subunits can have from about 1 to about 2, from about 1 to about 3, from about 1 to about 4, from about 1 to about 5, from about 1 to about 8, from about 1 to about 9, from about 1 to about 10, from about 1 to about 12, from about 1 to about 15, from about 1 to about 18, from about 1 to about 20, from about 2 to about 3, from about 2 to about 4, from about 2 to about 5, from about 2 to about 8, from about 2 to about 9, from about 2 to about 10, from about 2 to about 12, from about 2 to about 15, from about 2 to about 18, from about 2 to about 20, from about 3 to about 4, from about 3 to about 5, from about 3 to about 8, from about 3 to about 9, from about 3 to about 10, from about 3 to about 12, from about 3 to about 15, from about 3 to about 18, from about 3 to about 20, from about 4 to about 5, from about 4 to about 8, from about 4 to about 9, from about 4 to about 10, from about 4 to about 12, from about 4 to about 15, from about 4 to about 18, from about 4 to about 20, from about 5 to about 8, from about 5 to about 9, from about 5 to about 10, from about 5 to about 12, from about 5 to about 15, from about 5 to about 18, from about 5 to about 20, from about 8 to about 9, from about 8 to about 10, from about 8 to about 12, from about 8 to about 15, from about 8 to about 18, from about 8 to about 20, from about 9 to about 10, from about 9 to about 12, from about 9 to about 15, from about 9 to about 18, from about 9 to about 20, from about 10 to about 12, from about 10 to about 15, from about 10 to about 18, from about 10 to about 20, from about 12 to about 15, from about 12 to about 18, from about 12 to about 20, from about 15 to about 18, from about 15 to about 20 or from about 18 to about 20 charged amino acids.

[0135] In some embodiments, each of one or more subunits may have from 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, charged repeat units may be distributed between about every 2 repeat units to about every 50 repeat units. In some cases, charged repeat units may be distributed at least about every 2 repeat units, at least about every 5 repeat units, at least about every 10 repeat units, at least about every 15 repeat units, at least about every 20 repeat units, at least about every 25 repeat units, at least about every 30 repeat units, at least about every 40 repeat units, at least about every 45 repeat units, at least about every 50 repeat units, or more than every 50 repeat units. In some cases, charged repeat units may be distributed at most about every 50 repeat units, at most about every 45 repeat units, at most about every 40 repeat units, at most about every 35 repeat units, at most about every 30 repeat units, at most about every 25 repeat units, at most about every 20 repeat units, at most about every 15 repeat units, at most about every 10 repeat units, at most about every 5 repeat units, at most about every 2 repeat units, or less than every 2 repeat units. In some cases, charged repeat units may be distributed about every two repeat units, about every 5 repeat units, about every 10 repeat units, about every 15 repeat units, about every 20 repeat units, about every 25 repeat units, about every 30 repeat units, about every 35 repeat units, about every 30 repeat units, about every 35 repeat units, about every 40 repeat units, about every 45 repeat units, or about every 50 repeat units.

[0136] At least one negatively charged "flanking" residue may be positioned or introduced at the pore entrance, or at least one negatively charged residue may be positioned or introduced at the pore exit. In some embodiments, the spacing between the Cα atoms of at least one internally negatively charged amino acid and the Cα atoms of the negatively charged flanking amino acid may be at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about at least about or greater than about

[0137] In some embodiments, the spacing between the Cα atoms of at least one internally negatively charged amino acid and the Cα atoms of the negatively charged flanking amino acids can be from about to about In some embodiments, the spacing between the Cα atoms of at least one internally negatively charged amino acid and the Cα atoms of the negatively charged flanking amino acids can be from about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about or about to about

[0138] In some embodiments, the spacing between the Cα atom of at least one internally negatively charged amino acid and the Cα atom of the negatively charged flanking amino acid can be about about about about about about about about about about about about about about about about about about about about about about about about about about or about

[0139] The nanopore cavity can include separate charge groups oriented in a ring along the annular geometry of the nanopore. These charge groups can be arranged along the longitudinal length of the channel. In some embodiments, the nanopore can include 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 greater than about 20 separate charge groups. In some embodiments, the nanopore can include at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, or less than about 1 separate charge group.

[0140] In some embodiments, the nanopore can include from about 1 to about 20 separate charge groups. In some embodiments, the nanopore can include from about 1 to about 2, from about 1 to about 3, from about 1 to about 4, from about 1 to about 5, from about 1 to about 8, from about 1 to about 9, from about 1 to about 10, from about 1 to about 12, from about 1 to about 15, from about 1 to about 18, from about 1 to about 20, from about 2 to about 3, from about 2 to about 4, from about 2 to about 5, from about 2 to about 8, from about 2 to about 9, from about 2 to about 10, from about 2 to about 12, from about 2 to about 15, from about 2 to about 18, from about 2 to about 20, from about 3 to about 4, from about 3 to about 5, from about 3 to about 8, from about 3 to about 9, from about 3 to about 10, from about 3 to about 12, from about 3 to about 15, from about 3 to about 18, from about 3 to about 20, from about 4 to about 5, from about 4 to about 8, from about 4 to about 9, from about 4 to about 10, from about 4 to about 12, from about 4 to about 15, from about 4 to about 18, from about 4 to about 20, from about 5 to about 8, from about 5 to about 9, from about 5 to about 10, from about 5 to about 12, from about 5 to about 15, from about 5 to about 18, from about 5 to about 20, from about 8 to about 9, from about 8 to about 10, from about 8 to about 12, from about 8 to about 15, from about 8 to about 18, from about 8 to about 20, from about 9 to about 10, from about 9 to about 12, from about 9 to about 15, from about 9 to about 18, from about 9 to about 20, from about 10 to about 12, from about 10 to about 15, from about 10 to about 18, from about 10 to about 20, from about 12 to about 15, from about 12 to about 18, from about 12 to about 20, from about 15 to about 18, from about 15 to about 20, or from about 18 to about 20 separate charge groups.

[0141] In some embodiments, the nanopore may comprise 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 independent charge groups.

[0142] In some embodiments, the charge groups may each be spaced from one another along the longitudinal length of the channel by at least about 0.1 nanometer, at least about 0.2 nanometer, at least about 0.3 nanometer, at least about 0.4 nanometer, at least about 0.5 nanometer, at least about 0.6 nanometer, at least about 0.7 nanometer, at least about 0.8 nanometer, at least about 0.9 nanometer, 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 greater than about 10 nanometers. In some embodiments, the charge groups may each be spaced from one another along the longitudinal length of the channel by at most about 10 nanometers, at most about 9 nanometers, at most about 8 nanometers, at most about 7 nanometers, at most about 6 nanometers, at most about 5 nanometers, at most about 4 nanometers, at most about 3 nanometers, at most about 2 nanometers, at most about 1 nanometer, at most about 0.9 nanometer, at most about 0.8 nanometer, at most about 0.7 nanometer, at most about 0.6 nanometer, at most about 0.5 nanometer, at most about 0.4 nanometer, at most about 0.3 nanometer, at most about 0.2 nanometer, at most about 0.1 nanometer, or less than about 0.1 nanometer.

[0143] In some embodiments, the charge groups may each be spaced from one another along the longitudinal length of the channel by about 0.1 nanometer to about 5 nanometers. In some embodiments, the charge groups may each be spaced from one another along the longitudinal length of the channel by about 0.1 nanometer to about 0.2 nanometer, about 0.1 nanometer to about 0.3 nanometer, about 0.1 nanometer to about 0.4 nanometer, about 0.1 nanometer to about 0.5 nanometer, about 0.1 nanometer to about 1 nanometer, about 0.1 nanometer to about 1.5 nanometers, about 0.1 nanometer to about 2 nanometers, about 0.1 nanometer to about 2.5 nanometers, about 0.1 nanometer to about 3 nanometers, about 0.1 nanometer to about 4 nanometers, about 0.1 nanometer to about 5 nanometers, about 0.2 nanometer to about 0.3 nanometer, about 0.2 nanometer to about 0.4 nanometer, about 0.2 nanometer to about 0.5 nanometer, about 0.2 nanometer to about 1 nanometer, about 0.2 nanometer to about 1.5 nanometers, about 0.2 nanometer to about 2 nanometers, about 0.2 nanometer to about 2.5 nanometers, about 0.2 nanometer to about 3 nanometers, about 0.2 nanometer to about 4 nanometers, about 0.2 nanometer to about 5 nanometers, about 0.3 nanometer to about 0.4 nanometer, about 0.3 nanometer to about 0.5 nanometer, about 0.3 nanometer to about 1 nanometer, about 0.3 nanometer to about 1.5 nanometers, about 0.3 nanometer to about 2 nanometers, about 0.3 nanometer to about 2.5 nanometers, about 0.3 nanometer to about 3 nanometers, about 0.3 nanometer to about 4 nanometers, about 0.3 nanometer to about 5 nanometers, about 0.4 nanometer to about 0.5 nanometer, about 0.4 nanometer to about 1 nanometer, about 0.4 nanometer to about 1.5 nanometers, about 0.4 nanometer to about 2 nanometers, about 0.4 nanometer to about 2.5 nanometers, about 0.4 nanometer to about 3 nanometers, about 0.4 nanometer to about 4 nanometers, about 0.4 nanometer to about 5 nanometers, about 0.5 nanometer to about 1 nanometer, about 0.5 nanometer to about 1.5 nanometers, about 0.5 nanometer to about 2 nanometers, about 0.5 nanometer to about 2.5 nanometers, about 0.5 nanometer to about 3 nanometers, about 0.5 nanometer to about 4 nanometers, about 0.5 nanometer to about 5 nanometers, about 1 nanometer to about 1.5 nanometers, about 1 nanometer to about 2 nanometers, about 1 nanometer to about 2.5 nanometers, about 1 nanometer to about 3 nanometers, about 1 nanometer to about 4 nanometers, about 1 nanometer to about 5 nanometers, about 1.5 nanometers to about 2 nanometers, about 1.5 nanometers to about 2.5 nanometers, about 1.5 nanometers to about 3 nanometers, about 1.5 nanometers to about 4 nanometers, about 1.5 nanometers to about 5 nanometers, about 2 nanometers to about 2.5 nanometers, about 2 nanometers to about 3 nanometers, about 2 nanometers to about 4 nanometers, about 2 nanometers to about 5 nanometers, about 2.5 nanometers to about 3 nanometers, about 2.5 nanometers to about 4 nanometers, about 2.5 nanometers to about 5 nanometers, about 3 nanometers to about 4 nanometers, about 3 nanometers to about 5 nanometers, about 4 nanometers to about 5 nanometers, about 5 nanometers to about 6 nanometers, about 6 nanometers to about 7 nanometers, about 7 nanometers to about 8 nanometers, about 8 nanometers to about 9 nanometers, or about 9 nanometers to about 10 nanometers.

[0144] In some embodiments, the charge groups can be spaced from each other along the longitudinal length of the channel by about 0.1 nanometer, about 0.2 nanometer, about 0.3 nanometer, about 0.4 nanometer, about 0.5 nanometer, about 0.6 nanometer, about 0.7 nanometer, about 0.8 nanometer, about 0.9 nanometer, 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. In some embodiments, a group of charges can be present at the cis-side entrance of the nanopore. In some embodiments, a group of charges can be present at the trans-side entrance of the nanopore. In some embodiments, a group of charges can be present at the cis-side entrance of the nanopore and a group of charges can be present at the trans-side entrance of the nanopore.

[0145] In some embodiments, the solution on the cis-side or the trans-side of the fluid chamber can be configured to have a set pH value. The solution can have a pH value 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, which can be employed. The solution can have a pH value of at most about 13, at most about 12, at most about 11, at most about 10.5, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 4.5, at most about 4, at most about 3.8, at most about 3, at most about 2, at most about 1, or less than about 1, which can be employed.

[0146] The solution can have a pH value of from about 1 to about 13 that can be employed. The solution can have a pH value of from about 1 to about 2, from about 1 to about 3, from about 1 to about 4, from about 1 to about 6, from about 1 to about 7, from about 1 to about 8, from about 1 to about 9, from about 1 to about 10, from about 1 to about 11, from about 1 to about 12, from about 1 to about 13, from about 2 to about 3, from about 2 to about 4, from about 2 to about 6, from about 2 to about 7, from about 2 to about 8, from about 2 to about 9, from about 2 to about 10, from about 2 to about 11, from about 2 to about 12, from about 2 to about 13, from about 3 to about 4, from about 3 to about 6, from about 3 to about 7, from about 3 to about 8, from about 3 to about 9, from about 3 to about 10, from about 3 to about 11, from about 3 to about 12, from about 3 to about 13, from about 4 to about 6, from about 4 to about 7, from about 4 to about 8, from about 4 to about 9, from about 4 to about 10, from about 4 to about 11, from about 4 to about 12, from about 4 to about 13, from about 6 to about 7, from about 6 to about 8, from about 6 to about 9, from about 6 to about 10, from about 6 to about 11, from about 6 to about 12, from about 6 to about 13, from about 7 to about 8, from about 7 to about 9, from about 7 to about 10, from about 7 to about 11, from about 7 to about 12, from about 7 to about 13, from about 8 to about 9, from about 8 to about 10, from about 8 to about 11, from about 8 to about 12, from about 8 to about 13, from about 9 to about 10, from about 9 to about 11, from about 9 to about 12, from about 9 to about 13, from about 10 to about 11, from about 10 to about 12, from about 10 to about 13, from about 11 to about 12, from about 11 to about 13 or from about 12 to about 13 that can be employed.

[0147] The solution can have a pH value of about 1, about 2, about 3, about 3.8, about 4, about 4.5, about 6, about 7, about 8, about 9, about 10, about 10.5, about 11, about 12 or about 13 that can be employed.

[0148] In some embodiments, electroosmotic flow (also known as electroosmotic force) acts on the membrane in the cis-to-trans or trans-to-cis direction. Electroosmotic flow can be a flow resulting from the net flow of an ion migration layer along the surface caused by the application of an electric potential. For example, a charged surface may form a static layer of mobile ions with the opposite charge. Under the application of an electric potential, the charged mobile ions may be induced to move in the direction of higher electric potential (if it is a negative potential) or in the direction of lower electric potential (if it is a positive potential). The flow of charged ions creates a drag on the surrounding solvent (such as water) molecules, which may in turn result in a net flow that exerts a force on the surrounding charged and neutral molecules. For example, in a negatively charged nanopore cavity, electroosmotic flow may be caused by the net flow of positive ions from the cis to the trans direction (e.g., due to a lower electric potential on the trans side), causing the surrounding water to flow from the cis to the trans direction and exerting a force on the surrounding molecules. The ion flux and the corresponding magnitude of electroosmotic flow can be affected by the following parameters, including the transmembrane ion concentration difference, the potential difference, the net charge of the nanopore cavity, the geometry of the nanopore cavity, or a combination thereof. In some embodiments, electroosmotic flow can be a flow caused by one or more constriction structures present in the nanopore channel. In some embodiments, electroosmotic flow can be a flow caused by the net flow of mobile ions along the surface resulting from the application of an electric potential and one or more constriction structures present in the nanopore channel.

[0149] In some embodiments, electroosmotic flow can be generated or altered by the difference between the solution on the cis side of the membrane and the solution on the trans side of the membrane. This difference can be a difference in molecular concentration, including ions, electrolytes, or osmotic regulators.

[0150] In some embodiments, the difference between the solutions may be salt asymmetry or ion asymmetry, where the ion concentration on one side of the membrane (e.g., the cis side) is different from that on the other side (e.g., the trans side). Ion asymmetry affects the transmembrane ion current, as described by the Goldman-Hodgkin-Katz equation.

[0151]

[0152] where the ion current (I(S)) is related to the transmembrane ion species S and the applied potential (Vm): where P (S) is the membrane permeability of the ion species S, zs is the valence of the ion, F is the Faraday constant, R is the gas constant, T is the temperature, [S] cis and [S] transThey are the cis-side concentration and the trans-side concentration of ionic species S, respectively. Since the difference in ionic concentrations between the cis-side and the trans-side affects the ionic flux, the total ionic flux of different types of ions will affect the electroosmotic force when ions flow across the membrane in different directions. This can enhance or weaken the electroosmotic force through the difference in ionic concentrations between the cis-side and the trans-side, thereby minimizing or maximizing the contribution of the ionic current of species S to the net ionic flux.

[0153] The difference in molecular concentrations between the two sides of the membrane can change the electroosmotic flux by providing a competing or auxiliary osmotic flux. The transmembrane concentration difference can generate an osmotic gradient, where the solvent (e.g., water) may diffuse across the membrane in the direction of higher molecular concentration, thereby minimizing the concentration difference between the two sides of the membrane. The osmotic gradient can be oriented to drive the water flow in the same direction or a different direction as the electroosmotic force. For example, a higher ionic concentration on the cis-side relative to the trans-side can generate an osmotic gradient that competes with the cis-to-trans electroosmotic force because the osmotic gradient can drive the water flow in the trans-to-cis direction. If the ionic concentrations also provide an osmotic gradient, they may also support the cis-to-trans electroosmotic flow.

[0154] In some embodiments, the electroosmotic force can act in the same direction as the electrophoretic force or in the opposite direction to the electrophoretic force. In some embodiments, the electroosmotic force can be greater than the electrophoretic force. In some embodiments, the electroosmotic force can be less than the electrophoretic force.

[0155] In some embodiments, the cis-to-trans EOF can include a net ionic current flowing from the cis-side of the membrane to the trans-side of the membrane. In some embodiments, the trans-to-cis EOF can include a net ionic current flowing from the trans-side of the membrane to the cis-side of the membrane. In some cases, the nanopore system can contain a total ionic current. In some cases, the net ionic current can include less than the flow of all ions in the nanopore system. In some cases, the net ionic current can include less than the flow of all ions in the nanopore system in a specific direction. In some cases, the specific direction can be from the cis-side of the membrane to the trans-side of the membrane. In some cases, the specific direction can be from the trans-side of the membrane to the cis-side of the membrane. In some cases, the total ionic current can include the total flow of all ions in the nanopore system. In some cases, the total ionic current can include the total flow of all ions in the nanopore system in a specific direction. In some cases, the specific direction can be from the cis-side of the membrane to the trans-side of the membrane. In some cases, the specific direction can be from the trans-side of the membrane to the cis-side of the membrane.

[0156] In some embodiments, the net ionic current can be from about 0.001% to about 100% of the total ionic current. In some cases, the net ionic current can be from 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. In some cases, the net ionic current can be 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. In some cases, the net ionic current can be at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, at most about 0.05%, at most about 0.01%, at most about 0.005%, at most about 0.001%, or less than 0.001% of the total ionic current. In some cases, the net ionic current can be about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, 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%, about 98%, about 99%, about 99.5%, or about 100% of the total ionic current.

[0157] In some embodiments, the translocation rate can be from about 0.1 amino acids per second (aa / s) to about 1,000 aa / s. In some cases, the translocation rate can 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 greater than 1,000 aa / s. In some cases, the translocation rate can be at most about 1,000 aa / s, at most about 500 aa / s, at most about 100 aa / s, at most about 50 aa / s, at most about 10 aa / s, at most about 5 aa / s, at most about 1 aa / s, at most about 0.5 aa / s, at most about 0.1 aa / s, or less than 0.1 aa / s. In some cases, the translocation rate can be about 0.1 aa / s, about 0.5 aa / s, about 1 aa / s, about 5 aa / s, about 10 aa / s, about 50 aa / s, about 100 aa / s, about 500 aa / s, or about 1,000 aa / s.

[0158] In some embodiments, cis-to-trans EOF occurs when the ratio of the cis-to-trans net ionic current to the total ionic current (also referred to as the cis-to-trans relative net current) is greater than about 0.0, greater than about 0.1, greater than about 0.2, greater than about 0.3, greater than about 0.4, greater than about 0.5, greater than about 0.6, greater than about 0.7, greater than about 0.8, greater than about 0.9, greater than about 0.95, or greater than about 0.99. In some embodiments, cis-to-trans EOF occurs when the ratio of the trans-to-cis net ionic current to the total ionic current (also referred to as the cis-to-trans relative net current) 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.

[0159] In some embodiments, cis-to-trans EOF is generated when the ratio of the cis-to-trans net ionic current to the total ionic current (also referred to as the cis-to-trans relative net current) 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 greater than about 0.99. In some embodiments, cis-to-trans EOF is generated when the ratio of the cis-to-trans net ionic current to the total ionic current (also referred to as the cis-to-trans relative net current) is at most about 0.99, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, at most about 0.5, at most about 0.4, at most about 0.3, at most about 0.2, at most about 0.1, at most about 0.0, at most about -0.1, at most about -0.2, at most about -0.3, at most about -0.4, at most about -0.5, at most about -0.6, at most about -0.7, at most about -0.8, at most about -0.9, -0.95, at most about -0.99, or less than about -0.99. In some embodiments, cis-to-trans EOF is generated when the ratio of the cis-to-trans net ionic current to the total ionic current (also referred to as the cis-to-trans relative net current) is from about -0.99 to about 0.99.In some embodiments, cis-to-trans EOF is generated when the ratio of the cis-to-trans net ionic current to the total ionic current (also referred to as the cis-to-trans relative net current) is from about -0.99 to about -0.9, from about -0.99 to about -0.8, from about -0.99 to about -0.6, from about -0.99 to about -0.4, from about -0.99 to about -0.2, from about -0.99 to about 0, from about -0.99 to about 0.2, from about -0.99 to about 0.4, from about -0.99 to about 0.6, from about -0.99 to about 0.8, from about -0.99 to about 0.99, from about -0.9 to about -0.8, from about -0.9 to about -0.6, from about -0.9 to about -0.4, from about -0.9 to about -0.2, from about -0.9 to about 0, from about -0.9 to about 0.2, from about -0.9 to about 0.4, from about -0.9 to about 0.6, from about -0.9 to about 0.8, from about -0.9 to about 0.99, from about -0.8 to about -0.6, from about -0.8 to about -0.4, from about -0.8 to about -0.2, from about -0.8 to about 0, from about -0.8 to about 0.2, from about -0.8 to about 0.4, from about -0.8 to about 0.6, from about -0.8 to about 0.8, from about -0.8 to about 0.99, from about -0.6 to about -0.4, from about -0.6 to about -0.2, from about -0.6 to about 0, from about -0.6 to about 0.2, from about -0.6 to about 0.4, from about -0.6 to about 0.6, from about -0.6 to about 0.8, from about -0.6 to about 0.99, from about -0.4 to about -0.2, from about -0.4 to about 0, from about -0.4 to about 0.2, from about -0.4 to about 0.4, from about -0.4 to about 0.6, from about -0.4 to about 0.8, from about -0.4 to about 0.99, from about -0.2 to about 0, from about -0.2 to about 0.2, from about -0.2 to about 0.4, from about -0.2 to about 0.6, from about -0.2 to about 0.8, from -0.2 to about 0.99, from about 0 to about 0.2, from about 0 to about 0.4, from about 0 to about 0.6, from about 0 to about 0.8, from about 0 to about 0.99, from about 0.2 to about 0.4, from about 0.2 to about 0.6, from about 0.2 to about 0.8, from about 0.2 to about 0.99, from about 0.4 to about 0.6, from about 0.4 to about 0.8, from about 0.4 to about 0.99, from about 0.6 to about 0.8, from about 0.6 to about 0.99, or from about 0.8 to about 0.99.

[0160] In some embodiments, cis-to-trans EOF is generated when the ratio of the cis-to-trans net ionic current to the total ionic current (also referred to as the cis-to-trans relative net current) is about -0.99, about -0.95, about -0.9, about -0.8, about -0.7, about -0.6, about -0.5, about -0.4, about -0.3, about -0.2, about -0.1, 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.

[0161] In some embodiments, the absolute relative net electro-osmotic current (IrelV) with respect to the applied electric potential can 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 electro-osmotic current (IrelV) with respect to the applied electric potential can be at most about 1 pA / mV, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, at most about 0.5, at most about 0.4, at most about 0.3, at most about 0.2, at most about 0.15, at most about 0.10, at most about 0.09, at most about 0.08, at most about 0.07, at most about 0.06, at most about 0.05, at most about 0.04, at most about 0.03, at most about 0.02, at most about 0.01 or less than about 0.1 pA / mV.

[0162] In some embodiments, the absolute relative net electroosmotic current (IrelV) relative to the applied electric potential can be from about 0.01 pA / mV to about 1 pA / mV. In some embodiments, the absolute relative net electroosmotic current (IrelV) relative to the applied electric potential can be from about 0.01 pA / mV to about 0.02 pA / mV, from about 0.01 pA / mV to about 0.04 pA / mV, from about 0.01 pA / mV to about 0.06 pA / mV, from about 0.01 pA / mV to about 0.08 pA / mV, from about 0.01 pA / mV to about 0.1 pA / mV, from about 0.01 pA / mV to about 0.15 pA / mV, from about 0.01 pA / mV to about 0.2 pA / mV, from about 0.01 pA / mV to about 0.4 pA / mV, from about 0.01 pA / mV to about 0.6 pA / mV, from about 0.01 pA / mV to about 0.8 pA / mV, from about 0.01 pA / mV to about 1 pA / mV, from about 0.02 pA / mV to about 0.04 pA / mV, from about 0.02 pA / mV to about 0.06 pA / mV, from about 0.02 pA / mV to about 0.08 pA / mV, from about 0.02 pA / mV to about 0.1 pA / mV, from about 0.02 pA / mV to about 0.15 pA / mV, from about 0.02 pA / mV to about 0.2 pA / mV, from about 0.02 pA / mV to about 0.4 pA / mV, from about 0.02 pA / mV to about 0.6 pA / mV, from about 0.02 pA / mV to about 0.8 pA / mV, from about 0.02 pA / mV to about 1 pA / mV, from about 0.04 pA / mV to about 0.06 pA / mV, from about 0.04 pA / mV to about 0.08 pA / mV, from about 0.04 pA / mV to about 0.1 pA / mV, from about 0.04 pA / mV to about 0.15 pA / mV, from about 0.04 pA / mV to about 0.2 pA / mV, from about 0.04 pA / mV to about 0.4 pA / mV, from about 0.04 pA / mV to about 0.6 pA / mV, from about 0.04 pA / mV to about 0.8 pA / mV, from about 0.04 pA / mV to about 1 pA / mV, from about 0.06 pA / mV to about 0.08 pA / mV, from about 0.06 pA / mV to about 0.1 pA / mV, from about 0.06 pA / mV to about 0.15 pA / mV, from about 0.06 pA / mV to about 0.2 pA / mV, from about 0.06 pA / mV to about 0.4 pA / mV, from about 0.06 pA / mV to about 0.6 pA / mV, from about 0.06 pA / mV to about 0.8 pA / mV, from about 0.06 pA / mV to about 1 pA / mV, from about 0.08 pA / mV to about 0.1 pA / mV, from about 0.08 pA / mV to about 0.15 pA / mV, from about 0.08 pA / mV to about 0.2 pA / mV, 0.08 pA / mV to about 0.4 pA / mV, from about 0.from about 0.08 pA / mV to about 0.6 pA / mV, from about 0.08 pA / mV to about 0.8 pA / mV, from about 0.08 pA / mV to about 1 pA / mV, from about 0.1 pA / mV to about 0.15 pA / mV, from about 0.1 pA / mV to about 0.2 pA / mV, from about 0.1 pA / mV to about 0.4 pA / mV, from about 0.1 pA / mV to about 0.6 pA / mV, from about 0.1 pA / mV to about 0.8 pA / mV, from about 0.1 pA / mV to about 1 pA / mV, from about 0.15 pA / mV to about 0.2 pA / mV, from 0.15 pA / mV to about 0.4 pA / mV, from about 0.15 pA / mV to about 0.6 pA / mV, from about 0.15 pA / mV to about 0.8 pA / mV, from about 0.15 pA / mV to about 1 pA / mV, from about 0.2 pA / mV to about 0.4 pA / mV, from about 0.2 pA / mV to about 0.6 pA / mV, from about 0.2 pA / mV to about 0.8 pA / mV, from about 0.2 pA / mV to about 1 pA / mV, from about 0.4 pA / mV to about 0.6 pA / mV, from about 0.4 pA / mV to about 0.8 pA / mV, from about 0.4 pA / mV to about 1 pA / mV, from about 0.6 pA / mV to about 0.8 pA / mV, from about 0.6 pA / mV to about 1 pA / mV, or from about 0.8 pA / mV to about 1 pA / mV.

[0163] In some embodiments, the absolute relative electroosmotic current (IrelV) relative to the applied potential can be about 0.01 pA / mV, about 0.02 pA / mV, about 0.03 pA / mV, about 0.04 pA / mV, about 0.05 pA / mV, about 0.06 pA / mV, about 0.07 pA / mV, about 0.08 pA / mV, about 0.09 pA / mV, about 0.10 pA / mV, about 0.15 pA / mV, about 0.2 pA / mV, about 0.3 pA / mV, about 0.4 pA / mV, about 0.5 pA / mV, about 0.6 pA / mV, about 0.7 pA / mV, about 0.8 pA / mV, about 0.9 pA / mV or about 1 pA / mV. In some cases, the absolute relative electroosmotic current relative to the applied potential can include a value calculated by dividing the electroosmotic flow rate by the applied potential. In some cases, the electroosmotic flow rate can include the total flow rate of some ions or salts in the nanopore system.

[0164] In some embodiments, under an applied transmembrane potential difference, the relative ionic selectivity P(+) / P(-) of the pore can be 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 greater than about 5. In some embodiments, under an applied transmembrane potential difference, the relative ionic selectivity P(+) / P(-) of the pore is at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, at most about 0.5, at most about 0.4, at most about 0.3, at most about 0.2, at most about 0.1, or less than about 0.1.

[0165] In some embodiments, under an applied transmembrane potential difference, the pore can have a relative ionic selectivity P(+) / P(-) of from about 0.1 to about 5. In some embodiments, under an applied transmembrane potential difference, the relative ionic selectivity P(+) / P(-) of the pore is from about 0.1 to about 0.2, from about 0.1 to about 0.3, from about 0.1 to about 0.4, from about 0.1 to about 0.5, from about 0.1 to about 1, from about 0.1 to about 1.5, from about 0.1 to about 2, from about 0.1 to about 2.5, from about 0.1 to about 3, from about 0.1 to about 4, from about 0.1 to about 5, from about 0.2 to about 0.3, from about 0.2 to about 0.4, from about 0.2 to about 0.5, from about 0.2 to about 1, from about 0.2 to about 1.5, from about 0.2 to about 2, from about 0.2 to about 2.5, from about 0.2 to about 3, from about 0.2 to about 4, from about 0.2 to about 5, from about 0.3 to about 0.4, from about 0.3 to about 0.5, from about 0.3 to about 1, from about 0.3 to about 1.5, from about 0.3 to about 2, from about 0.3 to about 2.5, from about 0.3 to about 3, from about 0.3 to about 4, from about 0.3 to about 5, from about 0.4 to about 0.5, from about 0.4 to about 1, from about 0.4 to about 1.5, from about 0.4 to about 2, from about 0.4 to about 2.5, from about 0.4 to about 3, from about 0.4 to about 4, from about 0.4 to about 5, from about 0.5 to about 1, from about 0.5 to about 1.5, from about 0.5 to about 2, from about 0.5 to about 2.5, from about 0.5 to about 3, from about 0.5 to about 4, from about 0.5 to about 5, from about 1 to about 1.5, from about 1 to about 2, from about 1 to about 2.5, from about 1 to about 3, from about 1 to about 4, from about 1 to about 5, from about 1.5 to about 2, from about 1.5 to about 2.5, from about 1.5 to about 3, from about 1.5 to about 4, from about 1.5 to about 5, from about 2 to about 2.5, from about 2 to about 3, from about 2 to about 4, from about 2 to about 5, from about 2.5 to about 3, from about 2.5 to about 4, from about 2.5 to about 5, from about 3 to about 4, from about 3 to about 5, or from about 4 to about 5.

[0166] In some embodiments, the relative ionic selectivity P(+) / P(-) of the pores can be 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 an applied transmembrane potential difference.

[0167] In some embodiments, the solutions on the cis and trans sides of the fluid chamber are configured to generate an electroosmotic force. The electroosmotic force can be generated by a difference in solute concentration between the solution on the cis side and the solution on the cis side. The solute can be one or more ions or one or more osmotic regulators. In some cases, the one or more ions can include chloride ion, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium ion, sulfate, sulfide, calcium ion, fluoride, hydroxide, aluminum ion, barium ion, bismuth ion, cadmium ion, cesium ion, chromium ion, cobalt ion, copper ion, hydrogen ion, iron ion, lead ion, lithium ion, magnesium ion, mercury ion, nickel ion, potassium ion, rubidium ion, silver ion, sodium ion, strontium ion, tin ion, zinc ion, iodide ion, nitride ion, oxide ion, glutamate, acetate, formate, acetate, butyrate, benzoate, carboxylate, alkoxide ion, benzyl alcoholate, oxalate, ammonate, tartrate, malate, citrate, gluconate, maleate, sorbate, stearate, lactate, glycerate, urate, diazonium salt, imine salt, hypophosphite, organophosphate, methanesulfonate, bechgaard salt, picolinate, cocaine salt, morphine salt, sodium glutamate, triethanolamine salicylate, triphenylmethyl hexafluorophosphate, choline chloride, copper ibuprofenate, homatropine methylbromide, mellite, tetrapropylammonium perruthenate, collidinium p-toluenesulfonate, pyridine chloride, ethylenediaminetetraacetic acid tetrasodium, lithium diisopropylamide, lithium bis(trimethylsilyl)amide, potassium triphenylborohydride, redox salt, ferrocyanide, ferricyanide, or any combination thereof. In some embodiments, the one or more osmotic regulators can be one or more types of salts. In some cases, the one or more salts can include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc chloride hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, tetramine copper sulfate, zinc chloride hydroxide monohydrate, sodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof. These ions or osmotic regulators can flow across the nanopore membrane. These ions can be high mobility ions or low mobility ions.

[0168] In some embodiments, an EOF can be generated by an asymmetric salt distribution between the cis and trans sides of a membrane. In some cases, the concentration of one or more salts on the cis side of the membrane can be different 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 can 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 can 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 can 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 can be lower than the concentration of one or more salts on the cis side of the membrane.

[0169] In some cases, the concentration of one or more salts on the cis side of the membrane can be between about 1 nanomolar (nM) and about 1,000 nM. In some cases, the concentration of one or more salts on the cis side of the membrane can be between about 1 nM and about 10 nM, between about 10 nM and about 100 nM, or between about 100 nM and 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, 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 greater than 1,000 nM. In some cases, the concentration of one or more salts on the cis side of the membrane can be at most about 1,000 nM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 95 nM, at most about 90 nM, at most about 85 nM, at most about 80 nM, at most about 75 nM, at most about 70 nM, at most about 65 nM, at most about 60 nM, at most about 55 nM, at most about 45 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, at most about 5 nM, at most about 1 nM or less than 1 nM.In some cases, the concentration of one or more salts on the cis side of the membrane can be about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM or about 1,000 nM.

[0170] In some embodiments, the concentration of salt, ion, osmolyte or electrolyte on the cis side can 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 salt, ion, osmolyte or electrolyte on the cis side can be at most about 5 M, at most about 4.5 M, at most about 4 M, at most about 3.5 M, at most about 3 M, at most about 2.5 M, at most about 2 M, at most about 1.75 M, at most about 1.50 M, at most about 1.25 M, at most about 1 M, at most about 0.90 M, at most about 0.80 M, at most about 0.70 M, at most about 0.60 M, at most about 0.50 M, at most about 0.40 M, at most about 0.30 M, at most about 0.20 M, at most about 0.10 M, at most about 0.05 M, at most about 0.01 M or less than about 0.01 M.

[0171] In some embodiments, the concentration of the salt, ion, osmolyte, or electrolyte on the cis side can be from about 0.01 M to about 5 M. In some embodiments, the concentration of the salt, ion, osmolyte, or electrolyte on the cis side can be from about 0.01 M to about 0.1 M, from about 0.01 M to about 0.5 M, from about 0.01 M to about 1 M, from about 0.01 M to about 1.5 M, from about 0.01 M to about 2 M, from about 0.01 M to about 2.5 M, from about 0.01 M to about 3 M, from about 0.01 M to about 3.5 M, from about 0.01 M to about 4 M, from about 0.01 M to about 4.5 M, from about 0.01 M to about 5 M, from about 0.1 M to about 0.5 M, from about 0.1 M to about 1 M, from about 0.1 M to about 1.5 M, from about 0.1 M to about 2 M, from about 0.1 M to about 2.5 M, from about 0.1 M to about 3 M, from about 0.1 M to about 3.5 M, from about 0.1 M to about 4 M, from about 0.1 M to about 4.5 M, from about 0.1 M to about 5 M, from about 0.5 M to about 1 M, from about 0.5 M to about 1.5 M, from about 0.5 M to about 2 M, from about 0.5 M to about 2.5 M, from about 0.5 M to about 3 M, from about 0.5 M to about 3.5 M, from about 0.5 M to about 4 M, from about 0.5 M to about 4.5 M, from about 0.5 M to about 5 M, from about 1 M to about 1.5 M, from about 1 M to about 2 M, from about 1 M to about 2.5 M, from about 1 M to about 3 M, from about 1 M to about 3.5 M, from about 1 M to about 4 M, from about 1 M to about 4.5 M, from about 1 M to about 5 M, from about 1.5 M to about 2 M, from about 1.5 M to about 2.5 M, from about 1.5 M to about 3 M, from about 1.5 M to about 3.5 M, from about 1.5 M to about 4 M, from about 1.5 M to about 4.5 M, from about 1.5 M to about 5 M, from about 2 M to about 2.5 M, from about 2 M to about 3 M, from about 2 M to about 3.5 M, from about 2 M to about 4 M, from about 2 M to about 4.5 M, from about 2 M to about 5 M, from about 2.5 M to about 3 M, from about 2.5 M to about 3.5 M, from about 2.5 M to about 4 M, from about 2.5 M to about 4.5 M, from about 2.5 M to about 5 M, from about 3 M to about 3.5 M, from about 3 M to about 4 M, from about 3 M to about 4.5 M, from about 3 M to about 5 M, from about 3.5 M to about 4 M, from about 3.5 M to about 4.5 M, from about 3.5 M to about 5 M, from about 4 M to about 4.5 M, from about 4 M to about 5 M, or from about 4.5 M to about 5 M.

[0172] In some embodiments, the concentration of the salt, ion, osmolyte, or electrolyte on the cis side can 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.

[0173] In some cases, the concentration of one or more salts on the trans side of the membrane can be between about 1 nanomolar (nM) and about 1,000 nM. In some cases, the concentration of one or more salts on the trans side of the membrane can be between about 1 nM and about 10 nM, between about 10 nM and about 100 nM, or between about 100 nM and about 1,000 nM. In some cases, the concentration of one or more salts on the trans 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 greater than about 1,000 nM. In some cases, the concentration of one or more salts on the trans side of the membrane can be at most about 1,000 nM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 95 nM, at most about 90 nM, at most about 85 nM, at most about 80 nM, at most about 75 nM, at most about 70 nM, at most about 65 nM, at most about 60 nM, at most about 55 nM, at most about 45 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, at most about 5 nM, at most about 1 nM, or less than 1 nM.In some cases, the concentration of one or more salts on the trans side of the membrane can be about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM or about 1,000 nM.

[0174] In some embodiments, the concentration of salt, ion, osmolyte or electrolyte on the trans side can 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 salt, ion, osmolyte or electrolyte on the trans side can be at most about 5 M, at most about 4.5 M, at most about 4 M, at most about 3.5 M, at most about 3 M, at most about 2.5 M, at most about 2 M, at most about 1.75 M, at most about 1.50 M, at most about 1.25 M, at most about 1 M, at most about 0.90 M, at most about 0.80 M, at most about 0.70 M, at most about 0.60 M, at most about 0.50 M, at most about 0.40 M, at most about 0.30 M, at most about 0.20 M, at most about 0.10 M, at most about 0.05 M, at most about 0.01 M or less than about 0.01 M.

[0175] In some embodiments, the concentration of salts, ions, osmotic agents, or electrolytes on the trans side can be from about 0.01 M to about 5 M. In some embodiments, the concentration of salts, ions, osmotic agents, or electrolytes on the cis side can be from about 0.01 M to about 0.1 M, from about 0.01 M to about 0.5 M, from about 0.01 M to about 1 M, from about 0.01 M to about 1.5 M, from about 0.01 M to about 2 M, from about 0.01 M to about 2.5 M, from about 0.01 M to about 3 M, from about 0.01 M to about 3.5 M, from about 0.01 M to about 4 M, from about 0.01 M to about 4.5 M, from about 0.01 M to about 5 M, from about 0.1 M to about 0.5 M, from about 0.1 M to about 1 M, from about 0.1 M to about 1.5 M, from about 0.1 M to about 2 M, from about 0.1 M to about 2.5 M, from about 0.1 M to about 3 M, from about 0.1 M to about 3.5 M, from about 0.1 M to about 4 M, from about 0.1 M to about 4.5 M, from about 0.1 M to about 5 M, from about 0.5 M to about 1 M, from about 0.5 M to about 1.5 M, from about 0.5 M to about 2 M, from about 0.5 M to about 2.5 M, from about 0.5 M to about 3 M, from about 0.5 M to about 3.5 M, from about 0.5 M to about 4 M, from about 0.5 M to about 4.5 M, from about 0.5 M to about 5 M, from about 1 M to about 1.5 M, from about 1 M to about 2 M, from about 1 M to about 2.5 M, from about 1 M to about 3 M, from about 1 M to about 3.5 M, from about 1 M to about 4 M, from about 1 M to about 4.5 M, from about 1 M to about 5 M, from about 1.5 M to about 2 M, from about 1.5 M to about 2.5 M, from about 1.5 M to about 3 M, from about 1.5 M to about 3.5 M, from about 1.5 M to about 4 M, from about 1.5 M to about 4.5 M, from about 1.5 M to about 5 M, from about 2 M to about 2.5 M, from about 2 M to about 3 M, from about 2 M to about 3.5 M, from about 2 M to about 4 M, from about 2 M to about 4.5 M, from about 2 M to about 5 M, from about 2.5 M to about 3 M, from about 2.5 M to about 3.5 M, from about 2.5 M to about 4 M, from about 2.5 M to about 4.5 M, from about 2.5 M to about 5 M, from about 3 M to about 3.5 M, from about 3 M to about 4 M, from about 3 M to about 4.5 M, from about 3 M to about 5 M, from about 3.5 M to about 4 M, from about 3.5 M to about 4.5 M, from about 3.5 M to about 5 M, from about 4 M to about 4.5 M, from about 4 M to about 5 M, or from about 4.5 M to about 5 M.

[0176] In some embodiments, the concentration of salts, ions, osmotic agents, or electrolytes on the trans side can 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.

[0177] In some embodiments, the concentration difference of salts, ions or electrolytes between the cis side and the trans side can 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 the concentration of salts, ions or electrolytes between the cis side and the trans side can be at most about 5 M, at most about 4.5 M, at most about 4 M, at most about 3.5 M, at most about 3 M, at most about 2.5 M, at most about 2 M, at most about 1.75 M, at most about 1.50 M, at most about 1.25 M, at most about 1 M, at most about 0.90 M, at most about 0.80 M, at most about 0.70 M, at most about 0.60 M, at most about 0.50 M, at most about 0.40 M, at most about 0.30 M, at most about 0.20 M, at most about 0.10 M, at most about 0.05 M, at most about 0.01 M or less than about 0.01 M.

[0178] In some embodiments, the difference in salt, ion, or electrolyte concentration between the cis side and the trans side can be from about 0.01 M to about 5 M. In some embodiments, the difference in salt, ion, or electrolyte concentration between the cis side and the trans side can be from about 0.01 M to about 0.1 M, from about 0.01 M to about 0.5 M, from about 0.01 M to about 1 M, from about 0.01 M to about 1.5 M, from about 0.01 M to about 2 M, from about 0.01 M to about 2.5 M, from about 0.01 M to about 3 M, from about 0.01 M to about 3.5 M, from about 0.01 M to about 4 M, from about 0.01 M to about 4.5 M, from about 0.01 M to about 5 M, from about 0.1 M to about 0.5 M, from about 0.1 M to about 1 M, from about 0.1 M to about 1.5 M, from about 0.1 M to about 2 M, from about 0.1 M to about 2.5 M, from about 0.1 M to about 3 M, from about 0.1 M to about 3.5 M, from about 0.1 M to about 4 M, from about 0.1 M to about 4.5 M, from about 0.1 M to about 5 M, from about 0.5 M to about 1 M, from about 0.5 M to about 1.5 M, from about 0.5 M to about 2 M, from about 0.5 M to about 2.5 M, from about 0.5 M to about 3 M, from about 0.5 M to about 3.5 M, from about 0.5 M to about 4 M, from about 0.5 M to about 4.5 M, from about 0.5 M to about 5 M, from about 1 M to about 1.5 M, from about 1 M to about 2 M, from about 1 M to about 2.5 M, from about 1 M to about 3 M, from about 1 M to about 3.5 M, from about 1 M to about 4 M, from about 1 M to about 4.5 M, from about 1 M to about 5 M, from about 1.5 M to about 2 M, from about 1.5 M to about 2.5 M, from about 1.5 M to about 3 M, from about 1.5 M to about 3.5 M, from about 1.5 M to about 4 M, from about 1.5 M to about 4.5 M, from about 1.5 M to about 5 M, from about 2 M to about 2.5 M, from about 2 M to about 3 M, from about 2 M to about 3.5 M, from about 2 M to about 4 M, from about 2 M to about 4.5 M, from about 2 M to about 5 M, from about 2.5 M to about 3 M, from about 2.5 M to about 3.5 M, from about 2.5 M to about 4 M, from about 2.5 M to about 4.5 M, from about 2.5 M to about 5 M, from about 3 M to about 3.5 M, from about 3 M to about 4 M, from about 3 M to about 4.5 M, from about 3 M to about 5 M, from about 3.5 M to about 4 M, from about 3.5 M to about 4.5 M, from about 3.5 M to about 5 M, from about 4 M to about 4.5 M, from about 4 M to about 5 M, or from about 4.5 M to about 5 M.

[0179] In some embodiments, the difference in salt, ion, or electrolyte concentration between the cis side and the trans side can 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.

[0180] In some embodiments, one or more salts may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxychloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, tetraamine copper sulfate, zinc hydroxychloride monohydrate, sodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, potassium glutamate, sodium ferrocyanide, sodium ferricyanide, potassium ferrocyanide, potassium ferricyanide, or any combination thereof.

[0181] 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 hydroxychloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, tetraamine copper sulfate, zinc hydroxychloride monohydrate, sodium 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 hydroxychloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, tetraamine copper sulfate, zinc hydroxychloride monohydrate, sodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof.

[0182] 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 trans side of the membrane. In some cases, the types of one or more salts on the cis side of the membrane may be the same as the salts on the trans 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 trans side of the membrane. In some embodiments, the types of one or more salts on the cis side of the membrane may be different from one or more salts on the trans side of the membrane.

[0183] In some embodiments, the one or more salts can include from about 1 type of salt to about 10 types of salts. In some cases, the one or more salts can include at least about 1 type of salt, at least about 2 types of salts, at least about 3 types of salts, at least about 4 types of salts, at least about 5 types of salts, at least about 6 types of salts, at least about 7 types of salts, at least about 8 types of salts, at least about 9 types of salts, at least about 10 types of salts, or more than 10 types of salts. In some cases, the one or more types of salts can include at most about 10 types of salts, at most about 9 types of salts, at most about 8 types of salts, at most about 7 types of salts, at most about 6 types of salts, at most about 5 types of salts, at most about 4 types of salts, at most about 3 types of salts, at most about 2 types of salts, at most about 1 type of salt, or less than 1 type of salt. In some cases, the one or more types of salts can include one type of salt, about two types of salts, about three types of salts, about four types of salts, about five types of salts, about six types of salts, about seven types of salts, about eight types of salts, about nine types of salts, or about ten types of salts.

[0184] In some embodiments, the one or more salts on the cis side of the membrane can be of the same type as the one or more salts on the trans side of the membrane. In some cases, the same type of salts present on the cis and trans sides of the membrane can be present at the same concentration. In some cases, the same type of salts present on the cis and trans sides of the membrane can be present at different concentrations.

[0185] In some embodiments, the one or more salts on the cis side of the membrane can be of a different type from the 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 can have the same concentration. In some cases, the different types of salts present on the cis and trans sides of the membrane can have different concentrations.

[0186] In some embodiments, the concentration of one or more salts on the cis side of the membrane can 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 can 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% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490% or about 490% to about 500% higher than the concentration of one or more salts on the trans side of the membrane.

[0187] In some cases, the concentration of one or more salts on the cis side of the membrane can be 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 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 higher than 500% higher than the concentration of one or more salts on the trans side of the membrane.

[0188] In some cases, the concentration of one or more salts on the cis side of the membrane can be up to about 500%, up to about 490%, up to about 480%, up to about 470%, up to about 460%, up to about 450%, up to about 440%, up to about 430%, up to about 420%, up to about 410%, up to about 400%, up to about 390%, up to about 380%, up to about 370%, up to about 360%, up to about 350%, up to about 340%, up to about 330%, up to about 320%, up to about 310%, up to about 300%, up to about 290%, up to about 280%, up to about 270%, up to about 260%, up to about 250%, up to about 240%, up to about 230%, up to about 220%, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, 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 1%, up to about 0.5%, up to about 0.1% or less than 0.1% higher than the concentration of one or more salts on the trans side of the membrane.

[0189] In some cases, the concentration of one or more salts on the cis side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500% higher than the concentration of one or more salts on the trans side of the membrane.

[0190] In some embodiments, the concentration of one or more salts on the cis side of the membrane can be lower than the concentration of one or more salts on the trans side of the membrane by between about 0.1% and about 500%. In some cases, the concentration of one or more salts on the cis side of the membrane can be lower than the concentration of one or more salts on the trans side of the membrane by between about 0.1% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, between about 60% and about 65%, between about 65% and about 70%, between about 70% and about 75%, between about 75% and about 80%, between about 80% and about 85%, between about 85% and about 90%, between about 90% and about 95%, between about 95% and about 100%, between about 100% and about 110%, between about 110% and about 120%, between about 120% and about 130%, between about 130% and about 140%, between about 140% and about 150%, between about 150% and about 160%, between about 160% and about 170%, between about 170% and about 180%, between about 180% and about 190%, between about 190% and about 200%, between about 200% and about 210%, between about 210% and about 220%, between about 220% and about 230%, between about 230% and about 240%, between about 240% and about 250%, between about 250% and about 260%, between about 260% and about 270%, between about 270% and about 280%, between about 280% and about 290%, between about 290% and about 300%, between about 300% and about 310%, between about 310% and about 320%, between about 320% and about 330%, between about 330% and about 340%, between about 340% and about 350%, between about 350% and about 360%, between about 360% and about 370%, between about 370% and about 380%, between about 380% and about 390%, between about 390% and about 400%, between about 400% and about 410%, between about 410% and about 420%, between about 420% and about 430%, between about 430% and about 440%, between about 440% and about 450%, between about 450% and about 460%, between about 460% and about 470%, between about 470% and about 480%, between about 480% and about 490%, or between about 490% and about 500%.

[0191] In some cases, the concentration of one or more salts on the cis side of the membrane can be 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 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 more than 500% lower than the concentration of one or more salts on the trans side of the membrane.

[0192] In some cases, the concentration of one or more salts on the cis side of the membrane can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1% or less than 0.1% lower than the concentration of one or more salts on the trans side of the membrane.

[0193] In some cases, the concentration of one or more salts on the cis side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500% lower than the concentration of one or more salts on the trans side of the membrane.

[0194] In some embodiments, the concentration of one or more salts on the trans side of the membrane can 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 trans side of the membrane can 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% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490% or about 490% to about 500% higher.

[0195] In some cases, the concentration of one or more salts on the trans side of the membrane can be 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 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 higher than 500% higher than the concentration of one or more salts on the cis side of the membrane.

[0196] It should be noted that there seems to be a small issue in the English translation where the "higher than the concentration of one or more salts on the cis side of the membrane" at the end might be a bit misplaced in the original Chinese structure. It might be more clearly written in English as "higher than that of one or more salts on the cis side of the membrane" for better flow, but this is translated strictly according to the original text's structure.In some cases, the concentration of one or more salts on the trans side of the membrane can be up to about 500%, up to about 490%, up to about 480%, up to about 470%, up to about 460%, up to about 450%, up to about 440%, up to about 430%, up to about 420%, up to about 410%, up to about 400%, up to about 390%, up to about 380%, up to about 370%, up to about 360%, up to about 350%, up to about 340%, up to about 330%, up to about 320%, up to about 310%, up to about 300%, up to about 290%, up to about 280%, up to about 270%, up to about 260%, up to about 250%, up to about 240%, up to about 230%, up to about 220%, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, 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 1%, up to about 0.5%, up to about 0.1%, or less than 0.1% higher than the concentration of one or more salts on the cis side of the membrane.

[0197] In some cases, the concentration of one or more salts on the trans side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% higher than the concentration of one or more salts on the cis side of the membrane.

[0198] In some embodiments, the concentration of one or more salts on the trans side of the membrane can be lower than the concentration of one or more salts on the cis side of the membrane by between about 0.1% and about 500%. In some cases, the concentration of one or more salts on the trans side of the membrane can be lower than the concentration of one or more salts on the cis side of the membrane by between about 0.1% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, between about 60% and about 65%, between about 65% and about 70%, between about 70% and about 75%, between about 75% and about 80%, between about 80% and about 85%, between about 85% and about 90%, between about 90% and about 95%, between about 95% and about 100%, between about 100% and about 110%, between about 110% and about 120%, between about 120% and about 130%, between about 130% and about 140%, between about 140% and about 150%, between about 150% and about 160%, between about 160% and about 170%, between about 170% and about 180%, between about 180% and about 190%, between about 190% and about 200%, between about 200% and about 210%, between about 210% and about 220%, between about 220% and about 230%, between about 230% and about 240%, between about 240% and about 250%, between about 250% and about 260%, between about 260% and about 270%, between about 270% and about 280%, between about 280% and about 290%, between about 290% and about 300%, between about 300% and about 310%, between about 310% and about 320%, between about 320% and about 330%, between about 330% and about 340%, between about 340% and about 350%, between about 350% and about 360%, between about 360% and about 370%, between about 370% and about 380%, between about 380% and about 390%, between about 390% and about 400%, between about 400% and about 410%, between about 410% and about 420%, between about 420% and about 430%, between about 430% and about 440%, between about 440% and about about 450%, between about 450% and about 460%, between about 460% and about 470%, between about 470% and about 480%, between about 480% and about 490% or between about 490% and about 500%.

[0199] In some cases, the concentration of one or more salts on the trans side of the membrane can be 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 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 greater than 500% lower than the concentration of one or more salts on the cis side of the membrane.

[0200] In some cases, the concentration of one or more salts on the trans side of the membrane can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1% lower than the concentration of one or more salts on the cis side of the membrane.

[0201] In some cases, the concentration of one or more salts on the trans side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% lower than the concentration of one or more salts on the cis side of the membrane.

[0202] In some embodiments, EOF can be generated by an asymmetric salt distribution. An asymmetric salt distribution can 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.

[0203] Alternatively, EOF can be generated by a symmetric salt distribution between the cis and trans sides of the membrane. A symmetric salt distribution means that 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 can be the same as the concentration of one or more salts on the trans side of the membrane.

[0204] In some embodiments, EOF can be generated by an asymmetric ion distribution between the cis and trans sides of the membrane. An asymmetric ion distribution can 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 can 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 can 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 can 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 can be lower than the concentration of one or more ions on the cis side of the membrane.

[0205] In some cases, the concentration of one or more ions on the cis side of the membrane can be between about 1 nanomolar (nM) and about 1,000 nM. In some cases, the concentration of one or more ions on the cis side of the membrane can be between about 1 nM and about 10 nM, between about 10 nM and about 100 nM, or between about 100 nM and 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 greater than 1,000 nM. In some cases, the concentration of one or more ions on the cis side of the membrane can be at most about 1,000 nM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 95 nM, at most about 90 nM, at most about 85 nM, at most about 80 nM, at most about 75 nM, at most about 70 nM, at most about 65 nM, at most about 60 nM, at most about 55 nM, at most about 45 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, at most about 5 nM, at most about 1 nM or less than 1 nM.In some cases, the concentration of the salt on the cis side of the membrane can be about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM or about 1,000 nM.

[0206] In some cases, the concentration of one or more ions on the trans side of the membrane can be between about 1 nanomolar (nM) and about 1,000 nM. In some cases, the concentration of one or more ions on the trans side of the membrane can be between about 1 nM and about 10 nM, between about 10 nM and about 100 nM, or between about 100 nM and about 1,000 nM. In some cases, the concentration of one or more ions on the trans 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 greater than 1,000 nM. In some cases, the concentration of one or more ions on the trans side of the membrane can be at most about 1,000 nM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 95 nM, at most about 90 nM, at most about 85 nM, at most about 80 nM, at most about 75 nM, at most about 70 nM, at most about 65 nM, at most about 60 nM, at most about 55 nM, at most about 45 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, at most about 5 nM, at most about 1 nM, or less than 1 nM.In some cases, the concentration of one or more ions on the trans side of the membrane can be about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, or about 1,000 nM.

[0207] In some embodiments, one or more ions can include chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, 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.

[0208] In some embodiments, one or more ions on the cis side of the membrane can include chloride ion, carbonate ion, chlorite ion, chlorate ion, phosphate ion, bicarbonate ion, bromide ion, ammonium sulfate ion, ammonium ion, sulfate, 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.

[0209] In some embodiments, one or more ions on the trans side of the membrane can include chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, 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.

[0210] In some embodiments, one or more ions on the cis side of the membrane can be the same type of ions 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 can be different types of ions from one or more ions on the trans side of the membrane.

[0211] In some embodiments, one or more ions can include from about 1 ion to about 10 ions. In some cases, one or more ions can include 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 can include at most about 10 ions, at most about 9 ions, at most about 8 ions, at most about 7 ions, at most about 6 ions, at most about 5 ions, at most about 4 ions, at most about 3 ions, at most about 2 ions, at most about 1 ion, or less than 1 ion. In some cases, one or more ions can include about one ion, about two ions, about three ions, about four ions, about five ions, about six ions, about seven ions, about eight ions, about nine ions, or about ten ions.

[0212] In some embodiments, one or more ions on the cis side of the membrane can have the same concentration 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 can have different concentrations from one or more ions on the trans side of the membrane.

[0213] In some embodiments, the concentration of one or more ions on the cis side of the membrane can be higher than the concentration of one or more ions on the trans side of the membrane by about 0.1% to about 500%. In some cases, the concentration of one or more ions on the cis side of the membrane can be higher than the concentration of one or more ions on the trans side of the membrane by 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%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490% or about 490% to about 500%.

[0214] In some cases, the concentration of one or more ions on the cis side of the membrane can be 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 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 higher than 500% higher than the concentration of one or more ions on the trans side of the membrane.

[0215] In some cases, the concentration of one or more ions on the cis side of the membrane can be up to about 500%, up to about 490%, up to about 480%, up to about 470%, up to about 460%, up to about 450%, up to about 440%, up to about 430%, up to about 420%, up to about 410%, up to about 400%, up to about 390%, up to about 380%, up to about 370%, up to about 360%, up to about 350%, up to about 340%, up to about 330%, up to about 320%, up to about 310%, up to about 300%, up to about 290%, up to about 280%, up to about 270%, up to about 260%, up to about 250%, up to about 240%, 230%, up to about 220%, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, 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%, about 1%, up to about 0.5%, up to about 0.1% or less than 0.1% higher than the concentration of one or more ions on the trans side of the membrane.

[0216] In some cases, the concentration of one or more ions on the cis side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500% higher than the concentration of one or more ions on the trans side of the membrane.

[0217] In some embodiments, the concentration of one or more ions on the cis side of the membrane can be lower than the concentration of one or more ions on the trans side of the membrane by about 0.1% to about 500%. In some cases, the concentration of one or more ions on the cis side of the membrane can be lower than the concentration of one or more ions on the trans side of the membrane by 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%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490%, or about 490% to about 500%.

[0218] In some cases, the concentration of one or more ions on the cis side of the membrane can be 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 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 more than 500% lower than the concentration of one or more ions on the trans side of the membrane.

[0219] In some cases, the concentration of one or more ions on the cis side of the membrane can be up to about 500%, up to about 490%, up to about 480%, up to about 470%, up to about 460%, up to about 450%, up to about 440%, up to about 430%, up to about 420%, up to about 410%, up to about 400%, up to about 390%, up to about 380%, up to about 370%, up to about 360%, up to about 350%, up to about 340%, up to about 330%, up to about 320%, up to about 310%, up to about 300%, up to about 290%, up to about 280%, up to about 270%, up to about 260%, up to about 250%, up to about 240%, up to about 230%, up to about 220%, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, 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 1%, up to about 0.5%, up to about 0.1% or less than 0.1% lower than the concentration of one or more ions on the trans side of the membrane.

[0220] In some cases, the concentration of one or more ions on the cis side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500% lower than the concentration of one or more ions on the trans side of the membrane.

[0221] In some embodiments, the concentration of one or more ions on the trans side of the membrane can be about 0.1% to about 500% higher than the concentration of the salt on the cis side of the membrane. In some cases, the concentration of the salt on the trans side of the membrane can 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% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490% or about 490% to about 500% higher than the concentration of one or more ions on the cis side of the membrane.

[0222] In some cases, the concentration of one or more ions on the trans side of the membrane can be 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 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 greater than 500% higher than the concentration of one or more ions on the cis side of the membrane.

[0223] In some cases, the concentration of one or more ions on the trans side of the membrane can be up to about 500%, up to about 490%, up to about 480%, up to about 470%, up to about 460%, up to about 450%, up to about 440%, up to about 430%, up to about 420%, up to about 410%, up to about 400%, up to about 390%, up to about 380%, up to about 370%, up to about 360%, up to about 350%, up to about 340%, up to about 330%, up to about 320%, up to about 310%, up to about 300%, up to about 290%, up to about 280%, up to about 270%, up to about 260%, up to about 250%, up to about 240%, up to about 230%, up to about 220%, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, 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 1%, up to about 0.5%, up to about 0.1%, or less than 0.1% higher than the concentration of one or more ions on the cis side of the membrane.

[0224] In some cases, the concentration of one or more ions on the trans side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% higher than the concentration of one or more ions on the cis side of the membrane.

[0225] In some embodiments, the concentration of one or more ions on the trans side of the membrane can be lower than the concentration of one or more ions on the cis side of the membrane by about 0.1% to about 500%. In some cases, the concentration of one or more ions on the trans side of the membrane can be lower than the concentration of one or more ions on the cis side of the membrane by 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%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490% or about 490% to about 500%.

[0226] In some cases, the concentration of one or more ions on the trans side of the membrane can be 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 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 greater than 500% lower than the concentration of one or more ions on the cis side of the membrane.

[0227] In some cases, the concentration of one or more ions on the trans side of the membrane can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, 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 about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1% lower than the concentration of one or more ions on the cis side of the membrane.

[0228] In some cases, the concentration of one or more ions on the trans side of the membrane can be about 0.1%, about 0.5%, 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%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% lower than the concentration of one or more ions on the cis side of the membrane.

[0229] Alternatively, EOF can be generated by a symmetric ion distribution between the cis side and the trans side of the membrane. The symmetric ion distribution can be 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.

[0230] In some embodiments, EOF can be generated by an asymmetric concentration of one or more salts and an asymmetric concentration of one or more ions between the cis side and the trans side of the membrane.

[0231] In some embodiments, the electroosmotic force can act in the same direction as the electrophoretic force or in the opposite direction. In some embodiments, the electroosmotic force can be greater than the electrophoretic force. In some examples, the electroosmotic force can be less than the electrophoretic force.

[0232] The present invention relates to systems and methods for analyzing a target analyte using a nanopore-based sensor. More specifically, the present invention relates to methods, nanopore systems, and devices for single-molecule profiling of polymers such as polypeptides or polysaccharides.

[0233] Multiple studies have demonstrated that polypeptides (proteins that unfold during or prior to translocation through a narrow nanopore) can translocate freely or move through a narrow nanopore (typically less than 2 nm in diameter) under motor control. However, unlike polynucleotides with a fixed negative charge, which can be electrophoretically pulled into a nanopore by an electric field generated by applying a voltage, capturing and controlling the movement of peptides with different compositions remains a challenge. This is because different compositions result in a range of electrical and / or structural properties (e.g., a mixture of positively charged, negatively charged, neutral, hydrophilic, hydrophobic, and aromatic), which impede simple capture under electrophoretic conditions and translocation in the unfolded state.

[0234] Due to the complex composition of analytes (e.g., proteins, peptides, polypeptides), it was previously thought impossible to push / deliver them in their native form (e.g., without attachment to a DNA leader sequence or addition of other (e.g., polyanionic) tags to create an electrophoretic capture motif) from the cis side into a nanopore. Depending on the charge and / or applied voltage, unfolded peptides are sometimes attracted to and sometimes repelled by the nanopore due to their different charges, so it is impossible to translocate multiple complex peptide segments through the nanopore solely by electrophoretic methods. In fact, previous studies have only demonstrated the translocation of extremely short peptides with contour lengths shorter than the channel length of the nanopore or carefully selected (model) protein substrates whose charge, structure, or added electrophoretic tags facilitate electrophoretic capture and translocation through the nanopore. For example, see 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 2021, 2021.09.28.462155.

[0235] However, this in no way represents the wide amino acid composition of proteins found in nature. For example, see Motone et al. (iScience 24, September 24, 2021) who reviewed recent methods for driving protein chains and peptides through nanopores using a range of techniques. It was pointed out that nanopore protein sequencing is a challenging frontier that has not yet been achieved.

[0236] Bayat et al. (Nature Comm. 2022 Vol. 13, 5113) reported the label-free detection and analysis of highly anionic linear polysaccharides using protein nanopores. It was found that the wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, glycosidic linkages, and glucuronic acid residue epimers.

[0237] Robertson et al. (BBA - Biomembranes, Vol. 1863, No. 9, 2021) focused on the physical chemistry of nanopore sensing and reviewed the types of analytes that can be detected. Among them, size recognition of polyethylene glycol (PEG) with a length of approximately 48 repeat units based on modified α-hemolysin (aHL) or aerolysin-based nanopore systems was mentioned.

[0238] The present disclosure provides a simple and robust new method for transporting long non-nucleic acid-based polymers through nanopores, for example for sequencing or characterizing them. In some cases, the present disclosure can move the polymer against the direction of the dominant electrophoretic force (EPF) acting thereon, thereby preventing its translocation, and without labeling the polymer analyte.

[0239] It has been found that these goals can be achieved by exploiting a large and / or dominant cis-to-trans electroosmotic flow (EOF) generated by a large number of cis-to-trans ions flowing through the nanopore, which can transport and move various elongated and complex polymer substrates from cis to trans and through the nanopore, even against 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 solvent from the cis side to the trans side of the nanopore system.

[0240] The present disclosure provides a system that can capture and transport polymer analytes from the cis side of a nanopore using strong electroosmotic forces. In some embodiments, the strong electroosmotic forces exert a traction force on the polymer during its translocation through the nanopore, thereby measuring and / or characterizing current changes that depend on the structure and / or composition.

[0241] Thus, in one embodiment, the present invention relates to a method for translocating a non-nucleic acid-based polymer analyte through a nanopore, the nanopore being contained in a membrane that divides the fluid chambers of the nanopore system into a cis side and a trans side, the method comprising adding the analyte to the cis side and allowing it to translocate, wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) generated by a cis-to-trans net ionic current flow. A method for translocating a non-nucleic acid-based polymer analyte through a nanopore, the nanopore being contained in a membrane that divides the fluid chambers of the nanopore system into a cis side and a trans side, the method comprising adding the polymer analyte to the cis side of the nanopore and allowing the polymer analyte to translocate to the trans side of the nanopore, 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 wherein the nanopore system has a cis-to-trans EOF caused by a cis-to-trans net ionic current flow, and wherein the cis-to-trans EOF overcomes the trans-to-cis EPF acting on the polymer analyte.

[0242] For example, the nanopore system has a cis-to-trans EOF generated by a ratio of the cis-to-trans net ionic current to the total ionic current (also referred to herein as I rel ; see below) greater than 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, and most preferably greater than 0.35 or less than -0.35.

[0243] In some embodiments, the nanopore system has an ion selectivity with P(+) / P(−) greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, and most preferably greater than 3.0 or less than 0.33. The cis-to-trans EOF is opposite to the trans-to-cis EPF acting on the analyte. In one aspect, under a transmembrane voltage application, the nanopore system has an ion selectivity P(+) / P(−) greater than 3.0 or less than 0.3.

[0244] In some embodiments, under a transmembrane voltage application, the magnitude of the ion selectivity P(+) / P(−) can 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.

[0245] In some embodiments, under a transmembrane voltage application, the magnitude of the ion selectivity P(+) / P(−) can be at most about 0.5, at most about 0.45, at most about 0.42, at most about 0.40 mV, at most about 0.38, at most about 0.36, at most about 0.35, at most about 0.34, at most about 0.33, at most about 0.31, at most about 0.30, at most about 0.29, at most about 0.28, at most about 0.27, at most about 0.26, at most about 0.25, at most about 0.24, at most about 0.23, at most about 0.22, at most about 0.21, at most about 0.20, or less than 0.20.

[0246] In some embodiments, the magnitude of the ion selectivity P(+) / P(-) can be from about 2.0 to about 5.0. In some embodiments, the ion selectivity P(+) / P(-) can be from about 2.0 to about 2.2, from about 2.0 to about 2.4, from about 2.0 to about 2.6, from about 2.0 to about 2.8, from about 2.0 to about 3.0, from about 2.0 to about 3.3, from about 2.0 to about 3.6, from about 2.0 to about 3.8, from about 2.0 to about 4.0, from about 2.0 to about 4.3, from about 2.0 to about 4.5, from about 2.0 to about 4.7, from about 2.0 to about 5.0, from about 2.5 to about 2.6, from about 2.5 to about 2.8, from about 2.5 to about 3.0, from about 2.5 to about 3.3, from about 2.5 to about 3.6, from about 2.5 to about 3.8, from about 2.5 to about 4.0, from about 2.5 to about 4.3, from about 2.5 to about 4.5, from about 2.5 to about 4.7, from about 2.5 to about 5.0, from about 3.0 to about 3.2, from about 3.0 to about 3.3, from about 3.0 to about 3.4, from about 3.0 to about 3.5, from about 3.0 to about 3.6, from about 3.0 to about 3.7, from about 3.0 to about 3.8, from about 3.0 to about 4.0, from about 3.0 to about 4.3, from about 3.0 to about 4.5, from about 3.0 to about 4.7, from about 3.0 to about 5.0, from about 3.3 to about 3.4, from about 3.3 to about 3.5, from about 3.3 to about 3.6, from about 3.3 to about 3.7, from about 3.3 to about 3.8, from about 3.3 to about 4.0, from about 3.3 to about 4.3, from about 3.3 to about 4.5, from about 3.3 to about 4.7, from about 3.3 to about 5.0, from about 3.5 to about 3.7, from about 3.5 to about 3.8, from about 3.5 to about 4.0, from about 3.5 to about 4.3, from about 3.5 to about 4.5, from about 3.5 to about 4.7, from about 3.5 to about 5.0.

[0247] In some embodiments, the magnitude of the ion selectivity P(+) / P(-) can be from about 0.20 to about 0.5. In some embodiments, the ion selectivity P(+) / P(-) can be about 0.2 to about 0.22, about 0.2 to about 0.24, about 0.2 to about 0.26, about 0.20 to about 0.28, about 0.20 to about 0.3, about 0.2 to about 0.33, about 0.2 to about 0.36, about 0.20 to about 0.38, about 0.2 to about 0.4, about 0.2 to about 0.43, about 0.20 to about 0.45, about 0.20 to about 0.47, about 0.2 to about 0.48, about 0.25 to about 0.27, about 0.25 to about 0.28, about 0.25 to about 0.30, about 0.25 to about 0.33, about 0.25 to about 0.36, about 0.25 to about 0.38, about 0.25 to about 0.40, about 0.25 to about 0.43, about 0.25 to about 0.45, about 0.25 to about 0.47, about 0.25 to about 5.0, about 0.30 to about 0.32, about 0.30 to about 0.33, about 0.30 to about 0.34, about 0.30 to about 0.35, about 0.30 to about 0.36, about 0.30 to about 0.37, about 0.30 to about 0.38, about 0.30 to about 0.40, about 0.30 to about 0.43, about 0.30 to about 0.45, about 0.30 to about 0.47, about 0.30 to about 0.5.

[0248] In some embodiments, the magnitude of the ion selectivity P(+) / P(-) can be 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.

[0249] In some embodiments, the magnitude of the transmembrane applied voltage can 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 magnitude of the transmembrane applied voltage can be at most about 1000 mV, at most about 900 mV, at most about 800 mV, at most about 700 mV, at most about 600 mV, at most about 500 mV, at most about 450 mV, at most about 400 mV, at most about 350 mV, at most about 300 mV, at most about 250 mV, at most about 200 mV, at most about 150 mV, at most about 100 mV, at most about 90 mV, at most about 80 mV, at most about 70 mV, at most about 60 mV, at most about 50 mV, at most about 40 mV, at most about 30 mV, at most about 20 mV, at most about 10 mV, at most about 5 mV, at most about 1 mV, or less than about 1 mV.

[0250] In some embodiments, the magnitude of the transmembrane applied voltage can be from about 1 mV to about 100 mV. In some embodiments, the magnitude of the transmembrane applied voltage can be from about 1 mV to about 5 mV, from about 1 mV to about 10 mV, from about 1 mV to about 20 mV, from about 1 mV to about 30 mV, from about 1 mV to about 40 mV, from about 1 mV to about 50 mV, from about 1 mV to about 60 mV, from about 1 mV to about 70 mV, from about 1 mV to about 80 mV, from about 1 mV to about 90 mV, from about 1 mV to about 100 mV, from about 5 mV to about 10 mV, from about 5 mV to about 20 mV, from about 5 mV to about 30 mV, from about 5 mV to about 40 mV, from about 5 mV to about 50 mV, from about 5 mV to about 60 mV, from about 5 mV to about 70 mV, from about 5 mV to about 80 mV, from about 5 mV to about 90 mV, from about 5 mV to about 100 mV, from about 10 mV to about 20 mV, from about 10 mV to about 30 mV, from about 10 mV to about 40 mV, from about 10 mV to about 50 mV, from about 10 mV to about 60 mV, from about 10 mV to about 70 mV, from about 10 mV to about 80 mV, from about 10 mV to about 90 mV, from about 10 mV to about 100 mV, from about 20 mV to about 30 mV, from about 20 mV to about 40 mV, from about 20 mV to about 50 mV, from about 20 mV to about 60 mV, from about 20 mV to about 70 mV, from about 20 mV to about 80 mV, from about 20 mV to about 90 mV, from about 20 mV to about 100 mV, from about 30 mV to about 40 mV, from about 30 mV to about 50 mV, from about 30 mV to about 60 mV, from about 30 mV to about 70 mV, from about 30 mV to about 80 mV, from about 30 mV to about 90 mV, from about 30 mV to about 100 mV, from about 40 mV to about 50 mV, from about 40 mV to about 60 mV, from about 40 mV to about 70 mV, from about 40 mV to about 80 mV, from about 40 mV to about 90 mV, from about 40 mV to about 100 mV, from about 50 mV to about 60 mV, from about 50 mV to about 70 mV, from about 50 mV to about 80 mV, from about 50 mV to about 90 mV, from about 50 mV to about 100 mV, from about 60 mV to about 70 mV, from about 60 mV to about 80 mV, from about 60 mV to about 90 mV, from about 60 mV to about 100 mV, from about 70 mV to about 80 mV, from about 70 mV to about 90 mV, from about 70 mV to about 100 mV, from about 80 mV to about 90 mV, from about 80 mV to about 100 mV, or from about 90 mV to about 100 mV.

[0251] In some embodiments, the magnitude of the transmembrane applied voltage can be from about 100 mV to about 1,000 mV.In some embodiments, the magnitude of the transmembrane applied voltage can be from about 100 mV to about 150 mV, about 100 mV to about 200 mV, about 100 mV to about 250 mV, about 100 mV to about 300 mV, about 100 mV to about 400 mV, about 100 mV to about 500 mV, about 100 mV to about 600 mV, about 100 mV to about 700 mV, about 100 mV to about 800 mV, about 100 mV to about 900 mV, about 100 mV to about 1,000 mV, about 150 mV to about 200 mV, about 150 mV to about 250 mV, about 150 mV to about 300 mV, about 150 mV to about 400 mV, about 150 mV to about 500 mV, about 150 mV to about 600 mV, about 150 mV to about 700 mV, about 150 mV to about 800 mV, about 150 mV to about 900 mV, about 150 mV to about 1,000 mV, about 200 mV to about 250 mV, about 200 mV to about 300 mV, about 200 mV to about 400 mV, about 200 mV to about 500 mV, about 200 mV to about 600 mV, about 200 mV to about 700 mV, about 200 mV to about 800 mV, about 200 mV to about 900 mV, about 200 mV to about 1,000 mV, about 250 mV to about 300 mV, about 250 mV to about 400 mV, about 250 mV to about 500 mV, about 250 mV to about 600 mV, about 250 mV to about 700 mV, about 250 mV to about 800 mV, about 250 mV to about 900 mV, about 250 mV to about 1,000 mV, about 300 mV to about 400 mV, about 300 mV to about 500 mV, about 300 mV to about 600 mV, about 300 mV to about 700 mV, about 300 mV to about 800 mV, about 300 mV to about 900 mV, about 300 mV to about 1,000 mV, about 400 mV to about 500 mV, about 400 mV to about 600 mV, about 400 mV to about 700 mV, about 400 mV to about 800 mV, about 400 mV to about 900 mV, about 400 mV to about 1,000 mV, about 500 mV to about 600 mV, about 500 mV to about 700 mV, about 500 mV to about 800 mV, about 500 mV to about 900 mV, about 500 mV to about 1,000 mV, about 600 mV to about 700 mV, about 600 mV to about 800 mV, about 600 mV to about 900 mV, about 600 mV to about 1,000 mV, about 700 mV to about 800 mV, about 700 mV to about 900 mV, about 700 mV to about 1,000 mV, about 800 mV to about 900 mV, about 800 mV to about 1,000 mV or about 900 mV to about 1,000 mV.

[0252] In some embodiments, the magnitude of the transmembrane applied voltage can be about 1 mV, about 5 mV, about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, about 100 mV, about 150 mV, about 200 mV, about 250 mV, about 300 mV, about 350 mV, about 400 mV, about 450 mV, about 500 mV, about 600 mV, about 700 mV, about 800 mV, about 900 mV, or about 1000 mV. In some embodiments, the voltage is negative from cis to trans. In some embodiments, the voltage is positive from cis to trans.

[0253] In some embodiments, a signal is measured. The signal can include an electrical signal. The signal can be related to or caused by the translocation of an analyte. The signal can include an ion current or a change in ion current. The signal can include a voltage or a change in voltage across the membrane and / or nanopore. The signal can include a measurement of the change in current between different states of the nanopore. The states of the nanopore can include an open channel, capture of an analyte by the nanopore, or passage of a polymer through the nanopore from a captured state. In some embodiments, measuring the signal can include comparing the signals in different states of the nanopore.

[0254] In some embodiments, the electrode pair is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the trans side. In some embodiments, the applied voltage is a positive voltage on the trans 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 absolute relative net electroosmotic current under the applied voltage is greater than about 0.10 pA / mV. In some embodiments, the nanopore includes an inner pore constriction structure of about 0.5 nanometers to about 2 nanometers (nm).

[0255] Also provided is a nanopore system for translocating a non-nucleic acid-based polymeric analyte (e.g., an analyte) through a nanopore. The system includes a nanopore contained in a membrane that separates the fluid chambers of the nanopore system into a cis side and a trans side, wherein the analyte is to be added to the cis side, and wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) caused by cis-to-trans net ion current flow such that the target polymer is captured in the nanopore. The cis-to-trans EOF is dominant, which results from, for example, the ratio of the cis-to-trans net ion current to the total ion current being greater than 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, and most preferably greater than 0.35 or less than -0.35.

[0256] In a specific aspect, the nanopore system of the present invention has an ionic selectivity P(+) / 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, such as >3.5 or <0.28.

[0257] The method or nanopore system of the present invention relies on a dominant cis-to-trans EOF generated by a net ionic current flow from cis to trans, which has not been taught or suggested in the art.

[0258] In some cases, EPF may be the main process driving capture and / or translocation in the nanopore system. Therefore, all previous validations either used selected model polymers (with a net charge contributing to EPF) or modified the polymers with highly charged tags (such as adding polyanion tags) so that the EPF force acting on the polymer drives translocation in the cis-to-trans direction. When EOF was previously used in nanopore systems, it was most commonly either acting in the trans-to-cis direction to resist cis-to-trans EPF (slowing down EPF-driven translocation or trapping molecules in the nanopore) or combining with cis-to-trans EPF in the cis-to-trans direction to assist translocation. Although some previous studies have shown that neutral or weakly charged small molecules or small polymers can be trapped in nanopores by weak electroosmotic forces (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 evidence that cis-to-trans EOF can be used to capture and / or translocate long and / or complex polymers (contour length greater than the length of the pore) that can overcome the trans-to-cis EPF acting in the opposite direction.

[0259] Also see US2022 / 0283140A1, which discloses a method and system for single molecule proteomics using a nanopore sensor, using a reagent such as guanidine chloride to bind inside the nanopore and / or providing an electroosmotic force within the nanopore to measure the electronic characteristics of proteins or peptides transported through the nanopore. In this system, EOF is used to assist translocation, but it is set so that EPF is in the same direction. The present disclosure provides methods and systems in which cis-to-trans EOF can overcome reverse EPF.

[0260] Although some previous studies have shown that neutral or weakly charged small molecules or small polymers can be trapped in nanopores by weak electroosmotic forces, there is no conclusive evidence that using cis-to-trans EOF can overcome the trans-to-cis EPF acting in the opposite direction to capture and / or translocate long and / or complex polymers (whose contour length is greater than the length of the pore).

[0261] In some embodiments, the novel system relies on setting specific strong electroosmotic means in the translocation direction. This was not predictable before because the system EOF acting on the polymeric analyte may repel the analyte, thus preventing its capture and / or translocation. In addition, long polymeric analytes may clog the nanopore. In fact, this is exactly the most common way of EOF in the prior art nanopore systems, i.e., forming a trap to hold the analyte within the pore. In some cases, the nanopore captures the free end of the polymer because this end has no label to generate a strong EPF.

[0262] The polymeric analyte can be synthetic, semi-synthetic or of biological origin. For example, it is a biopolymer other than DNA. It can contain peptide units, sugar units or water-soluble plastic monomers and any combination thereof, or be composed of peptide units, sugar units or water-soluble plastic monomers and any combination thereof. Preferably, the polymeric analyte is a polypeptide, a polysaccharide or a water-soluble plastic, such as PEG or a PEGylated polypeptide.

[0263] In one embodiment, the polymeric analyte is an unmodified (unlabeled) analyte. Appropriately, in the method of the present invention, the ends of the polymer are unstructured, preferably where the polymer is denatured or partially denatured. In one aspect, the length of the elongated polymer is greater than the longitudinal axis of the central channel of the nanopore in the direction perpendicular to the membrane, preferably where the length of the polymer is greater than 50 monomer units, such as greater than 50 peptide units.

[0264] In a preferred embodiment, the present invention provides a method for translocating at least 30 peptide units and / or a non-nucleic acid-based polymeric analyte (e.g., analyte, polypeptide) comprising positively and / or negatively charged residues. The polypeptide can be in a denatured / unfolded state, preferably where the polypeptide is added in a pre-denatured state.

[0265] The method may further comprise (c) measuring the change in ionic current caused by the translocation of the target polymer through the nanopore, preferably where operation (c) comprises measuring the change in current in the following states: (i) open channel, (ii) nanopore capturing the polymer and / or (iii) the polymer from (ii) passing through the nanopore, more preferably where the measurement comprises detecting the difference between states (i), (ii) and / or (iii). In one embodiment, the measurement comprises measuring the difference caused by the composition and / or structure of the polymer passing through the nanopore during state (iii).

[0266] Cis-to-trans electroosmotic flow (EOF) can be achieved in a variety of ways. For example, it can be achieved by adjusting the pH, type and / or concentration of salt, and / or osmotic pressure on the membrane of the nanopore system, by changing the charge of the nanopore (e.g., through genetic engineering), or by any combination of the above. Preferably, the dominant EOF is achieved by modifying the asymmetric salt distribution between the cis and trans sides of the nanopore and / or the chamber.

[0267] In a specific aspect, the ion selectivity P(+) / P(-) of the system is greater than 2.0, preferably greater than 2.5, most preferably greater than 3.0. Preferably, the system comprises a cation-selective (mutant) nanopore.

[0268] In one embodiment, the nanopore is a solid-state nanopore or a biological nanopore, preferably having an inner pore constriction structure with a diameter in the range of 0.5 - 2 nm.

[0269] In some embodiments, the nanopore can be a biological nanopore, more preferably an α-helix or β-barrel oligomeric pore-forming toxin or porin. The nanopore is suitably selected from aerolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, fragaceatoxin C (FraC), lumbrokinase, phage-derived portal proteins (Phi29, G20c, etc.) or mutants thereof. In certain aspects, the nanopore is selected from the mutant CytK nanopores listed in Table 1. Those skilled in the art will understand that the nanopore can also be a nanopore constructed from elements of existing nanopores (see, for example, WO2021 / 101378) or a nanopore developed de novo using predictive protein engineering software (see, for example, Shimizu et al., Nature Nanotechnology, Vol. 17, pp. 67 - 75, 2022).

[0270] The method according to the invention may further comprise operation (c), measuring the change in ionic current caused by the translocation of a polymeric analyte through a nanopore. Operation (c) preferably comprises measuring the change in current in the following states: (i) open channel, (ii) nanopore capturing the polymer and / or (iii) the polymer from (ii) passing through the nanopore. For example, it comprises detecting the difference between states (i), (ii) and / or (iii). In a specific aspect, the measurement comprises measuring the difference caused by the composition and / or structure of a non-nucleic acid-based polymeric analyte (e.g., analyte, protein) passing through the nanopore during state (iii). The method suitably comprises performing one or more characteristic measurements on the target polymer. The one or more measurements may characterize one, two, three, four or five or more characteristics of the polymeric analyte. The one or more characteristics are preferably selected from: (i) the length of the polymer; (ii) the identity of the polymer; (iii) the polymer sequence; (iv) the secondary or tertiary structure of the polymer; and / or (v) whether the polymer is (post-translationally) modified. According to the invention, any combination of (i) to (v) can be measured.

[0271] Another embodiment of the invention relates to a nanopore system for translocating a polymeric analyte through a nanopore, the system comprising:

[0272] (a) a membrane having a nanopore therein, the membrane separating the chamber into a cis side and a trans side, wherein the polymeric analyte is added to the cis side and translocates through the nanopore to the trans side; (b) on the cis side of the chamber, a protein transporter that captures the polymeric analyte, the protein transporter being able to bind the polymeric analyte and translocate it sequentially through the nanopore; and (c) means for providing a voltage difference between the cis side and the trans side of the membrane.

[0273] In some embodiments, the nanopore system further has a cis-to-trans electroosmotic force (EOF) generated by the net ion current flow from cis to trans, thereby capturing the polymeric analyte in the nanopore. Preferably, the nanopore system has a cis-to-trans EOF generated by a ratio of the net ion current (Irel) from cis to trans to the total ion current 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.

[0274] In a specific aspect, 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, and even greater than 3.5 or less than 0.2.

[0275] The voltage difference can be provided in a variety of ways. For example, a circuit can apply a voltage and / or can measure a current; or the system includes a first circuit for applying a voltage and / or a second circuit for measuring a current. An asymmetric salt across a membrane can also be used to generate a voltage difference. For example, the device includes a circuit for providing a voltage between a cis side and a trans side and for measuring the ionic current flowing through the nanopore. See Figure 1. Preferably, a negative voltage is applied to the trans side.

[0276] The system can also include means for measuring a signal based on the ionic current flowing through the nanopore during translocation. The measuring means is arranged to detect a change in the signal that reflects the characteristics of the analyte (e.g., a protein) during translocation.

[0277] The system can use other methods to measure the voltage-current characteristics of the nanopore system, such as using an ion flux fluorescence probe or a field effect transistor system to measure voltage changes. However, there are also other suitable detection methods, such as tunneling, surface-enhanced Raman, plasmon, and / or other spectroscopic methods, which do not measure ionic current but directly measure the properties of the target analyte in the nanopore.

[0278] The present invention also provides an analysis device that includes one or more nanopore systems disclosed herein, for example, in the form of an array.

[0279] Another embodiment relates to the use of the method, nanopore system, or device of the present invention for characterizing at least one feature of a polymeric analyte, preferably for detecting and / or analyzing one or more polymeric analytes at the single-molecule level. The method is independent of the charge of the polymeric analyte to be analyzed and thus can, in principle, analyze any type of polymer. The method and its corresponding system provide a highly desirable single-molecule polymer sequencing method. Embodiments of the present invention can be applied to protein or glycan sequencing, single-molecule protein or glycan sequencing, proteomics, single-cell post-translational modification detection, glycopeptide analysis, detection of protein or glycan biomarkers and / or their post-translational modifications, and / or detection of disease biomarkers. A preferred application of the method is single-molecule protein sequencing and / or discovery and / or quantitative analysis of protein post-translational modifications.

[0280] Definitions

[0281] Electroosmotic force

[0282] According to the present invention, the nanopore system has cis-to-trans electroosmotic flow, or vice versa, which exerts a drag force (independent of its charge) on particles dispersed in solution, which is commonly referred to as electroosmotic force (EOF). EOF results from the net flow of ions (e.g., cis-to-trans), which exerts a strong force on the solvent itself (water) sufficient to move the fluid (Chinappi et al., 2020, ACS Nano, 14, 11, pp. 15816-15828), and this force exerts a significant force on any molecule within the flux. Electroosmosis can compete or act synergistically with electrophoresis (EPF).

[0283] According to the present invention, the nanopore system has cis-to-trans electroosmotic flow, where EOF is superior to EPF. Surprisingly, a sufficiently dominant cis-to-trans EOF is capable of capturing and / or translocating complex and / or charged polymers, thereby counteracting the EPF acting from trans-to-cis. This selected high and / or dominant electric field is believed to be capable of capturing and / or retaining the target analyte in the nanopore. The EOF acts directly on the polymer to move it through the nanopore.

[0284] In some embodiments, the cis side refers to the compartment in the sensor system to which the analyte is added and / or the compartment to which the nanopore is added in the case of a bio-derived nanopore (and assuming vectorial insertion, as most nanopores have a preferred insertion direction based on their insertion location). However, it should be noted that the terms "trans" and "cis" used herein are general conventions determined based on the electron / voltage polarity at the cross-electrode. For example, due to the existence of many options, we do not wish to be limited to any one type of circuit, and the cis chamber is grounded and the applied transmembrane potential is the potential of the trans side, i.e., the potential of the trans side minus the potential of the cis side. A positive current is defined as positive charges (e.g., K + ions) moving through the nanopore from the trans side to the cis side, or negative charges (e.g., Cl - ions) moving from the cis side to the trans side (see, for example, Maglia et al., Methods Enzymol, 2010; 475: 591-623).

[0285] In some embodiments, the present invention teaches that the direction of EOF depends on the polarity of the applied electric potential and / or the relative conditions of the cis-side compartment and the trans-side compartment, along with the ion selectivity of the nanopore. Additionally, the present invention teaches that the direction of EOF (whether from the cis-side to the trans-side or from the trans-side to the cis-side) determines the direction of the net force that translocates the polymeric analyte across the nanopore, and thus determines to which side the polymeric analyte is added in the context of the methods described herei...

Claims

1. A method, comprising: (a) Providing: (i) A nanopore system, wherein the nanopore system comprises (1) a fluid chamber, and (2) a membrane, the membrane comprising nanopores, the membrane separating the fluid chamber into a cis side and a trans side; and (ii) A non-nucleic acid-based polymeric analyte, wherein the linear length of the non-nucleic acid-based polymeric analyte is greater than the channel length of the nanopore; (b) Translocating the non-nucleic acid-based polymeric analyte from the cis side of the fluid chamber to the trans side, wherein the non-nucleic acid-based polymeric analyte comprises an elongated structure, wherein the nanopore system has a cis-to-trans electroosmotic force generated by a net cis-to-trans ionic current, wherein the cis-to-trans electroosmotic force resists the electrophoretic force acting in the opposite direction to the cis-to-trans electroosmotic force to translocate the non-nucleic acid-based polymeric analyte through the nanopore.

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 1-3, wherein the electroosmotic force is at least 100% greater than the electrophoretic force.

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

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

7. The method according to claim 6, wherein the solute comprises an ion or an osmolyte.

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 the cis-to-trans electroosmotic force upon application of an electric potential.

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

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

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

12. The method according to any one of claims 1-11, wherein when the non-nucleic acid-based polymeric analyte is stretched, the linear length of the non-nucleic acid-based polymeric analyte is greater than the channel length of the nanopore through the membrane.

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

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

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

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

17. The method according to claim 16, wherein the at least 30 peptide units comprise 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 - 18, wherein the polypeptide is provided in a folded state.

20. The method according to any one of claims 1 - 19, further comprising measuring a signal generated by translocation of the non-nucleic acid-based polymeric analyte through the nanopore.

21. The method according to claim 20, wherein said measurement comprises: Measuring the signal in the following states: (a) the open channel of the nanopore; (b) the capture of the non-nucleic acid-based polymeric analyte by the nanopore; or (c) the non-nucleic acid-based polymeric analyte passing through the nanopore.

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

23. The method according to claim 20, wherein the signal comprises an ionic current, a change in ionic current, or a deviation thereof.

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

25. The method according to any one of claims 1 - 24, wherein the linear length of the non-nucleic acid-based polymeric analyte is at most 4,000 kDa.

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

27. The method according to any one of claims 1 - 25, wherein the linear length of the non-nucleic acid-based polymeric analyte is at most twice the channel length of the nanopore.

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

29. The method according to any one of claims 1 - 28, wherein the cis-to-trans electroosmotic force comprises a cis-to-trans net ionic current.

30. The method according to any one of claims 1 - 29, wherein the cis-to-trans electroosmotic force is adjusted by the pH, the type of salt, the concentration of salt, the osmotic pressure, the modification of the nanopore, or any combination thereof on the membrane of the system.

31. The method according to any one of claims 1 - 30, wherein the cis-to-trans electroosmotic force is adjusted by changing the charge of the nanopore.

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

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

0.

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

50.

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

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

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

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

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

40. The method according to any one of claims 36 - 39, wherein the amplitude of the applied voltage is greater than 20 mV.

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

42. The method according to any one of claims 1 - 41, wherein the nanopore comprises an inner pore constriction structure of about 0.5 nanometers to about 2 nanometers (nm).

43. The method according to any one of claims 1 - 42, wherein the nanopore comprises an α - helical oligomeric pore structure.

44. The method according to any one of claims 1 - 43, wherein the nanopore comprises a β - barrel oligomeric pore structure.

45. The method according to any one of claims 1 - 44, wherein the nanopore comprises a recombinant nanopore.

46. The method according to any one of claims 1 - 45, wherein the nanopore comprises a protein such as aerolysin (Aer), cytolysin K (CytK), MspA, α - hemolysin (aHL), CsgG, fragaceatoxin C (FraC), lumbrokinase, OmpF, OmpG, FhuA, a phage - derived portal protein, a modified variant thereof, or an ion - selective mutant thereof.

47. The method according to any one of claims 1 - 46, wherein the nanopore comprises a biological nanopore.

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

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

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 - 51, wherein the nanopore comprises a mutant CytK nanopore.

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 method according to claim 52, wherein the mutant CytK nanopore comprises one of the following combinations of amino acid substitutions: (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) K128D, K155D, Q145D, and S151D.

57. The method according to claim 52, wherein the mutant CytK nanopore comprises one or more of the following combinations of amino acid substitutions: (i) S120D, G122D, or K155D; (ii) S120D in combination with K128F / K128D; (iii) Q122D or S151D; (iv) K128D or K128F; (v) S120D, K115D, and Q122D; (vi) K128F, S120D, and G122D; and (vii) K128F, S120D, G122D, and K155D.

58. A system, comprising: (a) a fluid chamber; (b) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into (i) a cis side containing a first solution and (ii) a trans side containing a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte through the nanopore by electroosmotic flow, wherein the non-nucleic acid-based polymer analyte comprises an elongated structure, and wherein the linear length of the non-nucleic acid-based polymer analyte is greater than the channel length of the nanopore; (c) a pair of electrodes, comprising a first electrode and a second electrode, wherein the first electrode is disposed on the cis side of the fluid chamber and the second electrode is disposed on the trans side of the fluid chamber, wherein the pair of electrodes is configured to generate an electrophoretic force opposite to the direction of electroosmotic flow.

59. A system, comprising: a fluid chamber; a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte by electroosmotic flow; a pair of electrodes, comprising a first electrode and a second electrode; and A controller operably coupled to the fluid chamber, the nanopore, and the pair of electrodes, wherein the controller: (a) generates an electrophoretic force acting in a direction opposite to electroosmotic flow using the pair of electrodes to translocate the non-nucleic acid-based polymer analyte through the nanopore, and (b) detects one or more signals related to at least one characteristic of the non-nucleic acid-based polymer analyte during or after the translocation of the non-nucleic acid-based polymer analyte through the nanopore, wherein the linear length of the non-nucleic acid-based polymer analyte is greater than the channel length of the nanopore.

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 characteristic of the non-nucleic acid-based 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 - 64, wherein the first solution contains a first concentration of solute and the second solution contains a second concentration of solute.

66. The system according to claim 65, wherein the solute comprises an ion or an osmolyte.

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 upon application of an electric potential.

68. The system according to any one of claims 58 - 67, wherein the electroosmotic flow comprises a cis-to-trans net ion current.

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

70. The system according to any one of claims 58 - 69, wherein the electroosmotic flow is regulated by a change in the charge of the nanopore.

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

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

0.

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

50.

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

75. The system according to claim 74, wherein the applied voltage is a negative voltage on the trans side.

76. The system according to claim 74, wherein the applied voltage is a positive voltage on the trans side.

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

78. The system according to any one of claims 74-77, wherein the amplitude of the applied voltage is greater than 20 mV.

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

80. The system according to any one of claims 58-79, wherein the nanopore comprises an inner pore constriction structure of about 0.5 nanometers to about 2 nanometers (nm).

81. The system according to any one of claims 58-80, wherein the nanopore comprises an α-helical oligomeric pore structure.

82. The system according to any one of claims 58-81, wherein the nanopore comprises a β-barrel oligomeric pore structure.

83. The system according to any one of claims 58-82, wherein the nanopore comprises a recombinant nanopore.

84. The system according to any one of claims 58-83, wherein the nanopore comprises a protein selected from the group consisting of aerolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, fragaceatoxin C (FraC), lumbrokinase, OmpF, OmpG, FhuA, a phage-derived portal protein, a modified variant thereof, or an ion-selective mutant thereof.

85. The system according to any one of claims 58-84, wherein the nanopore comprises a biological nanopore.

86. The system according to claim 85, wherein the biological nanopore is modified to restrict the passage of one or more ions through the channel of the nanopore.

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

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 58-89, wherein the nanopore comprises a mutant CytK nanopore.

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 system according to claim 90, wherein the mutant CytK nanopore comprises one of the following combinations of amino acid substitutions: (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) K128D, K155D, Q145D and S151D.

95. The system according to claim 90, wherein the mutant CytK nanopore comprises one or more of the following combinations of amino acid substitutions: (i) S120D, G122D or K155D; (ii) S120D in combination with K128F / K128D, (iii) Q122D or S151D; (iv) K128D or K128F, (v) S120D, K115D and Q122D; (vi) K128F, S120D and G122D; and (vii) K128F, S120D, G122D and K155D.

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

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

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

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

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

101. The system according to any one of claims 58 - 100, wherein the non-nucleic acid-based 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-based polymer analyte comprises a polypeptide, a polysaccharide, or a water-soluble plastic.

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

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

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

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

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

108. The system according to any one of claims 58 - 107, wherein the linear length of the non - nucleic acid - based polymeric analyte is at most 4,000 kDa.

109. The system according to any one of claims 58 - 108, wherein the linear length of the non - nucleic acid - based polymeric analyte is at least 2 times the channel length of the nanopore.

110. The system according to any one of claims 58 - 108, wherein the linear length of the non - nucleic acid - based polymeric analyte is at most 2 times the channel length of the nanopore.

111. An apparatus comprising an array having the system according to any one of claims 58 - 110.

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

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

114. Use of the method according to any one of claims 1 - 57 for detecting and analyzing one or more non - nucleic acid - based polymeric analytes at the single - molecule level.

115. Use of the system according to any one of claims 58 - 110 for detecting and analyzing one or more non - nucleic acid - based polymeric analytes at the single - molecule level.

116. Use of the method according to any one of claims 1 - 57 for detecting and analyzing one or more polypeptides.

117. Use of the system according to any one of claims 58 - 110 for detecting and analyzing one or more polypeptides.

118. A method for translocating a non - nucleic acid - based polymeric analyte through a nanopore, the nanopore being contained in a membrane that separates the fluidic chamber of the nanopore system into a cis - side and a trans - side, the method comprising adding the polymeric analyte to the cis - side of the nanopore system and allowing the polymeric analyte to translocate, wherein the length of the elongated polymeric analyte is greater than the length of the central channel of the nanopore in a direction perpendicular to the membrane, and wherein the nanopore system has a cis - to - trans electroosmotic force (EOF) generated by a net cis - to - trans ionic current, and wherein the cis - to - trans EOF overcomes the trans - to - cis electrophoretic force (EPF) acting on the polymeric analyte.

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

120. The method according to claim 118 or 119, wherein the ends of the polymer are unstructured, preferably wherein the polymer is denatured or partially denatured.

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

122. The method according to any one of claims 118 - 121, wherein the polymeric analyte is synthetic, semi - synthetic or of biological origin, such as a biopolymer, preferably comprising peptide units, sugar units and water - soluble plastic monomers and any combination thereof or consisting of peptide units, sugar units and water - soluble plastic monomers and any combination thereof.

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

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

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

126. The method according to any one of claims 1-125 further comprises: (c) Measuring the change in ionic current caused by the translocation of the target polymer through the nanopore, preferably, wherein (c) comprises measuring the current change in the following states: (i) open channel, (ii) capture of the polymer by the nanopore, and (iii) translocation of the polymer from (ii) through the nanopore, more preferably, wherein the measurement comprises detecting the difference between states (i), (ii) and (iii).

127. A nanopore system for translocating a polymeric analyte through a nanopore, the system comprising a nanopore contained within a membrane that separates the fluid chambers of the nanopore system into a cis side and a trans side, wherein, The analyte will be added to the cis - side, and the nanopore system has a cis - to - trans electroosmotic force (EOF) generated by the net cis - to - trans ionic current, wherein the cis - to - trans EOF overcomes the trans - to - cis electrophoretic force (EPF) acting on the polymeric analyte.

128. The method according to any one of claims 118 - 126 or the nanopore system according to claim 127, wherein the nanopore system has a cis - to - trans EOF generated due to the ratio of the net cis - to - trans ionic current to the total ionic current being 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.

129. The method or nanopore system according to any one of claims 118 - 128, wherein the cis - to - trans EOF is set by adjusting the pH, the type and / or concentration of salt and / or the osmotic pressure on the membrane of the nanopore system, by changing (e.g., genetically engineering) the charge of the nanopore or any combination thereof.

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

131. The method or nanopore system according to any one of claims 118 - 130, wherein the ion selectivity P(+) / P(-) of the nanopore system is greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, and most preferably greater than 3.0 or less than 0.

33.

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

133. The method or nanopore system according to any one of claims 118 - 132, wherein the nanopore is a biological nanopore, preferably having an inner pore constriction structure in the range of 0.5 - 2 nm.

134. The method or nanopore system according to any one of claims 118 - 133, wherein the nanopore is an α - helical or β - barrel oligomeric pore - forming toxin or porin, preferably wherein the nanopore is selected from the group consisting of aerolysin (Aer), cytolysin K (CytK), MspA, α - hemolysin (aHL), CsgG, fragaceatoxin C (FraC), lumbrokinase, a phage - derived portal protein, a modified variant thereof, or an ion - selective mutant thereof.

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

136. The method or system according to any one of claims 118 - 135, wherein the nanopore is a mutant CytK nanopore comprising one or more amino acid substitutions selected from the group consisting of K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, and S151D, wherein the numbering corresponds to the CytK amino acids available under accession number A0A2S1A9G3_9BACI in UniProt, preferably, wherein the CytK mutant nanopore comprises 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; K128D, K155D, Q145D, and S151D.

137. An analysis device comprising an array of nanopore systems according to any one of claims 127 - 136. Use of the method, nanopore system or device according to any one of claims 118 - 137 for characterizing at least one feature of a target polymer, preferably for detecting and analyzing one or more target polymers at the single molecule level, more preferably for detecting and analyzing one or more target polypeptides.

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