Nanopore-based analysis of proteins

Abstract

The present disclosure relates to systems and methods for protein analysis, and more particularly to nanopore systems, devices, and methods for single molecule protein analysis and sequencing. There is provided a method of translocating a target protein through a nanopore included in a membrane separating a fluid chamber of a nanopore system into a cis-side and a trans-side, comprising: (a) allowing a protein transferase in solution to capture and form a complex with the target protein to be translocated; (b) contacting a transposase-target protein complex with the cis-side of the nanopore and allowing the target protein to translocate to the trans-side; wherein the nanopore system has an electroosmotic force (EOF) from cis to trans, which is generated from a large net ionic current with respect to the total ionic current from cis to trans, so that the target protein is captured in the nanopore, and there is a transposase at the top of the nanopore that controls translocation.

Description

[0001] Incorporation by Reference

[0002] This application claims the benefit of European application EP22204590.8 filed on October 28, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Determining the properties of analytes is an important aspect of scientific research. The characteristics of analytes are important for further scientific research or clinical aspects. Summary of the Invention

[0004] In one aspect, the present disclosure provides a method comprising: (a) providing: a nanopore system, wherein the nanopore system comprises a fluid chamber and a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side and a trans side; (b) contacting a complex comprising a non-nucleic acid-based polymer analyte and a translocase with the cis side of the nanopore; and (c) translocating the non-nucleic acid-based polymer analyte to the trans side of the fluid chamber using a cis-side to trans-side electroosmotic force, wherein the cis-side to trans-side electroosmotic force maintains the translocation of the complex at the cis-side entrance of the nanopore channel.

[0005] In certain embodiments, before (c), the method further includes contacting the non-nucleic acid-based polymer analyte with a translocase to generate a complex. In certain embodiments, the complex is produced on the cis side of the fluid chamber. In certain embodiments, the cis side to trans side electroosmotic force includes a net ion current from the cis side to the trans side. In certain embodiments, the electroosmotic force from the cis side to the trans side is regulated by pH value, salt type, salt concentration, transmembrane osmotic pressure, modification of the nanopore, or any combination thereof. In certain embodiments, the modification of the nanopore includes modification of the nanopore charge. In certain embodiments, the electroosmotic force from the cis side to the trans side is regulated by an asymmetric salt distribution between the cis side and the trans side of the fluid chamber. In certain embodiments, the complex is formed in the solution on the cis side of the fluid chamber. In certain embodiments, the complex is formed before the complex contacts the cis side of the nanopore.

[0006] In some embodiments, the translocase comprises an adenosine triphosphate (ATP) driven unfolding enzyme. In some embodiments, the translocase comprises a nucleotide triphosphate (NTP) driven unfolding enzyme. In some embodiments, the translocase comprises an ATPase associated with various cell active (AAA+) enzymes. In some embodiments, the AAA+ enzyme is selected from the group consisting of ATP-dependent Clp protease ATP binding subunit ClpX (ClpX) and ClpX-like protease, ATP-dependent Clp protease ATP binding subunit ClpA (ClpA), proteasome activating nucleotidase (PAN), LON protease, VCP-like ATPase (VAT), AMA, 854, membrane-bound AAA (MBA), archaeal ubiquitin-like modified protein (SAMP), ATP-dependent Clp protease ATP binding subunit ClpC (ClpC), ATP-dependent Clp protease ATP binding subunit ClpC (ClpC), The invention also comprises the following proteins: the synthesis subunit ClpE (ClpE), the ATP-dependent ATPase subunit HslU (HslU), the caseinolytic mitochondrial matrix peptidase partner subunit Y (ClpY), LonA, LonB, the ATP-dependent zinc metalloprotease FtsH, the proteasome-associated ATPase (Mpa), the actinomycete cell division cycle protein 48 (Cdc48, also known as p97 and VCP) and the Cdc48-like protein (Cpa), the mitochondrial outer transmembrane helical translocase (Msp1), the protein translocase subunit SecA (SecA), and functional homologs, orthologs or analogs thereof.

[0007] In some embodiments, the system further comprises a pair of electrodes. In some embodiments, the electrode pair is configured to provide an applied voltage to generate the 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.

[0008] 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 over the applied voltage is greater than about 0.10 pA / mV (picoampere / millivolt).

[0009] In some embodiments, the non-nucleic acid-based polymer analyte includes a main branch structure located at the N-terminus or the C-terminus. In some embodiments, the main branch structure is configured to couple one or more transpositions to the non-nucleic acid-based polymer analyte. In some embodiments, the main branch structure is configured to block one or more transpositions. In some embodiments, the main branch structure includes a recognition motif. In some embodiments, the main branch structure further includes a capture motif, a blocking motif, an interception motif, or a combination thereof.

[0010] In another aspect, the present disclosure provides a system comprising: a fluid chamber; and a membrane comprising a nanopore, the nanopore separating the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, wherein the first solution and the second solution are configured to generate an electroosmotic force, wherein the electroosmotic force is configured to couple a translocase of a complex at a cis-side inlet of a channel of the nanopore, wherein the complex comprises a non-nucleic acid-based polymer analyte and the translocase.

[0011] In some embodiments, the system further comprises a translocase. In some embodiments, the translocase comprises an ATP-driven unfolding enzyme. In some embodiments, the translocase comprises an NTP-driven unfolding enzyme. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, the AAA+ enzyme is selected from ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, homologs, and analogs thereof.

[0012] In some embodiments, the translocase is configured to sequentially translocate a non-nucleic acid-based polymer analyte through a nanopore. In some embodiments, the first solution comprises a solute at a first concentration, and the second solution comprises a solute at a second concentration. In some embodiments, the solute comprises an ion or an osmotic agent. In some embodiments, the difference between the solute at the first concentration and the solute at the second concentration is configured to generate an electroosmotic force. In some embodiments, the electroosmotic force comprises a net ionic current flowing from the cis side to the trans side. In some embodiments, the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the system membrane, modification of the nanopore, or any combination thereof. In some embodiments, the electroosmotic force is regulated by modification of the nanopore charge. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

[0013] In some embodiments, the system further comprises a pair of electrodes. In some embodiments, the first electrode of the pair of electrodes is arranged on the cis side of the membrane, and the second electrode of the pair of electrodes is arranged on the trans side of the membrane. In some embodiments, the electrode pair is configured to detect signals during the translocation of the non-nucleic acid-based polymer analyte. In some embodiments, the signal is associated with the characteristics of the non-nucleic acid-based polymer analyte. 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 amplitude of the applied voltage is less than 300mV. In some embodiments, the amplitude of the applied voltage is greater than about 20mV. In some embodiments, the absolute relative net electroosmotic current on the applied voltage is greater than about 0.10pA / mV. In some embodiments, the signal comprises an ion current or a variation thereof.

[0014] In another aspect, the present disclosure provides a method comprising: providing: a nanopore system, wherein the nanopore system comprises a fluid chamber and a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side and a trans side; a non-nucleic acid-based polymer analyte, wherein the non-nucleic acid-based polymer analyte is coupled to a main branch structure comprising a blocking motif, an interception motif, a coupling motif, or a combination thereof; a translocase; and translocating the non-nucleic acid-based polymer analyte from the cis side to the trans side of the fluid chamber.

[0015] In some embodiments, the main branch structure comprises a nucleic acid. In some embodiments, the main branch structure comprises a peptide. In some embodiments, the main branch structure comprises a nucleic acid and a peptide. In some embodiments, the blocking motif is configured to destroy the interaction between the translocase and the non-nucleic acid-based polymer analyte. In some embodiments, the blocking motif comprises an amino acid sequence. In some embodiments, the amino acid sequence comprises n repeats of (glycine) n, (serine-glycine) n, (glycine-serine) n, (alanine-serine) n, (valine) n, (alanine-serine) n, (valine-serine) n or (serine-valine) n. In some embodiments, n is greater than about 2, 3, 6, 9, 12, 15, 18 or 21. In some embodiments, the blocking motif comprises a non-amino acid chemical region. In some embodiments, the non-amino acid chemical region comprises polyethylene glycol.

[0016] In some embodiments, the interception motif is configured to prevent a translocase from translocating a non-nucleic acid-based polymer analyte through the interception motif. In some embodiments, the interception motif is configured to prevent a translocase from translocating the non-nucleic acid-based polymer analyte through the main branch structure. In some embodiments, the interception motif is configured to prevent a translocase from translocating the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, the interception motif comprises a steric hindrance. In some embodiments, the steric hindrance comprises one or more bulky amino acids. In some embodiments, one or more bulky amino acids comprise histidine, phenylalanine, tyrosine, or tryptophan. In some embodiments, the steric hindrance comprises at least about one bulky amino acid. In some embodiments, the steric hindrance comprises at least about five bulky amino acids. In some embodiments, the steric hindrance comprises at least about a portion of an anti-unfolding protein. In some embodiments, the anti-unfolding protein comprises maltose binding protein, titin, dihydrofolate reductase, a nuclease, or a combination thereof. In some embodiments, the anti-unfolding protein comprises a disulfide bond. In some embodiments, the steric hindrance comprises a large tethering molecule. In some embodiments, the large tethering molecule comprises a carbohydrate, a polycyclic molecule, a branched dextran, biotin, streptavidin, a nanobody, an antibody, or a small antigenic element.

[0017] In some embodiments, the coupling motif is configured to couple the main branch structure to a non-nucleic acid-based polymer analyte. In some embodiments, the non-nucleic acid-based polymer analyte comprises a peptide. In some embodiments, the coupling motif is connected to the C-terminus of the peptide. In some embodiments, the coupling motif is connected to the N-terminus of the peptide. In some embodiments, the coupling motif comprises a recognition sequence that can be recognized by an enzyme with peptide ligase activity. In some cases, the enzyme can interact with the recognition sequence in the coupling motif. In some embodiments, the coupling motif comprises a chemical group. In some embodiments, the chemical group comprises maleimide, iodoacetamide, 2-thiopyridine, 3-arylpropionitrile, NHS-ester, isocyanate, isothiocyanate, benzoyl fluoride, diazonium salt or PTAD. In some embodiments, the coupling motif comprises an enzyme coupling region. In some embodiments, the enzyme coupling region connects the coupling motif to an enzyme. In some cases, the enzyme can interact with the coupling motif.

[0018] In some embodiments, the enzyme comprises a peptidase, a holoacylase, or a sortase. In some embodiments, the coupling motif of the main branch structure is coupled to the non-nucleic acid-based polymer analyte via a covalent bond. In some embodiments, the coupling motif of the main branch structure is coupled to the non-nucleic acid-based polymer analyte via a connector.

[0019] In some embodiments, the main branch structure further comprises at least one of the following: a recognition motif; or a capture motif.

[0020] In some embodiments, the capture motif comprises a polycationic tag. In some embodiments, the polycationic tag comprises n repeats of (serine-glycine-arginine)n, (serine-arginine)n, (lysine)n, or (arginine)n. In some cases, the polycationic tag may comprise serine, glycine, arginine, lysine, or any combination thereof. In some embodiments, the capture motif comprises a polyanionic tag. In some embodiments, the polyanionic tag comprises n repeats of (serine-glycine-aspartic acid)n, (serine-aspartic acid)n, (aspartic acid)n, (serine-glycine-glutamate)n, (serine-glutamate)n, or (glutamate)n. In some cases, the polyanionic tag may comprise serine, glycine, aspartic acid, glutamate, or any combination thereof. In some embodiments, the recognition motif comprises a portion of ssrA, a prokaryotic ubiquitin-like protein, SulA, a peroxisomal membrane protein (Pex15), or a combination thereof. In some embodiments, the sequence of the recognition motif comprises one or more of SEQ ID NOs: 201-206.

[0021] In some embodiments, the main branch structure is connected to the C-terminus or N-terminus of the non-nucleic acid-based polymer analyte. In some embodiments, the non-nucleic acid-based polymer analyte includes a polypeptide. In some embodiments, the main branch structure is coupled to the N-terminus of the polypeptide. In some embodiments, the main branch structure is coupled to the C-terminus of the polypeptide. In some embodiments, the non-nucleic acid-based polymer analyte includes another main branch structure. In some embodiments, the main branch structure and the other main branch structure are configured to translocate the non-nucleic acid-based polymer analyte through the nanopore in the C-terminus to N-terminus direction, the N-terminus to C-terminus direction, or the C-terminus to N-terminus direction and the N-terminus to C-terminus direction. In some embodiments, the non-nucleic acid-based polymer analyte is translocated using electroosmotic force.

[0022] In some embodiments, the method further comprises providing an electrophoretic force exerted in a direction opposite to the electroosmotic force. In some embodiments, the electroosmotic force propels the non-nucleic acid-based polymer analyte through the nanopore against the electrophoretic force. In some embodiments, the electroosmotic force comprises a net ionic current from the cis side to the trans side. In some embodiments, the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the nanopore system membrane, modification of the nanopore, or any combination thereof. In some embodiments, the electroosmotic force is regulated by modification of the nanopore charge. In some cases, the charge of the nanopore can be modified at the cis entrance of the channel. In some cases, the charge of the nanopore can be modified at the trans entrance of the channel. In some cases, the charge of the nanopore can be modified in the central channel of the nanopore. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

[0023] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the electrode pair is configured to provide an applied voltage to generate the 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 about 20 mV. In some embodiments, the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

[0024] In some embodiments, a translocase is used to translocate a non-nucleic acid-based polymer analyte. In some embodiments, the translocase comprises an ATP-driven unfolding enzyme. In some embodiments, the translocase comprises an NTP-driven unfolding enzyme. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, the AAA+ enzyme is selected from ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, homologs, and analogs thereof.

[0025] In another aspect, the present disclosure provides a system comprising: a fluid chamber; a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte; a translocase; and a main branch structure comprising at least about one of a blocking motif, an intercepting motif, or a coupling motif, or a combination thereof, wherein the main branch structure is configured to couple to the non-nucleic acid-based polymer analyte.

[0026] In another aspect, the present disclosure provides a system comprising: a fluid chamber; a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte; and a controller operably coupled to the fluid chamber and the nanopore, wherein the controller is configured to detect one or more signals associated with at least about one feature of the main branch structure and one or more signals associated with at least about one feature of the non-nucleic acid-based polymer analyte during or after translocation of the non-nucleic acid-based polymer analyte coupled to the main branch structure through the nanopore using a translocase; wherein the main branch structure comprises at least about one of a blocking motif, an intercepting motif, a coupling motif, or a combination thereof.

[0027] In some embodiments, the controller is further configured to detect, using a pair of electrodes, one or more signals associated with at least one feature of the main branch structure and one or more signals associated with at least one feature of the non-nucleic acid-based polymer analyte. In some embodiments, the controller is further configured to separate the one or more signals associated with at least one feature of the main branch structure from the one or more signals associated with at least one feature of the non-nucleic acid-based polymer analyte.

[0028] In some embodiments, the main branch structure includes one or more nucleic acid molecules. In some embodiments, the main branch structure includes one or more peptides. In some embodiments, the main branch structure includes one or more nucleic acid molecules and one or more peptides. In some embodiments, the blocking motif is configured to destroy the interaction between the translocase and the non-nucleic acid-based polymer analyte. In some embodiments, the blocking motif includes an amino acid sequence. In some embodiments, the amino acid sequence includes n repeats of (glycine) n, (serine-glycine) n, (glycine-serine) n, (alanine-serine) n, (valine) n, (alanine-serine) n, (valine-serine) n or (serine-valine) n. In some embodiments, n can be about 1 to about 50. In some embodiments, n can be from about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 2 to about 25, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 3 to about 4, about 3 to about 5, about 3 to about 10, about 3 to about 15, about 3 to about 20, about 3 to about 25, about 3 to about 30, about 3 to about 40, about 3 to about 50, about 4 to about 5, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 4 to about about 25, about 4 to about 30, about 4 to about 40, about 4 to about 50, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 10 to about 40, about 10 to about 50, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 50, about 20 to about 25, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 25 to about 30, about 25 to about 40, about 25 to about 50, about 30 to about 40, about 30 to about 50, about 40 to about 50.

[0029] In some embodiments, n can be 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, about 20, about 25, about 30, about 40, about 45, or about 50. In some embodiments, the blocking motif comprises a non-amino acid chemical region. In some embodiments, the non-amino acid chemical region comprises polyethylene glycol.

[0030] In some embodiments, the interception motif is configured to prevent a translocase from translocating a non-nucleic acid-based polymer analyte through the interception motif. In some embodiments, the interception motif is configured to prevent a translocase from translocating the non-nucleic acid-based polymer analyte through the main branch structure. In some embodiments, the interception motif is configured to prevent the translocase from translocating the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, the interception motif comprises a steric hindrance. In some embodiments, the steric hindrance comprises one or more bulky amino acids. In some embodiments, one or more bulky amino acids comprise histidine, phenylalanine, tyrosine, or tryptophan. In some embodiments, the steric hindrance comprises at least about one bulky amino acid. In some embodiments, the steric hindrance comprises at least about five bulky amino acids. In some embodiments, the steric hindrance comprises at least about a portion of an anti-unfolding protein. In some cases, the anti-unfolding protein may comprise any folding protein. In some cases, the anti-unfolding protein may comprise an α-helix, a β-strand, a β-turn, a helix-hairpin-helix motif, or any combination thereof. In some cases, the anti-unfolding protein may comprise one or more anti-unfolding domains. In some cases, the one or more anti-unfolding domains may include an α-helix, a β-strand, a β-turn, a helix-hairpin-helix motif, or any combination thereof. In some cases, the anti-unfolding protein may have about 1 to 10 anti-unfolding domains. In some cases, the anti-unfolding protein may have at least about one anti-unfolding domain, at least about two anti-unfolding domains, at least about three anti-unfolding domains, at least about four anti-unfolding domains, at least about five anti-unfolding domains, at least about six anti-unfolding domains, at least about seven anti-unfolding domains, at least about eight anti-unfolding domains, at least about nine anti-unfolding domains, at least about ten anti-unfolding domains, or more than ten anti-unfolding domains. In some cases, the unfolding resistance protein can have a maximum of about ten anti-unfolding domains, a maximum of about nine anti-unfolding domains, a maximum of about eight anti-unfolding domains, a maximum of about seven anti-unfolding domains, a maximum of about six anti-unfolding domains, a maximum of about five anti-unfolding domains, a maximum of about four anti-unfolding domains, a maximum of about three anti-unfolding domains, a maximum of about two anti-unfolding domains, a maximum of about one anti-unfolding domain or less than one anti-unfolding domain. In some cases, the anti-unfolding protein can have about one anti-unfolding domain, about two anti-unfolding domains, about three anti-unfolding domains, about four anti-unfolding domains, about five anti-unfolding domains, about six anti-unfolding domains, about seven anti-unfolding domains, about eight anti-unfolding domains, about nine anti-unfolding domains or about ten anti-unfolding domains. In some embodiments, the anti-unfolding protein includes maltose binding protein, titin, dihydrofolate reductase, nuclease or a combination thereof. In some embodiments, the anti-unfolding protein comprises a disulfide bond. In some embodiments, the steric hindrance includes a large tethering molecule.In some embodiments, the large tethering molecule comprises a carbohydrate, a polycyclic molecule, a branched dextran, biotin, streptavidin, a nanobody, an antibody, or a small antigenic element.

[0031] In some embodiments, the coupling motif is configured to couple the main branch structure to a non-nucleic acid-based polymer analyte. In some embodiments, the non-nucleic acid-based polymer analyte comprises a peptide. In some embodiments, the coupling motif is connected to the C-terminus of the peptide. In some embodiments, the coupling motif is connected to the N-terminus of the peptide. In some embodiments, the coupling motif comprises an enzyme having peptide ligase activity. In some embodiments, the coupling motif comprises a recognition sequence that can be recognized by an enzyme having peptide ligase activity. In some cases, the enzyme can interact with the coupling motif. In some embodiments, the coupling motif comprises a chemical group. In some embodiments, the chemical group comprises maleimide, iodoacetamide, 2-thiopyridine, 3-arylpropionitrile, NHS-ester, isocyanate, isothiocyanate, benzoyl fluoride, diazonium salt, or PTAD. In some embodiments, the coupling motif comprises an enzyme coupling region. In some embodiments, the enzyme coupling region connects the coupling motif to the enzyme. In some cases, the enzyme can interact with the coupling motif.

[0032] In some embodiments, the enzyme comprises a peptidase, a holoacylase, or a sortase. In some embodiments, the coupling motif of the main branch structure is coupled to the non-nucleic acid-based polymer analyte via a covalent bond. In some embodiments, the coupling motif of the main branch structure is coupled to the non-nucleic acid-based polymer analyte via a connector.

[0033] In some embodiments, the main branch structure further comprises at least one of the following: a recognition motif; or a capture motif.

[0034] In some embodiments, the capture motif comprises a polycationic tag. In some embodiments, the polycationic tag comprises n repeats of (serine-glycine-arginine)n, (serine-arginine)n, (lysine)n, or (arginine)n. In some cases, the polycationic tag may comprise serine, glycine, arginine, lysine, or any combination thereof. In some embodiments, the capture motif comprises a polyanionic tag. In some embodiments, the polyanionic tag comprises n repeats of (serine-glycine-aspartic acid)n, (serine-aspartic acid)n, (aspartic acid)n, (serine-glycine-glutamate)n, (serine-glutamate)n, or (glutamate)n. In some cases, the polyanionic tag may comprise serine, glycine, aspartic acid, glutamate, or any combination thereof. In some embodiments, the recognition motif comprises a portion of ssrA, a prokaryotic ubiquitin-like protein, SulA, a peroxisomal membrane protein (Pex15), or a combination thereof. In some embodiments, the sequence of the recognition motif comprises one or more of SEQ ID NOs: 201-206.

[0035] In some embodiments, the main branch structure is attached to the C-terminus or N-terminus of the non-nucleic acid-based polymer analyte. In some embodiments, the non-nucleic acid-based polymer analyte comprises a polypeptide, wherein the main branch structure is attached to the N-terminus of the polypeptide. In some embodiments, the non-nucleic acid-based polymer analyte comprises a polypeptide, wherein the main branch structure is attached to the C-terminus of the polypeptide. In some embodiments, the non-nucleic acid-based polymer analyte comprises a second main branch structure.

[0036] In some embodiments, the first solution and the second solution are configured to generate a transmembrane electroosmotic force. 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 osmotic agent. In some embodiments, the difference between the solute of the first concentration and the solute of the second concentration is configured to generate a transmembrane electroosmotic force. In some embodiments, the transmembrane electroosmotic force comprises a net ionic current flowing from the cis side of the membrane to the trans side. In some embodiments, the transmembrane electroosmotic force is regulated by pH value, salt type, salt concentration, transmembrane osmotic pressure of the system membrane, modification of the nanopore, or any combination thereof. In some embodiments, the transmembrane electroosmotic force is regulated by the charge of the modified nanopore. In some embodiments, the transmembrane electroosmotic force is regulated by the asymmetric salt distribution between the cis side and the trans side of the membrane.

[0037] In some embodiments, the system further comprises a pair of electrodes comprising a first electrode and a second electrode. In some embodiments, the first electrode is arranged on the cis side of the fluid chamber, and the second electrode is arranged on the trans side of the fluid chamber. In some embodiments, the electrode pair is configured to provide an applied voltage to generate an electrophoretic force through the membrane in a direction opposite to the electroosmotic force. In some embodiments, the electroosmotic force is strong enough to allow the non-nucleic acid-based polymer analyte to translocate against the electrophoretic force through the nanopore. 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 300mV. In some embodiments, the amplitude of the applied voltage is greater than about 20mV. In some embodiments, the absolute relative net electroosmotic current on the applied voltage is greater than about 0.10pA / mV.

[0038] In another aspect, the present disclosure provides a method comprising: providing: a nanopore system, wherein the nanopore system comprises a fluid chamber and; (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side and a trans side; a non-nucleic acid-based polymer analyte; and translocating the non-nucleic acid-based polymer analyte from the cis side to the trans side of the fluid chamber, wherein the nanopore comprises an adaptor protein, wherein at least about a portion of the adaptor protein is located within a channel of the nanopore.

[0039] In some embodiments, the adaptor protein is configured to modify the geometry of the nanopore channel. In some embodiments, the adaptor protein is configured to constrict the nanopore channel. In some embodiments, the adaptor protein is configured to modify the charge of the nanopore channel. In some embodiments, the adaptor protein is configured to modify the nanopore channel or a portion thereof to have a positive charge. In some embodiments, the adaptor protein is configured to modify the nanopore channel or a portion thereof to have a net negative charge. In some embodiments, the adaptor protein comprises a protein adaptor protein or a chemical adaptor protein. In some embodiments, the protein adaptor protein comprises a CsgF subunit, a CsgF subunit truncation, or a CsgF subunit homolog, analog, or homolog. In some embodiments, the chemical adaptor protein comprises a cyclodextrin, a cucurbituril, a crown ether, a calixarene, a porphyrin, cyclosporine, cyclam, or 1,4,8,11-tetraazacyclotetradecane. In some embodiments, the adaptor protein is coupled to the nanopore channel. In some embodiments, the adaptor protein is coupled to the nanopore channel via a covalent bond. In some embodiments, the adaptor protein is coupled to the nanopore channel via a non-covalent bond. In some embodiments, the adaptor protein is coupled to the channel of the nanopore via a linker. In some embodiments, the nanopore system comprises a cis-to-trans electroosmotic force generated by a net ionic current cis-to-trans.

[0040] In some embodiments, the method further comprises providing an electrophoretic force acting in a direction opposite to the cis-to-trans electroosmotic force. In some embodiments, the cis-to-trans electroosmotic force is sufficiently strong to overcome the electrophoretic force and propel the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, the electroosmotic force is utilized to translocate the non-nucleic acid-based polymer analyte through the nanopore.

[0041] In some embodiments, the method further comprises providing an electrophoretic force exerted in a direction opposite to the electroosmotic force. In some embodiments, the electroosmotic force acts in opposition to the electrophoretic force to propel the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, the electroosmotic force comprises a net ionic current from the cis side to the trans side. In some embodiments, the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the nanopore system membrane, modification of the nanopore, or any combination thereof. In some embodiments, the electroosmotic force is regulated by the charge of the modified nanopore. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

[0042] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the electrode pair is configured to provide an applied voltage to generate the 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 about 20 mV. In some embodiments, the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

[0043] In some embodiments, a translocase is used to translocate a non-nucleic acid-based polymer analyte. In some embodiments, the translocase comprises an ATP-driven unfolding enzyme. In some embodiments, the translocase comprises an NTP-driven unfolding enzyme. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, the AAA+ enzyme is selected from ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, homologs, and analogs thereof.

[0044] In another aspect, the present disclosure provides a system comprising: a fluid chamber; and a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte; wherein the nanopore comprises an adaptor protein within a channel of the nanopore.

[0045] In some embodiments, the adaptor protein comprises a protein adaptor protein or a chemical adaptor protein. In some embodiments, the protein adaptor protein comprises a CsgF subunit, a CsgF subunit truncation, or a CsgF subunit homolog, analog, or homolog. In some embodiments, the chemical adaptor protein comprises a cyclodextrin, a cucurbituril, a crown ether, a calixarene, a porphyrin, a cyclosporine, a cyclem, or 1,4,8,11-tetraazacyclotetradecane. In some embodiments, the adaptor protein is coupled to the channel of the nanopore. In some embodiments, the adaptor protein is coupled to the channel of the nanopore via a covalent bond. In some embodiments, the adaptor protein is coupled to the channel of the nanopore via a non-covalent bond. In some embodiments, the adaptor protein is coupled to the channel of the nanopore via a linker.

[0046] In some embodiments, the first solution comprises a solute of a first concentration, and the second solution comprises a solute of a second concentration. In some embodiments, the solute comprises an ion or an osmotic agent. In some embodiments, the difference between the solute of the first concentration and the solute of the second concentration is configured to generate an electroosmotic force. In some embodiments, the first solution and the second solution are configured to generate an electroosmotic force across the membrane. In some embodiments, the electroosmotic force is generated by a net ionic current flowing from the cis side of the membrane to the trans side.

[0047] In some embodiments, the system further comprises an electrophoretic force in the opposite direction to the electroosmotic force, wherein the electroosmotic force is strong enough to propel the non-nucleic acid-based polymer analyte through the nanopore against the electrophoretic force. In some embodiments, the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the system membrane, modification of the nanopore, or any combination thereof. In some embodiments, the electroosmotic force is regulated by the charge of the modified nanopore. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

[0048] In some embodiments, the system further comprises a pair of electrodes. In some embodiments, the first electrode of the pair of electrodes is arranged on the cis side of the membrane, and the second electrode of the pair of electrodes is arranged on the trans side of the membrane. In some embodiments, the electrode pair is configured to detect signals during the translocation of the non-nucleic acid-based polymer analyte. In some embodiments, the signal is associated with the characteristics of the non-nucleic acid-based polymer analyte. 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 amplitude of the applied voltage is less than 300mV. In some embodiments, the amplitude of the applied voltage is greater than about 20mV. In some embodiments, the absolute relative net electroosmotic current on the applied voltage is greater than about 0.10pA / mV.

[0049] In another aspect, the present disclosure provides a method comprising: (a) providing: a nanopore system, wherein the nanopore system comprises a fluid chamber and a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side and a trans side; (b) adding a combined solution to the cis side of the fluid chamber, wherein the combined solution comprises a non-nucleic acid-based polymer analyte and a preload solution; and (c) translocating the non-nucleic acid-based polymer analyte from the cis side to the trans side of the fluid chamber.

[0050] In some embodiments, prior to (b), the step further comprises combining a sample comprising a non-nucleic acid-based polymer analyte with a preload solution. In some embodiments, the preload solution comprises translocation. In some embodiments, the non-nucleic acid-based polymer analyte is translocated using a translocase. In some embodiments, the translocase comprises an ATP-driven unfolding enzyme. In some embodiments, the translocase comprises an NTP-driven unfolding enzyme. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, the AAA+ enzyme is selected from ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, homologs, and analogs thereof.

[0051] In some embodiments, the sample and the preload solution are combined to form a non-nucleic acid based polymer analyte-translocase complex. In some embodiments, the sample and the preload solution are combined to form a non-nucleic acid based polymer analyte-main branch structure complex. In some embodiments, the preload solution includes a main branch structure. In some embodiments, the preload solution includes a chemical substance that enhances the binding of the non-nucleic acid based polymer analyte to the components of the preload solution. In some embodiments, the binding of the non-nucleic acid based polymer analyte to the components of the preload solution is higher than the binding of the non-nucleic acid based polymer analyte to the components in the fluid chamber. In some embodiments, the preload solution includes one or more auxiliary factors. In some embodiments, the one or more auxiliary factors include NTP, M 2+ , NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, polysucrose, manganese iron, cobalt, copper, penicillamine, trientine, ethylenediaminetetraacetic acid.

[0052] In some embodiments, the non-nucleic acid-based polymer analyte is translocated using an electroosmotic force. In some embodiments, the translocation includes providing an electrophoretic force in the opposite direction of the electroosmotic force. In some embodiments, the electroosmotic force pushes the non-nucleic acid-based polymer analyte through the nanopore against the electrophoretic force. In some embodiments, the electroosmotic force includes a net ionic current from the cis side to the trans side.

[0053] In some embodiments, the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the nanopore system membrane, modification of the nanopore, or any combination thereof. In some embodiments, the electroosmotic force is regulated by modifying the charge of the nanopore. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis and trans sides of the membrane.

[0054] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the electrode pair is configured to provide an applied voltage to generate the 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 about 20 mV. In some embodiments, the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

[0055] In another aspect, the present disclosure provides a system comprising: a fluid chamber; and a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side comprising a first solution and a trans side comprising 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; and a preload solution configured to interact with the non-nucleic acid-based polymer analyte.

[0056] In some embodiments, the first solution comprises a solute at a first concentration and the second solution comprises a solute at a second concentration. In some embodiments, the solute comprises an ion or an osmotic agent. In some embodiments, the difference between the solute at the first concentration and the solute at the second concentration is configured to generate an electroosmotic force. In some embodiments, the first solution and the second solution are configured to generate a transmembrane electroosmotic force. In some embodiments, the electroosmotic force is generated by a net ionic current flowing from the cis side of the membrane to the trans side. In some embodiments, the electroosmotic force is regulated by pH value, salt type, salt concentration, transmembrane osmotic pressure of the system membrane, modification of the nanopore, or any combination thereof. In some embodiments, the electroosmotic force is regulated by the charge of the modified nanopore. In some embodiments, the electroosmotic force is regulated by the asymmetric salt distribution between the cis side and the trans side of the membrane.

[0057] In some embodiments, the system further comprises a pair of electrodes disposed on the cis side and the trans side of the membrane, wherein the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force across the membrane in a direction opposite to the electroosmotic flow. 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 about 300 mV. In some embodiments, the absolute relative net electroosmotic current over the applied voltage is greater than about 0.10 pA / mV. In some embodiments, the preload solution comprises one or more cofactors. In some embodiments, the one or more cofactors comprise divalent metal ions, NTPs, M 2+, NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, polysucrose, manganese iron, cobalt, copper, penicillamine, trientine, calcium sodium acetate, betaine, or ethylenediaminetetraacetic acid.

[0058] In some embodiments, the preload solution comprises a translocase. In some embodiments, the translocase comprises an ATP-driven unfolding enzyme. In some embodiments, the translocase comprises an NTP-driven unfolding enzyme. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, the AAA+ enzyme is selected from ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, or a functional homolog, homolog, or analog thereof.

[0059] In some embodiments, the preload solution comprises a main branch structure. In some embodiments, the preload solution comprises a chemical that enhances binding of the non-nucleic acid-based polymer analyte to a component of the preload solution relative to binding in the solution on the cis side of the fluid chamber. In some embodiments, the nanopore has an ion selectivity P greater than about 2.0. (+) / P (-) In some embodiments, the nanopore has an ion selectivity P of less than 0.50. (+) / P (-) In some embodiments, the non-nucleic acid based polymer analyte is an unmodified (unlabeled) non-nucleic acid based polymer analyte.

[0060] In some embodiments, the termini of the non-nucleic acid-based polymer analyte lack a three-dimensional structure. In some embodiments, at least about a portion of the non-nucleic acid-based polymer analyte is denatured. In some embodiments, the non-nucleic acid-based polymer analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof. In some embodiments, the non-nucleic acid-based polymer analyte comprises a polypeptide, a polysaccharide, or a water-soluble plastic. In some embodiments, the non-nucleic acid-based polymer analyte comprises a polypeptide. In some embodiments, the polypeptide comprises at least about 30 peptide units. In some embodiments, at least about 30 peptide units comprise positively charged residues. In some embodiments, at least about 30 peptide units comprise negatively charged residues. In some embodiments, at least about 30 peptide units comprise both positively charged residues and negatively charged residues. In some embodiments, at least about 30 peptide units comprise positively charged residues. In some embodiments, at least about 30 peptide units comprise negatively charged residues. In some embodiments, at least about 30 peptide units comprise both positively charged residues and negatively charged residues. In some embodiments, the polypeptide is in a denatured state. In some embodiments, the polypeptide is provided in a folded state.

[0061] In some embodiments, the method further comprises measuring a signal generated by translocation of the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, measuring comprises measuring a signal for (a) an open channel state of the nanopore; (b) capture of the non-nucleic acid-based polymer analyte by the nanopore; or (c) passage of the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, measuring comprises detecting a difference between states (a), (b), and (c).

[0062] In some embodiments, the signal comprises an ionic current, a change in ionic current, or a derivative thereof. In some embodiments, the nanopore comprises an inner pore constriction of about 0.5 nM to about 2 nM. In some embodiments, the inner pore constriction is from about 1 nM to about 2 nM. 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 comprising hemolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, a phage-derived portal protein, a modified variant thereof, or an ion-selective mutant thereof.

[0063] 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. In some embodiments, the biological nanopore restricts the passage of one or more ions through the nanopore by modifying the charge of the nanopore channel. In some embodiments, the net charge is negative. In some embodiments, the net charge is positive.

[0064] In some embodiments, the nanopore is a mutant CytK nanopore. In some embodiments, the mutant CytK comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions comprise K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof. In some embodiments, the one or more amino acid substitutions comprise 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, T116D; (c) T147D or S151D; (d) K128D, K155D, S120D; (e) Q122D, T147D or S155D; (f) K128D, K155D, Q145D, S151D; or (g) a combination of both. In some embodiments, the mutant CytK nanopore comprises one or more combinations of the following 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, Q122D; (f) K128F, S120D, G122D; (g) K128F, S120D, G122D, K155D; or (h) a combination of the two.

[0065] In some embodiments, the nanopore has an ion selectivity P greater than about 2.0. (+) / P (-) In some embodiments, the nanopore has an ion selectivity P of less than 0.50. (+) / P (-). In some embodiments, the non-nucleic acid-based polymer analyte is an unmodified (label-free) non-nucleic acid-based polymer analyte. In some embodiments, the end of the non-nucleic acid-based polymer analyte lacks a three-dimensional structure. In some embodiments, at least about a portion of the non-nucleic acid-based polymer analyte is denatured. In some embodiments, the non-nucleic acid-based polymer analyte comprises a peptide unit, a sugar unit, a water-soluble plastic monomer, or any combination thereof. In some embodiments, the non-nucleic acid-based polymer analyte comprises a polypeptide, a polysaccharide, or a water-soluble plastic. In some embodiments, the non-nucleic acid-based polymer analyte comprises a polypeptide. In some embodiments, the polypeptide comprises at least about 30 peptide units. In some embodiments, at least about 30 peptide units comprise positively charged residues. In some embodiments, at least about 30 peptide units comprise negatively charged residues. In some embodiments, at least about 30 peptide units comprise positively charged residues and negatively charged residues. In some embodiments, the polypeptide is in a denatured state. In some embodiments, the polypeptide is provided in a folded state.

[0066] In some embodiments, the system further comprises measuring a signal generated by translocation of the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, measuring comprises measuring a signal for (a) an open channel state of the nanopore; (b) capture of the non-nucleic acid-based polymer analyte by the nanopore; or (c) passage of the non-nucleic acid-based polymer analyte through the nanopore. In some embodiments, measuring comprises detecting a difference between states (a), (b), and (c).

[0067] In some embodiments, the signal comprises an ionic current, a change in an ionic current, or a derivative thereof. In some embodiments, the nanopore comprises an inner pore constriction from about 0.5 nM to about 2 nM. In some embodiments, the inner pore constriction is from about 1 nM to about 2 nM. 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 comprising hemolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, a phage-derived portal protein, a modified variant thereof, or an ion-selective mutant thereof.

[0068] 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. In some embodiments, the biological nanopore restricts the passage of one or more ions through the nanopore by modifying the charge of the nanopore channel. In some embodiments, the net charge is negative. In some embodiments, the net charge is positive.

[0069] In some embodiments, the nanopore is a mutant CytK nanopore. In some embodiments, the mutant CytK comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions comprise K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof. In some embodiments, the one or more amino acid substitutions comprise 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, T116D; (c) T147D or S151D; (d) K128D, K155D, S120D; (e) Q122D, T147D or S155D; (f) K128D, K155D, Q145D, S151D; or (g) a combination of both. In some embodiments, the mutant CytK nanopore comprises one or more combinations of the following 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, Q122D; (f) K128F, S120D, G122D; (g) K128F, S120D, G122D, K155D; or (h) a combination of the two.

[0070] In another aspect, the present disclosure provides an apparatus comprising a system array comprising the system disclosed herein.

[0071] In another aspect, the present disclosure provides the use of any method, kit, or device disclosed herein for characterizing at least about one structural feature of a non-nucleic acid-based polymer analyte.

[0072] In another aspect, the present disclosure provides the use of any of the methods, kits, or devices disclosed herein for analyzing the amino acid sequence or amino composition of one or more non-nucleic acid-based polymer analytes at the single molecule level.

[0073] In another aspect, the present disclosure provides use of any of the systems disclosed herein for characterizing at least about one structural feature of a non-nucleic acid based polymer analyte.

[0074] In another aspect, the present disclosure provides the use of any of the disclosed systems for analyzing the amino acid sequence or amino acid composition of one or more non-nucleic acid based polymer analytes at the single molecule level.

[0075] In another aspect of the present disclosure, a method for translocating a target protein through a nanopore is provided, the nanopore being included in a membrane separating a fluid chamber of the nanopore system into a cis side and a trans side, comprising: (a) allowing a protein translocase in solution (optionally in the presence of NTPs) to capture and form a complex with a target protein to be translocated, (b) contacting the translocase-target protein complex with the cis side of the nanopore and allowing the target protein to translocate to the trans side, wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) resulting from a net cis-to-trans ionic current. The target protein is captured in the nanopore, and the protein translocase at the top of the nanopore controls the translocation.

[0076] In some embodiments, where the nanopore system has a cis-to-trans EOF, the ratio of the net ionic current cis-to-trans to the total ionic current (I rel ) is greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, greater than 0.35 or less than -0.35.

[0077] In some embodiments of any of the foregoing embodiments, the cis-to-trans EOF is arranged by adjusting the pH, type and / or concentration of salts and / or osmotic pressure on the nanopore system membrane, by modifying (e.g., genetic engineering) the nanopore charge, or any combination thereof, such as by modifying the nanopore and / or asymmetric salt distribution between the cis and trans sides of the chamber.

[0078] In some embodiments of any of the foregoing embodiments, the translocase-target protein complex is formed in solution on the cis side of the fluid chamber.

[0079] In some embodiments of any of the foregoing embodiments, the translocase-target protein complex is formed in solution in a separate step prior to adding the complex to the cis side of the fluid chamber to contact the nanopore.

[0080] In some embodiments of any of the foregoing embodiments, the target protein comprises a main branch structure at its N- and / or C-terminus to allow for preloading and optionally blocking translocation of one or more proteins. In some embodiments, the main branch structure comprises (i) a recognition motif for a protein translocase, and further comprises one or more of the following elements: (ii) a capture motif; (iii) a blocking motif; (iv) an interception motif.

[0081] In another aspect of the present disclosure, a nanopore system for translocating a target protein through a nanopore is provided, comprising: (a) a membrane having a nanopore therein, the membrane dividing a chamber into a cis side and a trans side, wherein the target protein will be added to the cis side and transported to the trans side through the nanopore, (b) on the cis side of the chamber, the target protein captured by a protein translocase, which can sequentially bind and transport the target protein through the nanopore; (c) a mechanism for providing a voltage difference between the cis and trans sides of the membrane, wherein the nanopore system has a cis to trans electroosmotic force (EOF), which is generated by a net ionic current from cis to trans, so that the target protein is captured in the nanopore, and the translocase at the top of the nanopore controls the translocation; wherein the nanopore system has a cis to trans EOF, which is generated by a net ionic current cis to trans passing through a total ionic current greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, greater than 0.35 or less than -0.35.

[0082] In some embodiments, the system further comprises a method for measuring a signal based on ionic current flowing through the nanopore during a translocation time, wherein the measuring method detects a change in the signal reflecting a property of the protein upon translocation.

[0083] In some embodiments of any of the foregoing embodiments, the ion selectivity of the nanopore system is (+) / P (-) Greater than 2.0 or less than 0.5, greater than 2.5 or less than 0.4, greater than 3.0 or less than 0.33.

[0084] In some embodiments of any of the foregoing embodiments, the nanopore is a biological nanopore having an internal pore constriction in the range of 0.5-2 nM, wherein the nanopore is an α-helical or β-barrel oligomeric pore-forming toxin or porin. In some embodiments, the nanopore is selected from the group consisting of aerolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, phage-derived portal proteins, and modified variants thereof, wherein the nanopore is modified to have a net charge in the b>21, b>28, and b>35 regions facing the lumen, wherein the net charge is negative. In some embodiments, the nanopore is selected from the group consisting of aerolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, phage-derived portal proteins, and modified variants thereof, wherein the nanopore is modified to have a net charge in the lumen-facing regions b>21, b>28, and b>35, wherein the net charge is positive. In some embodiments, any amino acid residue in the lumen-facing domain of the nanopore can be mutated. In some cases, the mutated amino acid residue can be mutated to a negatively charged amino acid. In some cases, the mutated amino acid residue can be mutated to a positively charged amino acid. In some cases, the mutated amino acid residue can be mutated to a neutrally charged amino acid.

[0085] In some embodiments, the nanopore can be an oligomer. In some cases, the oligomer nanopore can include one or more subunits. In some cases, each subunit of the one or more subunits can include about 20 to about 40 charges in the region of the subunit facing the lumen. In some cases, each unit of the one or more subunits can include at least about 20 charges, at least about 21 charges, at least about 22 charges, at least about 23 charges, at least about 24 charges, at least about 25 charges, at least about 26 charges, at least about 27 charges, at least about 28 charges, at least about 29 charges, at least about 30 charges, at least about 31 charges, at least about 32 charges, at least about 33 charges, at least about 34 charges, at least about 35 charges, at least about 36 charges, at least about 37 charges, at least about 38 charges, at least about 39 charges, at least about 40 charges, or more than 40 charges in the lumen facing the subunit. In some cases, each unit of one or more subunits may contain at most about 40 charges, at most about 39 charges, at most about 38 charges, at most about 37 charges, at most about 36 charges, at most about 35 charges, at most about 34 charges, at most about 33 charges, at most about 32 charges, at most about 31 charges, at most about 30 charges, at most about 29 charges, at most about 28 charges, at most about 27 charges, at most about 26 charges, at most about 25 charges, at most about 24 charges, at most about 23 charges, at most about 22 charges, at most about 21 charges, at most about 20 charges, or less than 20 charges in the lumen facing the subunit. In some cases, each unit of one or more subunits may include about 20 charges, about 21 charges, about 22 charges, about 23 charges, 24 charges, about 25 charges, about 26 charges, about 27 charges, about 28 charges, about 29 charges, about 30 charges, about 31 charges, about 32 charges, about 33 charges, about 34 charges, about 35 charges, about 36 charges, about 37 charges, about 38 charges, about 39 charges or about 40 charges in the inner cavity facing the subunit.

[0086] In some embodiments, the nanopore is a mutant CytK nanopore comprising one or more amino acid substitutions selected from the group consisting of K128D, K128F, K115D, S120D, Q122D, and S151D, comprising one of the following combinations of amino acid substitutions: S120D, G122D, and / or K155D; S120D combined with K128F / K128D, further comprising Q122D or S151D; K128D / K128F, S120D, K115D, Q122D; K128F, S120D, G122D, may be used in combination with K155D.

[0087] In some embodiments of any of the foregoing embodiments, the protein translocase is an NTP-driven unfolding enzyme, an AAA+ enzyme. In some embodiments, the AAA+ enzyme is selected from ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, (ClpY), LonA, LonB, FtsH, Mpa, Cpa, Mspl, SecA, and functional homologs, homologs, or analogs thereof.

[0088] In some embodiments of any of the foregoing embodiments, the nanopore system has an ion-selective (+) / P (-) Greater than 2.0, greater than 2.5, greater than 3.0, wherein there is a negative applied voltage on the trans side. In some embodiments, the system comprises a cation-selective (mutant) nanopore.

[0089] One aspect of the present disclosure provides an analytical device comprising a nanopore system array according to any one of the above embodiments.

[0090] One aspect of the present disclosure provides the use of a nanopore system or device according to any one of the above embodiments for characterizing at least about one structural feature of a target protein, for analyzing the amino acid sequence or amino acid composition of one or more target proteins at the single molecule level.

[0091] Another aspect of the present disclosure provides a non-transitory computer-readable medium containing machine-executable code that, when executed by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0092] Another aspect of the present disclosure provides a system comprising 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 herein.

[0093] Other aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative rather than restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1: Schematic diagram of a strong electroosmotic nanopore system for improved analyte transport and characterization through the nanopore. As shown, the nanopore exhibits a strong net electroosmotic force (EOF) in the cis-to-trans direction, indicated by the arrows. The electrophoretic force (EPF) acting on the analyte will depend on the charge composition of the target analyte within and near the nanopore channel and can therefore sometimes act in a net cis-to-trans or a net trans-to-cis direction. Regardless of the net direction of the EPF, the strong cis-to-trans EOF enables the capture, stretching, and efficient translocation of long polymer analytes from the cis compartment to the trans compartment. A) Schematic diagram illustrating the strong cis-to-trans EOF across the system, enabling the capture and translocation of protein analytes in the cis-to-trans direction. The magnitude of the ion flow in each direction is visually represented by the arrows in the pore, indicating that the EOF is generated by the large flow of ions from the cis side of the membrane to the trans side. The net flux results from a large cis-to-trans flow that dominates any cis-to-cis ion flow (e.g., low flow or counter-charged ions under applied potential). B) A strong cis-to-trans EOF can be established in a system by applying a positive voltage across the transcompartment of the membrane and using nanopores with a net positive internal charge to restrict cation flow from the trans side of the membrane to the cis side. C) A strong cis-to-trans EOF can be established in a system by applying a negative voltage across the transcompartment of the membrane and using nanopores with a net negative internal charge to restrict anion flow from the trans side of the membrane to the cis side.

[0095] Figure 2 : An exemplary nanopore system for characterizing and / or translocating analytes, such as proteins composed of mixed amino acids. A system consisting of a nanopore on a membrane, wherein the protein analyte is translocated from the cis chamber to the trans chamber through the nanopore with the help of a translocase motor, which advances in the direction of the arrow of the sub-subset of the protein analyte (moving from the terminal PA to the terminal PB of the protein analyte). Depending on the charge composition of the portion of the protein analyte in or near the central channel of the nanopore at any time, and the direction of the applied voltage, the net direction of the EPF acting on the protein may be cis to trans or trans to cis or effectively zero when the protein passes through the nanopore (as indicated by the dotted arrow marked EPF). In order to improve the characterization and / or translocation of protein analytes, the system is configured so that the net electroosmotic force (EOF) acting on the protein is in the cis to trans direction (as indicated by the arrow marked EOF) and is greater than the magnitude of the EPF.

[0096] Figure 3: An exemplary system for characterizing and translocating polymeric analytes, such as protein analytes of mixed amino acid composition, through a nanopore in a membrane. A) A protein analyte comprising a main branch protein, loaded with a protein translocase motor (NTP-driven translocase functioning in a direction away from the main branch protein, as indicated by the subset arrows), forms a protein:translocase complex and is added to the cis side of the system containing the nanopore in the membrane. The protein:translocase complex is captured by the nanopore, for example, by the main branch structure. B) The entire complex is pulled into the nanopore by a combination of cis-to-trans EPFs and / or EOFs until the translocase motor encounters the top of the nanopore, preventing further uncontrolled translocation. C) Cis-to-trans EPF and / or EOF forces act on the primary motif of the polymer region within the nanopore, pulling the polymer past the translocase, enabling the translocase to overcome the primary region's block and / or blocking region. The translocase will then continue along the polymer analyte under chemical energy-driven NTP hydrolysis, unfolding the protein structure ahead. Thus, the extruded polypeptide chain is fed cis-trans into the nanopore in a controlled manner. D) The protein analyte is fully processed by the translocase, which unbinds upon encountering the molecular terminus, releasing the polypeptide, which is then translocated to the transcompartment of the system. The nanopore can then be used to capture and process another protein: the translocase complex (A).

[0097] Figure 4: A) Components of a (peptide-based) "backbone structure" (6) for attachment to a target protein substrate, which can host / bind a protein translocase motor to unfold and control the translocation of the target protein substrate through the nanopore. This structure contains many possible elements:

[0098] 1. Recognition motif

[0099] 2. Capture Motif

[0100] 3. Blocking motif

[0101] 4. Interception Motif

[0102] 5. Coupled Motifs

[0103] B) Illustrative schematic of a main branch structure (6) attached to a target protein substrate (7), such as a folded or structural protein.

[0104] Figure 5: Example of loading a protein translocase (8) onto a main branch structure (6). The translocase first binds to the main branch structure at or near the recognition motif (B), then translocates along the structure in the direction of the subset arrow (C) through NTP hydrolysis until it encounters an obstacle and / or blocking motif, which hinders / pauses the translocase's progress (D).

[0105] Figure 6: Example process for loading multiple protein translocases onto a main branch construct. A) Schematic diagram of a substrate designed to load and arrest a single protein translocase. The combination of capture (2) and / or arrest (3) motifs has a footprint long enough to accommodate a single translocase site. B) Schematic diagram of a substrate designed to load and arrest n multiple protein translocases, including a longer combination of capture (2) and / or arrest (3) motifs that can effectively arrest and accommodate the binding footprints of n multiple translocases such that the tail translocase motor cannot push the main branch translocase past the arrest / arrest motifs.

[0106] Figure 7: Exemplary methods for loading a main branch structure (6) with a protein translocase (8) and attaching the main branch structure to a protein of interest (7). a) The main branch structure can first be coupled to the target protein analyte, then loaded with the translocase. b) The main branch substrate can be pre-loaded with the translocase and then coupled to the target protein analyte.

[0107] Figure 8: Electrical recordings of wild-type α-hemolysin nanopore (WT αHL) capture of a maltose-binding protein substrate (MBP-1). Measurements were performed using cis- and trans-solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5. The cis-chamber concentration was 0.1 μM MBP-1. Selected representative regions recorded at (A) -80 mV, (B) -120 mV, (C) -160 mV, and (D) -180 mV (counter electrode) are shown. Little MBP-1 capture was observed at lower voltages. As the voltage increased, a blockade of almost 0 pA was observed due to MBP-1 capture. Some captures spontaneously cleared the pore, but many had to be removed by briefly reversing the applied voltage (marked by arrows).

[0108] Figure 9: Same as Figure 8, but using wild-type CytK nanopores (WTCytK).

[0109] Figure 10: Representative examples of current vs. time traces of the wheat gluten binding protein substrate:ClpX translocase complex (MBP-1:ClpX) tested in weak EOF wild-type α-hemolysin (panels a and b) or wild-type CytK (WTCytK) (c). Measurements were performed in a system similar to that described in Figure 3 (except for the low or zero EOF nanopore). Preloaded MBP-1:ClpX complex (preloaded according to Figure 5) was added to the cis side of the nanopore system (concentrations of 0.2 μM ClpX, 0.1 μM MBP-1, and 2.5 mM ATP) (cis and trans solutions were 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, respectively).

[0110] Voltage-dependent trapping of the MBP-1:ClpX complex was observed in these low-EOF nanopores, resulting in a blockade of the ionic current of nearly 0 pA. However, no events progressed to the translocating peptide stage, and events remained blocked indefinitely unless ejected by a brief reversal of the applied voltage (arrowheads).

[0111] Figure 11: Representative examples of current vs. time traces of the maltose binding protein substrate:ClpX translocase complex (MBP-1:ClpX) tested in a strong EOF CytK K128D K155D S120D Q122D (CytK 4D2E) nanopore, according to the system described in Figure 3. Preloaded MBP-1:ClpX complexes (preloaded according to Figure 5) were added to the cis side of the nanopore system (concentrations of 0.2 μM ClpX, 0.1 μM MBP-1, and 2.5 mM ATP) (cis and trans solutions were 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, respectively). A) and B) are representative cross-sections at -80 mV from separate experiments. Typical ClpX-controlled MBP-1 translocation is marked with numbered arrows. Analyte translocation occurs via similar current patterns and has similar characteristics: all events begin with an immediate, almost complete blockade of the ionic current, from an open-pore level (IO) of approximately -65 pA to a blocked level (IB) of almost 0 pA, due to the capture of the MBP-1:ClpX complex. A short time later, the translocase overcomes the blockade, begins unfolding the protein, and passes the peptide into the nanopore, generating a change in current level any time the peptide passes through the pore due to variations in amino acid composition within the nanopore. Upon reaching the terminus of the protein, the translocase releases the substrate through the nanopore, resulting in a return to the unoccupied open-pore current, IO.

[0112] Figure 12 Figure 3: Representative amplified single-case events of ClpX controlling MBP-1 translocation through the CytK K128D K155D S120D Q122D (CytK 4D2E) nanopore. The event begins with a blockade (S1) from the open pore level (state i) to a level close to 0 pA (state ii). Within a short period of time, the translocase overcomes the blockade (S2) and begins to advance along the protein, in the process transporting the extruded polypeptide through the nanopore from cis to trans in a controlled manner, which leads to changes in current levels (section iii) depending on the different amino acid compositions in the nanopore. When ClpX reaches the end of MBP-1, the event terminates (S3) and returns to the open pore current level (state iv).

[0113] Figure 13: Further representative zoomed-in examples of ClpX-controlled MBP-1 translocation events through the CytK K128D K155D S120D Q122D (CytK4D2E) nanopore. This figure illustrates the consistent and characteristic current patterns observed between the S2 and S3 segments, which are due to the amino acid composition within the nanopore changing in the same sequential manner under translocation control as the MBP-1 polypeptide moves through the nanopore (dotted lines connect similar pattern motifs in separate events).

[0114] Figure 14: ClpX controls MBP-1 translocation events in a high-EOF nanopore system using highly ion-selective nanopores. Events were obtained from a CytK_4D2E nanopore (CytK K128D K155D Figure 5. A) CytK_3D1F2E nanopore (CytKK128F_S120D_Q122D_K155D) system at -80 mV, C) CytK_4D2E_Alt nanopore (CytKK128DK155DS120DS151D) system at -80 mV, D) CytK_2D1F2E nanopore (CytKK128FS120DQ122D) system at -120 mV. All cis- and trans-containing solutions were 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, preloaded with MBP-1:ClpX cis compartment (0.2 μM ClpX concentration, 0.1 μM MBP-1 and 2.5 mM ATP). Panels include a subset of schematic diagrams showing the location of net negative charge in the respective nanopores (where negative residues are balanced by adjacent positive residues, neither of which are shown).

[0115] Figure 15: Representative amplified example events of ClpX-controlled translocation of A) MBP-1 substrate and B) GFP-1 substrate through the CytKK128DK155DS120DQ122D (CytK4D2E) nanopore at -80 mV.

[0116] Figure 16: Example current vs. time traces of the maltose binding protein:ClpX translocase complex (MBP-1:ClpX) tested in a strongly EOFCytKK128DK155DS120DQ122D (CytK4D2E) nanopore with non-hydrolyzable γ-s-ATP. Preloaded MBP-1:ClpX complex (preloaded with a standard 10 mM ATP as described herein) was added to the cis side of the nanopore system at -80 mV (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, containing 0.2 μM ClpX: 0.1 μM MBP-1, 2.5 mM γ-s-ATP, and 0.25 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5). A) Blockade events were observed for the trapped MBP-1:ClpX complex; a typical zoomed-in example is shown in B). The event displays a typical initial blockade of nearly 0 pA (i), which then increases to -20 pA (ii). However, the event never progresses to the translocase-controlled polypeptide phase shift observed under conventional ATP conditions, resulting in an indefinite blockade that must be cleared by a brief reversal of the applied voltage (iii).

[0117] Figure 17 : Histogram of the duration of 35 full-length ClpX-controlled MBP-1 translocations through a CytK4D2E nanopore at -80 mV (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5 containing 0.2 μM ClpX, 0.1 μM MBP-1, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5).

[0118] Figure 18: Formation of the MBP-1:ClpX complex via the CytK4D2E nanopore system (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5) without (A) and with (B) preload incubation. A) ClpX (0.2 μM concentration) and MBP-1 (0.1 μM concentration) were added to the cis compartment. B) ClpX and MBP-1 were preincubated in a 10 μL volume (10 μM ClpX, 5 μM ClpX, 10 mM ATP, 25 mM MgCl2), followed by addition of the cis compartment (0.2 μM ClpX, 0.1 μM MBP-1 concentration). Arrows indicate voltage reversals, and asterisks indicate ClpX-controlled translocation.

[0119] Figure 19Typical capture and ClpX-controlled translocation of MBP-1 from cis to cis through a CytK4D2E nanopore inserted into the cis chamber (cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2). Preloaded MBP-1:ClpX complexes were added to the trans chamber (at a concentration of 0.2 μM ClpX, 0.1 μM MBP-1, and 2.5 mM ATP) and captured at the trans entrance of the nanopore at +80 mV and translocated under ClpX control, as evidenced by the strong trans-to-cis EOF generated under positive applied voltage.

[0120] Figure 20: A) Gel showing the results of ClpX / ClpP degradation assay of a GFP-ssrA substrate (GFP-0) at various KCl concentrations. B) Gel showing the results of degradation assay of GFP-0 at various potassium glutamate (KGlu) concentrations (65 nM ClpX, 65 nM ClpP, 2800 nM GFP).

[0121] Figure 21 : A typical ClpX-controlled translocation of MBP-1 at -120 mV through a CytK4d2e nanopore contains 1 M KGlu in the cis chamber and 1 M KCl in the trans chamber (cis: 0.2 μM ClpX, 0.1 μM MBP-1, 2.5 mM ATP in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5; trans: 1 M KCl, 50 mM Tris, 25 mM MgCl2, pH 7.5).

[0122] Figure 22: Schematic diagram showing an exemplary "Out Mode" method by capturing a target protein from the cis side into a nanopore with a system setup that has a high net cis to trans EOF and binding to a protein translocase that targets the protein, thereby pulling the polypeptide back out of the same nanopore to the cis side. A) Protein translocase (8) binds to the target protein (7), which is modified with a terminal tag to enable direct binding to the translocase (1) and is captured in the nanopore (2), thereby forming a translocase:target protein complex. B) The translocase:target protein complex is captured in the nanopore by a combination of EPF and / or EOF forces via the terminal capture tag, thereby causing the target protein portion of the complex to partially translocate from the cis side to the trans side through the nanopore. Until the bound translocase encounters the top of the nanopore (C). The net force of the cis to trans EOF acting on the polypeptide passing through the nanopore pulls the translocase:target protein complex and retains the translocase at the top of the pore. D) Under NTP-driven hydrolysis, the translocase continues to move along the target protein in the direction of the sub-arrow (cis to trans), pulling the peptide out of the nanopore from the trans to the cis direction. E) Upon reaching the end of the target protein molecule, the translocase dissociates from the target protein, releasing both the translocase and the target protein back to the cis side.

[0123] Figure 23 : Schematic diagram of the current and time translocation events caused by the translocation of the polypeptide through the nanopore controlled by the translocase, as shown in Figure 22.

[0124] Figure 24: A) Structural model of the wild-type CytK nanopore mapped with structural homology to the α-hemolysin nanopore. The model shows a low net charge within the nanopore, which is attributed to water-facing residues. B) Schematic representation of the residues within each beta strand of the wild-type CytK transmembrane barrel region, with the water-facing residues of the downward and upward strands most suitable for mutagenesis labeled. C) Model of the CytK4D2E nanopore (CytK K128D K155D S120DQ122D), showing a very high net negative internal charge due to mutations. D) Schematic representation of the locations of mutations in negative residues within the barrel region of the CytK 4D2E nanopore.

[0125] Figure 25: Recordings of different substrates in CytK nanopores. A) Schematic diagram of substrate design. B) Example electrophysiological readouts obtained from {GFP}-{MBP-1}, {LIVBP}-{MBP-1}, {SpuE}-{MBP-1}, and {GBP}-{MBP-1} substrates. Each substrate exhibited similar ionic current patterns in the region corresponding to MBP, as indicated by the lower arrow, and a unique ionic current signature specific to the attached protein in the second section, as indicated by the lower arrow. Measurements were performed with the CytK_4D2E nanopore in 1 M potassium glutamate, 50 mM Tris, 25 MgCl2, 10 mM DTT, and 1 mM EDTA, buffered to pH 7.5 at -80 mV. The ClpX concentration in the cis compartment was 0.2 μM, the indicated substrate concentration was 0.1 μM, and the ATP concentration was 2.5 mM.

[0126] Figure 26 Figure 2: Example single-molecule readout of an 88 kDa MBP-MBP fusion protein. Measurements were performed using a CytK_4D2E nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT, and 1 mM EDTA, buffered to pH 7.5 at -80 mV. The ClpX concentration in the cis chamber was 0.2 μM, the MBP-MBP concentration was 0.1 μM, and the ATP concentration was 2.5 mM.

[0127] FIG27 : Delaying ClpX at 37° C. using a blocking domain. A) Unfolding of GFP-1 with (+ClpX) and without ClpX (-ClpX), B) mNG with an α-helical blocking domain, C) mNG with a helix-turn helix blocking domain, and D) mNG with a hairpin blocking domain. Measurements were performed in the presence of 10 mM ATP at a 1:4 substrate:unfolding enzyme molar ratio (0.065 μM substrate and 0.26 μM ClpX) in a solution containing 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl 2 , 10 mM DTT, and 1 mM EDTA, buffered to pH 7.5.

[0128] Figure 28: Depiction of maltose binding protein (MBP) monomers and maltose binding protein dimers used for analysis in the present disclosure.

[0129] Figure 29Figure 2: Example electrophysiological recording of an MBP protein lacking the ssrA recognition motif, tagged at the C-terminus to allow for ClpX binding. ClpX-controlled MBP translocation (marked by arrows) is evident from the characteristic pattern of changes in the ionic current signal. Measurements were performed using a CytK_4D2E nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT, and 1 mM EDTA, buffered to pH 7.5 at -80 mV. The cis compartment contained 0.2 μM ClpX, 0.1 μM labeled MBP, and 2.5 mM ATP.

[0130] Figure 30 : Description of a computer system that is programmed or otherwise configured to implement the methods provided herein. DETAILED DESCRIPTION

[0131] Incorporation by Reference

[0132] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent such publications and patents or patent applications contradict the disclosure contained in this specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0133] The present disclosure relates to systems and methods for analyzing non-nucleic acid-based polymer analytes (e.g., analytes) using nanopore-based sensors. The present disclosure provides nanopore systems, devices, and methods for analysis and sequencing of single-molecule non-nucleic acid polymer analytes (e.g., single-molecule proteins).

[0134] Various studies have demonstrated the free translocation and controlled mobility of peptides (e.g., proteins that unfold during or before translocation through narrow nanopores) through narrow nanopores (typically <2 nM in diameter). However, unlike polynucleotides, which have a fixed negative charge and can be electrophoretically attracted to nanopores by an applied voltage field, capturing and controlling the mobility of peptides of varying compositions remains a challenge. This is because the varying compositions result in a range of electrical and structural properties (e.g., a mix of positive, negative, neutral, hydrophilic, hydrophobic, aromatic), which prevent simple capture under electrophoretic conditions and translocation in the unfolded state.

[0135] Due to their complex composition, it was previously thought impossible to push the analyte into the pore from the cis side in its native form (e.g., without attaching or coupling a DNA backbone or adding other (e.g., polyanionic) tags to create an electrophoretic capture motif). Different charges cause unfolded peptides to be sometimes attracted to the nanopore and sometimes repelled by the nanopore, depending on the charge and applied voltage, so it is impossible to translocate a variety of complex peptides through the nanopore using electrophoretic mechanisms alone. In fact, previous studies have demonstrated translocation of either very short peptides with a contour length shorter than the nanopore channel length or very carefully selected (model) analytes whose charge, structure, or added electrophoretic tags favor capture and translocation through the nanopore by electrophoresis. However, this is by no means representative of the broad amino acid composition of proteins found in nature. For example, see Motone et al. (iScience24, September 24, 2021) for a review of recent methods using a range of technologies designed to drive protein chains and peptides through nanopores. It is noted that nanopore protein sequencing is a challenging frontier that has not yet been achieved.

[0136] The present disclosure provides a novel method that may be simple and / or provide a robust mechanism for feeding non-nucleic acid-based polymer analytes (e.g., full-length proteins) through nanopores (e.g., for sequencing and / or characterizing them). In one example, the methods and systems disclosed herein may not require additional components (e.g., proteins or polynucleotides) to be fused, conjugated, and / or otherwise attached to the nanopore.

[0137] The study found that these goals can be achieved by using a large cis-to-trans dominant conductive osmotic flow (EOF), which is generated by a large cis-to-trans excess of ions flowing through the nanopore, combined with a translocase on the cis side of the nanopore, which can controllably transfer individual analytes from the cis to the trans and through the nanopore, opposing the direction of the electrophoretic force (EPF). In some embodiments, the cis-to-trans osmotic flow can be generated by the flow of ions and solvent from the cis side of the nanopore system to the trans side of the nanopore system.

[0138] The present disclosure provides a system that can capture and feed analytes (e.g., peptides, nucleic acid molecules, oligosaccharides, lipids, proteins) from the cis side of a nanopore using strong electroosmotic forces without coupling a motor translocase to the nanopore. In some embodiments, the force in the system is sufficient to keep the translocase motor at the top of the pore. For example, a strong electroosmotic pore combined with a translocase protein (e.g., a molecular motor protein) on the cis side can first unfold and then feed the analyte of various components through the nanopore, and then unbind to allow the system to process the next molecule. This can be achieved by pulling at least about a portion of the analyte in or near the nanopore through a strong EOF, because the translocase can diffuse out of the nanopore after unbinding to allow the new complex to bind.

[0139] In some embodiments, as the analyte translocates through the pore, a strong electroosmotic force pulls the analyte and, in turn, transmits this force upward to the bound translocase motor protein above, acting to hold the translocase at the top of the pore during the controlled translocation of the substrate. In turn, under nucleotide triphosphate (NTP)-controlled translocase activity, the translocase moves through the analyte, unfolding any three-dimensional structure in the analyte encountered by the translocase, thereby controlling the movement of the analyte into the nanopore and enabling changes in the current to be measured and characterized.

[0140] Thus, in one embodiment, the present disclosure provides a method of translocating an analyte through a nanopore included in a membrane separating a fluid chamber of a nanopore system into a cis side and a trans side, comprising:

[0141] (a) allowing the protein translocase to be captured in solution (optionally in the presence of NTPs) and to form a complex with the analyte to be translocated;

[0142] (b) contacting the translocase target protein complex with the cis side of the nanopore and allowing the analyte to translocate to the trans side;

[0143] The nanopore system has a cis-to-trans electroosmotic force (EOF) generated by a net cis-to-trans ionic current, so the analyte is trapped in the nanopore, and a translocase can be located on top of the nanopore, which controls the translocation.

[0144] For example, a nanopore system has a cis-to-trans EOF, which is determined by the ratio of the net ionic current cis-to-trans to the total ionic current (also referred to herein as I rel ) is greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, greater than 0.35 or less than -0.35.

[0145] In some embodiments, the cis to trans EOF is from the net ionic current cis to trans across the total ionic current, also referred to as the cis to trans relative net current, 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, at least about 0.99, or greater than about 0.99. In some embodiments, the cis to trans EOF is from the net ionic current cis to trans across the total ionic current, also referred to as the cis to trans relative net current, 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.

[0146] In some embodiments, the cis to trans EOF results from a net ionic current cis to trans across the total ionic current (also referred to as a cis to trans relative net current) ranging from about -0.99 to about 0.99. In certain embodiments, the cis to trans EOF results from a net ionic current cis to trans across the total ionic current (also referred to as a cis to trans relative net current) ranging from about -0.99 to about -0.9, about -0.99 to about -0.8, about -0.99 to about -0.6, about -0.99 to about -0.4, about -0.99 to about -0.2, about -0.99 to about 0, about -0.99 to about 0.2, about -0.99 to about 0.4, about -0.99 to about 0.6, about -0.99 to about 0.8, about -0.99 to about 0.99, about -0.9 to about about -0.8, about -0.9 to about -0.6, about -0.9 to about -0.4, about -0.9 to about -0.2, about -0.9 to 0, about -0.9 to about 0.2, about -0.9 to about 0.4, about -0.9 to about 0.6, about -0.9 to about 0.8, about -0.9 to about 0.99, about -0.8 to about -0.6, about -0.8 to about -0.4, about -0.8 to about -0.2, about -0.8 to about 0, about -0.8 to about 0.2, about -0.8 to about 0.4, about -0.8 to about 0.6, about -0.8 to about 0.8 , about -0.8 to about 0.99, about -0.6 to about -0.4, about -0.6 to about -0.2, about -0.6 to about 0, about -0.6 to about 0.2, about -0.6 to about 0.4, about -0.6 to about 0.6, about -0.6 to about 0.8, about -0.6 to about 0.99, about -0.4 to about -0.2, about -0.4 to about 0, about -0.4 to about 0.2, about -0.4 to about 0.4, about -0.4 to about 0.6, about -0.4 to about 0.8, about -0.4 to about 0.99, about -0.2 to about 0, about -0.2 to About 0.2, about -0.2 to about 0.4, about -0.2 to about 0.6, about -0.2 to about 0.8, about -0.2 to about 0.99, about 0 to about 0.2, about 0 to about 0.4, about 0 to about 0.6, about 0 to about 0.8, about 0 to about 0.99, about 0.2 to about 0.4, about 0.2 to about 0.6, about 0.2 to about 0.8, about 0.2 to about 0.99, about 0.4 to 0.6, about 0.4 to about 0.8, about 0.4 to about 0.99, about 0.6 to about 0.8, about 0.6 to about 0.99, about 0.8 to about 0.99.

[0147] In some embodiments, the cis to trans EOF is from the net ionic current cis to trans through the total ionic current, also referred to as the cis to trans relative net current, 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, about 0.99.

[0148] Suitably, the ion selectivity of the nanopore system P (+) / P (-) It may be greater than 2.0 or less than 0.5, greater than 2.5 or less than 0.4, greater than 3.0 or less than 0.33.

[0149] In some embodiments, the pores may comprise a relatively ion-selective P (+) / P (-) 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 under an applied transmembrane voltage difference. In some embodiments, the pore may comprise a relatively ion-selective P (+) / P (-) 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 under applied transmembrane voltage difference.

[0150] In some embodiments, the pore may comprise a relative ion selectivity P of from about 0.1 to about 5 under an applied voltage difference across the membrane. (+) / P (-) In some embodiments, the pores may form a relatively ion-selective P (+) / P (-)from about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 to about 0.5, about 0.1 to about 1, about 0.1 to about 1.5, about 0.1 to about 2, about 0.1 to about 2.5, about 0.1 to about 3, about 0.1 to about 4, about 0.1 to about 5, about 0.2 to about 0.3, about 0.2 to about 0.4, about 0.2 to about 0.5, about 0.2 to about 1, about 0.2 to about 1.5, about 0.2 to about 2, about 0.2 to about 2.5, about 0.2 to about 3, about 0.2 to about 4, about 0.2 to about 5, about 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 1, about 0.3 to about 1.5, about 0.3 to about 2, about 0.3 to about 2.5, about 0.3 to about 3, about 0.3 to about 4, about 0.3 to about 5, about 0.4 to about 0.5, about 0.4 to about 1, about 0.4 to about 1.5, about 0.4 to about 2, about 0.4 to about 2.5, about 0.4 to about 3, about 0.4 to about 4, about 0.4 to about 5, about 0.5 to about 1, about 0.5 to about 1.5, about 0.5 to about 2, about 0.5 to about 2.5, about 0.5 to about 3, about 0.5 to about 4, about 0.5 to about 5, about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1.5 to about 2, about 1.5 to about 2.5, about 1.5 to about 3, about 1.5 to about 4, about 1.5 to about 5, about 2 to about 2.5, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2.5 to about 3, about 2.5 to about 4, about 2.5 to about 5, about 3 to about 4, about 3 to about 5, or about 4 to about 5.

[0151] In some embodiments, under an applied voltage difference across the membrane, the pore may comprise a relative ion selectivity P of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5. (+) / P (-) .

[0152] Also provided is a nanopore system for transporting an analyte through the nanopore, comprising:

[0153] (a) A membrane having a nanopore therein, said membrane separating a chamber into a cis side and a trans side, wherein an analyte is added to the cis side and translocated to the trans side through the nanopore;

[0154] (b) on the cis side of the chamber, the analyte is captured by a protein translocase that can sequentially bind and translocate the analyte through the nanopore;

[0155] (c) a mechanism that provides a voltage difference between the cis and trans sides of the membrane;

[0156] The nanopore system has a cis-to-trans electroosmotic force (EOF), which is generated by the net ionic current passing through the cis-to-trans direction, so that the analyte is captured in the nanopore, and the nanopore can be located at the top of the nanopore, for example, rel In some embodiments, the transposase controls the translocation caused by a concentration greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, greater than 0.35 or less than -0.35. rel Can be from about -0.4 to about 0.4. In some cases, I rel It can be at least about -0.4, at least about -0.35, at least about -0.3, at least about -0.25, at least about -0.2, at least about -0.15, at least about -0.10, at least about -0.05, at least about 0, at least about 0.05, at least about 0.10, at least about 0.15, at least about 0.20, at least about 0.25, at least about 0.30, at least about 0.35, at least about 0.40, or greater than 0.40. In some cases, I rel At most about 0.40, at most about 0.35, at most about 0.30, at most about 0.25, at most about 0.20, at most about 0.15, at most about 0.10, at most about 0.05, at most about 0, at most about -0.05, at most about -0.10, at most about -0.15, at most about -0.20, at most about -0.25, at most about -0.30, at most about -0.35, at most about -0.40, or less than -0.40. In some cases, I rel It can be about -0.4, about -0.35, about -0.30, about -0.25, about -0.20, about -0.15, about -0.10, about -0.05, about 0, about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, or about 0.40.

[0157] In certain aspects, the nanopore systems of the present disclosure have an ion selectivity P greater than 2.0 or less than 0.5, greater than 2.5 or less than about 0.4, greater than 3.0 or less than 0.33. (+) / P (-) .

[0158] The present disclosure provides a method, system or device that can rely on a dominant cis to trans EOF and a translocase on the cis side of the nanopore.

[0159] In some cases, EPF may be the primary process driving capture and translocation in nanopore systems. Therefore, all previous demonstrations have either used selected model peptides (with a net charge that contributes to EPF) or modified the peptides with highly charged tags (e.g., adding polyanionic tags) so that the EPF force acting on the peptide drives translocation in the cis-to-trans direction.

[0160] For example, WO2021 / 111125 relates to a method for characterizing a target polypeptide by forming a conjugate of a target polypeptide with a highly charged polynucleotide and using a polynucleotide processing protein to control the movement of the conjugate relative to a nanopore. Therefore, in this method, a conjugate comprising a polypeptide portion is extended along a polynucleotide by a polynucleotide processing protein (such as a helicase) to move the nanopore. The present disclosure provides a method utilizing a protein translocase (consistent with electroosmotic flow). In some cases, the peptide may not be coupled to a charged portion.

[0161] In some embodiments, EOF can be used in the nanopore system of the present invention. EOF has been used in nanopore systems before, and it usually acts on the cis-to-trans EPF in the cis-to-cis direction (slowing down the translocation driven by the EPF) or combines with the cis-to-trans EPF in the cis-to-trans direction to help translocation. Some previous studies have shown that neutral or weakly charged small molecules or small polymers in nanopores can be captured 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). The present disclosure provides a method for capturing and / or translocating polymer analytes (e.g., long and / or complex polymers) using a cis-to-trans EOF that can overcome the trans-to-cis EPF. The polymer analyte may include a contour length greater than about the length of the nanopore.

[0162] Furthermore, to date, it has been impossible to control the delivery of analytes into a nanopore using motor proteins (e.g., translocases) in solution on the cis side of the nanopore, as the chains may not be pulled by the force and may clog the nanopore or be ejected back to the cis side of the membrane. This approach may be similar to trying to stuff cooked spaghetti into a plugged hole. For example, see WO 2013 / 123379, which proposes a nanopore system for transporting proteins from cis to trans through a nanopore, wherein a protein translocase is present on at least about one side of the nanopore. However, it is worth noting that WO 2013 / 123379 only provides experimental data for a system containing a translocase on the trans side, which corresponds to Nivala et al. (Nat. biotechnology. 2013; 31(3): 247-250), which reported a nanopore sensor based on α-hemolysin (aHL), in which the ClpX translocase was present in the trans solution. Theoretical examples proposed in WO2013 / 123379 have a protein translocase (ClpX) located on the cis side, requiring the aHL nanopore to be fused with a so-called "docking" protein (ClpP) to allow the translocase to dock non-covalently with the nanopore subunit. In order for this system to work, the axial pore of the translocase and the nanopore can be aligned in the correct direction. That is, ClpX can bind to aHL in such a way that when the protein substrate is captured from the solution and passes through the central cavity of ClpX, it enters directly into the upper cavity of the aHL hemolysin and is eventually forced to pass through the entire nanopore.

[0163] Thus, the present disclosure provides, for the first time, an analyte sensing system in which a translocase acts on the top of a nanopore ("top" refers to the entrance side or face of the nanopore on the membrane side to which the translocase is added), without the need to modify the nanopore with docking proteins or other types of auxiliary elements to transport a diverse and complex library of peptides. Instead, this new system relies on a strong electroosmotic mechanism specifically arranged in the direction of translocation.

[0164] In one embodiment, the present invention provides a method for translocating a target protein through a nanopore, comprising:

[0165] (a) providing a device comprising a nanopore in a membrane that separates a fluid chamber into a cis side and a trans side;

[0166] (b) allowing the protein translocase in solution to capture and form a complex with the target protein to be translocated;

[0167] (c) contacting the translocase target protein complex with the cis side of the nanopore;

[0168] (d) The nanopore system has an ion selectivity P+ / P- greater than about 3.0 or less than 0.3, so that the target protein is trapped in the nanopore and the translocase at the top of the nanopore controls the translocation.

[0169] In some embodiments, the force-generating electroosmotic flow can pull the analyte through the nanopore against any opposing EPF, thereby retaining the protein translocase on top of the nanopore until the analyte is released. Presumably, in one method of the present invention, the analyte is pulled through the nanopore against any opposing EPF, while the protein translocase is retained on top of the nanopore for the duration of the translocation event and then released so that another analyte can bind.

[0170] In another aspect of the present disclosure, the methods provided herein include providing a nanopore system. The nanopore system may include a membrane comprising a nanopore. In some cases, the membrane may separate a fluid chamber into a cis side and a trans side. A non-nucleic acid-based polymer analyte may also be provided. The non-nucleic acid-based polymer analyte may contact a translocase on the cis side of the fluid chamber. The analyte and the translocase may form a complex. The non-nucleic acid-based polymer analyte may be translocated from the cis side to the trans side using electroosmotic forces. The electroosmotic forces may maintain the translocation of the complex at the cis side entrance of the nanopore channel.

[0171] In another aspect of the present disclosure, provided herein is a system comprising: providing a nanopore system. The nanopore system may include a membrane comprising a nanopore. In some cases, the membrane may separate a fluid chamber into a cis side and a trans side. A non-nucleic acid-based polymer analyte may also be provided. The non-nucleic acid-based polymer analyte may contact a translocase on the cis side of the fluid chamber. The analyte and the translocase may form a complex. The non-nucleic acid-based polymer analyte may be translocated from the cis side to the trans side using electroosmotic forces. The electroosmotic forces may couple the translocation of the complex at the cis-side entrance of the nanopore channel. In some embodiments, the nanopore may include additional structures on the cis side of the membrane. In some embodiments, the nanopore may include additional structures on the trans side of the membrane. In some cases, the nanopore may include additional structures on both the cis and trans sides of the membrane. In some cases, the additional structures may include nucleic acid scaffold molecules. In some cases, the nucleic acid scaffold may be a DNA scaffold. In some cases, the nucleic acid scaffold may be an RNA scaffold. In some cases, the additional structures may include a protease. In some cases, the protease can include a serine protease, thrombin, a cysteine protease, a metalloproteinase, chymotrypsin, trypsin, papain, subtilisin or any combination thereof. In some cases, the additional structure can include a docking protein. In some cases, the docking protein can include ClpP, TatA, TatB, TatC, Tim50, Tim23, Tim17 or any combination thereof.

[0172] On the one hand, the translocase-analyte complex is formed in the solution on the cis side of the fluid chamber. Therefore, both operations b and c can be performed in the cis compartment.

[0173] In one embodiment, the net cis-to-trans EOF is achieved by adjusting: (i) pH, (ii) salt type and / or concentration and / or osmotic pressure across the membrane of the nanopore system, (iii) by modification (e.g., genetic engineering) or design of the nanopore charge, or any combination thereof. The dominant EOF is achieved by modifying the nanopore and / or by asymmetric salt distribution between the cis and trans sides of the chamber.

[0174] In some embodiments, the system has a cation-based relative current EOF of at least about 3.0 in the cis to trans direction. There may be a negative applied voltage on the trans side (e.g., where the system comprises a cation-selective (mutated) nanopore).

[0175] On the other hand, before the complex is added to the cis side of the fluid chamber to contact the nanopore, a translocase-analyte complex is formed in the solution during a separate operation. This method allows the use of optimal conditions to combine the translocase and analyte (complex formation). For example, in this premixing (preloading) operation, two components of higher concentrations can be selected, different salt conditions, temperature, pH value, cofactors, etc., rather than the (cis chamber) commonly used in nanopore sensor systems. The premix can be a part of a test kit that can be coupled to the analyte of interest. The premix can be added to the cis chamber in a diluted form.

[0176] In one embodiment, the nanopore is a solid-state nanopore or a biological nanopore having an internal pore constriction with a diameter in the range of 0.5-2 nm. In some cases, the solid-state nanopore can be a nanopore made of a synthetic material. In some cases, the biological nanopore can be a nanopore found in nature.

[0177] In some embodiments, the nanopore can be a biological nanopore. The nanopore can be an α-helical or β-barrel oligomeric pore, forming a toxin or porin. The nanopore is appropriately selected from the group consisting of aerolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, 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 2.

[0178] In some cases, nanopores can be constructed from elements of existing nanopores (e.g., WO 2021 / 101378) or developed de novo using predictive protein engineering software (e.g., Shimizu et al., 2022, Nature Nanotechnology volume 17, pp. 67-75).

[0179] In some embodiments, the nanopore systems of the present invention can utilize any biological nanopore, synthetic nanopore, recombinant nanopore, or any combination thereof. In some cases, a biological nanopore, synthetic nanopore, recombinant nanopore, or any combination thereof can function in the nanopore systems of the present disclosure without modification.

[0180] In one embodiment, the protein translocase is an NTP-driven unfolding enzyme, an AAA+ enzyme. The protein translocase can be selected from the group consisting of ATP-dependent Clp protease ATP-binding subunit clpX (ClpX), ATP-dependent Clp protease ATP-binding subunit clpA (clpA), Pan, LON, VAT, AMA, 854, MBA, SAMP, ATP-dependent Clp protease ATP-binding subunit clpC (ClpC), ATP-dependent Clp protease ATP-binding subunit clpE (ClpE), HsIU, (ClpY), LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, homologs, or analogs thereof. In some cases, translocation may include ClpX and / or ClpA. In some cases, the translocase may comprise a translocase specific for a polypeptide, a protein, a polypeptide, or any combination thereof. In some cases, the translocase may comprise a translocase specific for a nucleic acid molecule.

[0181] In some embodiments, the non-nucleic acid-based polymer analyte can be a target protein or peptide. The analyte can be in its original, unmodified form. For example, a hybrid or modified translocase that has been modified to remove binding specificity can be mixed with the unmodified analyte to achieve binding / loading. This loading method may result in a mixture of analyte-translocase complexes, some of which are loaded at the C-terminus of the analyte and move in the direction from the C-terminus to the N-terminus, and / or some of which are loaded at the N-terminus of the analyte and move in the direction from the N-terminus to the C-terminus. The translocase has binding specificity for either the N-terminus or the C-terminus of the analyte, so the complex has translocases that move in a common direction. The translocase acting on the unmodified analyte can increase capture in the nanopore by unfolding the analyte and generating free ends. Complexes based on loading the unmodified analyte with a translocase can have a single translocase or multiple translocases at random points along the analyte during nanopore capture. When the complex is trapped in the nanopore, the bound translocase can either feed the analyte into the nanopore or pull the analyte out of the nanopore, depending on which end of the analyte is captured and the orientation of the translocase at the top of the nanopore.

[0182] In another aspect of the present disclosure, the methods provided herein include providing a nanopore system. The nanopore system may include a membrane comprising nanopores. In some cases, the membrane may separate the fluid chamber into a cis side and a trans side. A non-nucleic acid-based polymer analyte may also be provided. The non-nucleic acid-based polymer analyte may include a main branch structure. The main branch structure may include a blocking motif, an interception motif, a coupling motif, a recognition motif, a capture motif, or any combination thereof. In some cases, translocation may also be provided. The non-nucleic acid-based polymer analyte may be translocated from the cis side to the trans side of the fluid chamber.

[0183] In another aspect of the present disclosure, provided herein is a system comprising: providing a nanopore system. The nanopore system may include a membrane containing nanopores. In some cases, the membrane may separate the fluid chamber into a cis side and a trans side. Non-nucleic acid-based polymer analytes may also be provided. The non-nucleic acid-based polymer analyte may include a main branch structure. The main branch structure may include a blocking motif, an interception motif, a coupling motif, a recognition motif, a capture motif or any combination thereof. In some cases, the main branch (or tail) structure may include a membrane binding motif. In some cases, translocation may also be provided. The non-nucleic acid-based polymer analyte may be translocated from the cis side of the fluid chamber to the trans side.

[0184] In another aspect of the present disclosure, provided herein is a system comprising: providing a nanopore system. The nanopore system may include a membrane comprising a nanopore. In some cases, the membrane may separate a fluid chamber into a cis side and a trans side. A non-nucleic acid-based polymer analyte may also be provided. In some cases, a first solution may be provided on the cis side. In some cases, a second solution may be provided on the trans side. The first solution and the second solution may be configured to translocate the non-nucleic acid-based polymer analyte. The system may also include a controller. In some cases, the controller may be operatively coupled to the fluid chamber and the nanopore. The controller may be configured to detect one or more signals associated with at least one feature of the main branch structure. The controller may be configured to detect one of a plurality of signals associated with at least one feature of the main branch structure, as well as one or more signals associated with at least one feature of the non-nucleic acid-based polymer analyte. In some cases, the one or more signals may be detected during translocation of the non-nucleic acid-based polymer analyte. In some cases, the one or more signals may be detected after translocation of the non-nucleic acid-based polymer analyte. In some cases, one or more signals can be detected during or after the non-nucleic acid based polymer analyte translocation. In some cases, the main branch structure can be coupled to the non-nucleic acid based polymer analyte. In some cases, the non-nucleic acid based polymer analyte can be transported by nanopore using nanopore. In some cases, the main branch structure can include a blocking motif, an interception motif, a coupling motif, a recognition motif, a capture motif or any combination thereof.

[0185] In other embodiments of the present invention, the analyte includes a "main branch" and / or "tail" extension located at the end of the protein. In certain aspects of the present disclosure, the analyte includes a main branch structure that can preload and / or optionally block a protein translocase. For example, preloading is performed outside the (cis chamber) of the nanopore system, and the analyte-translocase complex is then introduced into the cis chamber. In another embodiment, the analyte can be coupled to a main branch structure that can load and / or optionally block a protein translocase when mixed together in the cis chamber of the system. In some embodiments, the analyte can be coupled to a main branch structure that can load and / or optionally block a protein translocase when the two are mixed together in the translocase chamber of the system. The main branch structure may be an exogenous sequence. It includes (i) a recognition motif for a protein translocase to guide binding to a specific location and / or enable more efficient binding and loading. It may also include one or more of the following elements: (ii) a capture motif; (iii) a blocking motif; (iv) an interception motif; (v) a coupling motif.

[0186] In another embodiment, the translocase is coupled to the nanopore. In the disclosed system, the translocase may not be coupled to the top of the nanopore to optimally deliver the analyte into the nanopore. Instead, the disclosed strong cis-to-trans EOF causes the portion of analyte extruded from the translocase to be captured in the nanopore and translocated, which in turn pulls the translocase to the top of the pore, where it will continue to control the movement of the extruded analyte. In this embodiment, because the extruded analyte is close to the nanopore entrance, the analyte may not have a stagnation or capture motif.

[0187] The method according to the present disclosure may also include measuring the change in ionic current caused by the translocation of the analyte through the nanopore. The current change of the following states can be measured: (i) open channel, (ii) analyte captured by the nanopore, and / or (iii) analyte passing through the nanopore from (ii). For example, the method of measuring the change in ionic current includes detecting the difference between states (i), (ii) and (iii). In a specific aspect, the measurement includes measuring the difference caused by the amino acid composition or structure of the analyte passing through the nanopore during state (iii). The method appropriately includes performing one or more measurements on the characteristics of the analyte. One or more measurements can be characteristics of one, two, three, four or five or more features of the analyte. One or more features are selected from (i) the length of the analyte; (ii) the characteristics of the analyte; (iii) the sequence of the analyte; (iv) the secondary or tertiary structure of the analyte; (v) whether the analyte is modified. Any combination of (i) to (v) can be measured according to the present invention.

[0188] Further embodiments of the present disclosure relate to a nanopore system for transporting an analyte through a nanopore, comprising:

[0189] (a) A membrane having a nanopore therein, the membrane separating a chamber into a cis side and a trans side, wherein an analyte is added to the cis side and translocated to the trans side through the nanopore; (b) On the cis side of the chamber, the analyte is captured by a protein translocase, which can sequentially bind and translocate the analyte through the nanopore; and (c) an element that provides a voltage difference between the cis side and the trans side of the membrane. In some cases, the element in (c) can include a pair of electrodes.

[0190] In some embodiments, the nanopore system is further characterized by a cis-to-trans electroosmotic force (EOF) generated by a net cis-to-trans ionic current, whereby the analyte is trapped in the nanopore, and a translocase is present at the top of the nanopore to control translocation. The nanopore system has a cis-to-trans EOF due to a ratio of the net ionic current cis-to-trans to the total ionic current greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, greater than 0.35 or less than -0.35.

[0191] In certain aspects, the ion selectivity of the nanopore system is (+) / P (-) Greater than 2.0 or less than 0.5, greater than 2.5 or less than 0.4, greater than 3.0 or less than 0.33, or even greater than 3.5 or less than 0.2.

[0192] In some embodiments, the voltage differential can be provided in various ways, such as by a single circuit that both applies the voltage and measures the current, or by a system comprising a first circuit for applying the voltage and a second circuit for measuring the current. Asymmetric salts can also generate a voltage differential across the membrane. For example, the device includes circuits for applying a voltage between the cis and trans sides and for measuring the ionic current flowing through the nanopore. See Figure 1. A negative voltage is applied to the trans side.

[0193] In some embodiments, the system may further include methods for measuring a signal based on the ionic current flowing through the nanopore during a translocation period. These measurement mechanisms are configured to detect changes in the signal that reflect characteristics of the analyte during translocation. In some embodiments, the analyte measured may be a protein. In some cases, the properties of the protein measured may include the amino acid sequence of the protein, one or more post-translational modifications of the protein, amino acid mutations in the protein sequence, domain structure of the protein, length of the protein, net charge of the protein, or conformation of the protein. In some embodiments, the analyte measured may be a nucleic acid molecule. In some cases, the properties of the nucleic acid molecule measured may include the nucleotide sequence of the nucleic acid molecule, nucleotide mutations in the nucleic acid molecule sequence, methylation of the nucleic acid molecule, acetylation of the nucleic acid molecule, length of the nucleic acid molecule, net charge of the nucleic acid molecule, or conformation of the nucleic acid molecule. In some embodiments, the analyte measured may be an oligosaccharide. In some cases, the properties of the oligosaccharide measured may include the sequence of the oligosaccharide, the length of the oligosaccharide, the net charge of the oligosaccharide, the presence or absence of a lipid conjugate, the presence or absence of a peptide conjugate, or the structure of the oligosaccharide. In some embodiments, the analyte measured may be a lipid molecule. In some cases, the property of the lipid measured can include the length of the lipid, the net charge of the lipid, or the structure of the lipid.

[0194] The system can employ alternative mechanisms to measure the voltage-current properties of the nanopore system, such as those that use ion flux fluorescence probes or field-effect transistor systems rather than measuring voltage changes. However, there are other suitable detection methods, such as tunneling, surface-enhanced Raman, plasmonics, and other spectroscopic methods that do not measure ionic currents but instead directly measure the properties of the analyte in the nanopore.

[0195] Also provided is an analytical device comprising one or more nanopore systems disclosed herein, for example in the form of an array.

[0196] Another embodiment relates to the use of a method, nanopore system, or device according to the present disclosure for characterizing at least one characteristic of an analyte for detecting and analyzing one or more analytes at the single molecule level. In one aspect, the use comprises characterizing the amino acid sequence of a native analyte or a mixture of different native analytes.

[0197] In some embodiments, methods related to analysis of analytes are provided.

[0198] In some embodiments, methods related to analysis of analytes are provided.

[0199] In some embodiments, changes in ionic current can be measured as the analyte translocates through the nanopore. In some cases, changes in ionic current can be measured using a voltage-based chip. In some cases, a voltage-based chip can measure changes in voltage and / or current across the nanopore. In some cases, the voltage-based chip can be a counter electrode (e.g., an electrode close to the membrane / nanopore that measures the voltage across the nanopore).

[0200] Characterization methods can include measuring ion flow through the pore, typically by measuring current. Alternatively, ion flow through the pore can be measured optically, as disclosed in Heron et al.; J. Am. Chem. Soc. 9 Vol. 131 No. 5, 2009. Thus, the device can also include circuitry capable of applying a potential and measuring the electrical signal across the membrane and pore. Characterization methods can be performed using patch clamping or voltage clamping. Characterization methods may involve the use of voltage clamping.

[0201] In certain embodiments, analyte includes polymer analyte.Described analyte may include polymer analyte based on nucleic acid or non-nucleic acid based polymer analyte.Analyte can be synthetic, semi-synthetic or biological origin.For example, synthetic analyte may include analyte constructed by non-biological chemical process, such as polyethylene glycol (PEG) or synthetic DNA molecule of construction.For example, synthetic analyte may include analyte constructed by non-biological chemical process, such as polyethylene glycol (PEG), synthetic protein peptide or synthetic DNA molecule of construction.Biological analyte may include analyte produced by biological process, such as protein produced by cell or by system (such as enzyme in vitro translation system) adopting cell (or cell-derived) component.Biological analyte may include analyte produced by biological process, such as protein produced by cell.Semi-synthetic analyte may include part produced by biological and non-biological source, such as, protein produced by the organism that is bonded to PEG molecule. Possible electrical measurements include current measurement, impedance measurement, tunneling, electron tunneling measurement (Ivanov AP et al., NanoLett. 2011 Jan 12;(1)(1):279-85), FET measurement (International Appl WO 2005 / 124888), voltage FET measurement, or any combination thereof. In some embodiments, the signal can be electron tunneling through the solid-state nanopore or voltage FET measurement through the solid-state nanopore.

[0202] Characterization methods can include measuring ion flow through the pore by measuring current. Alternatively, ion flow through the pore can be measured optically, as disclosed in Heron et al., J. Am. Chem. Soc. 9, Vol. 131, No. 5, 2009. Thus, the device can also include circuitry capable of applying a potential and measuring the electrical signal across the membrane and pore. Characterization methods can be performed using patch clamping or voltage clamping. Preferably, the characterization method involves the use of voltage clamping.

[0203] The characterization method can be performed on arrays of nanopores or pores, wherein each array comprises 128, 256, 512, 1,024, 2,000, 3,000, 4,000, 6,000, 10,000, 12,000, 15,000 or more nanopores or pores.

[0204] Characterization methods may include measuring the current flowing through the pore. This method is typically performed by applying a voltage across the membrane and the pore. The voltage used is typically between +2V and -2V, typically between -400mV and +400mV. The voltage used is preferably within a range with a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, 20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. The voltage used is preferably within the range of 20mV to 240mV, most preferably within the range of 120mV to 220mV. By using increased applied potentials, the ability of the pore to differentiate between different characteristics of the analyte can be increased.

[0205] In certain embodiments, analyte comprises polymer analyte.Described analyte may comprise polymer analyte based on nucleic acid or non-nucleic acid based polymer analyte.Analyte can be synthetic, semi-synthetic or biological origin.For example, synthetic analyte may comprise analyte constructed by non-biological chemical process, for example polyethylene glycol (PEG) or synthetic DNA molecule of construction.Biological analyte may comprise analyte produced by biological process, for example, protein produced by cell.Semi-synthetic analyte may comprise part produced by biological and non-biological source, for example, protein produced by the organism that is bonded to PEG molecule.

[0206] In certain embodiments, analyte comprises protein (such as polypeptide) or peptide.Protein or peptide may comprise folded state, unfolded state or its intermediate state (such as, partially unfolded state).Folded state comprises the state of protein or peptide, wherein polymer is in low energy state, makes protein or peptide maintain two-dimensional or three-dimensional structure.This low energy state can be based on the amino acid interaction of peptide or protein.Unfolded state can comprise the state of protein or peptide, wherein polymer is in high energy state, makes protein or peptide not maintain two-dimensional or three-dimensional structure.The intermediate state between folded state and unfolded state can be the energy state that part or part of peptide or protein can maintain two-dimensional or three-dimensional structure, and other parts of peptide or protein can not maintain two-dimensional or three-dimensional structure.In certain embodiments, protein (such as polypeptide) can comprise folded protein structure.In certain embodiments, peptide can comprise linear structure.In some cases, peptide can comprise a part of protein.

[0207] In some embodiments, the analyte may comprise a nucleic acid molecule. In some cases, the nucleic acid molecule may be a DNA molecule. In some cases, the DNA molecule may be genomic DNA, mitochondrial DNA, or any combination thereof. In some cases, the nucleic acid molecule may be an RNA molecule. In some cases, the RNA molecule may be a translocation RNA (tRNA), a messenger RNA (mRNA), a ribosomal RNA (rRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a piwi-interacting RNA (piRNA), a small interfering RNA (siRNA), a microRNA (miRNA), or any combination thereof. In some embodiments, the analyte may comprise a lipid. In some cases, the lipid may be oleic acid, linoleic acid, palmitic acid, docosahexaenoic acid, eicosapentaenoic acid, or any combination thereof. In some embodiments, the analyte may comprise an oligosaccharide. In some cases, the oligosaccharide may be a glycoprotein, inulin, lactose, mannose, sucrose, oligofructose, a monosaccharide, a carbohydrate, maltose, oligogalactose, a polysaccharide, chitosan, a pentasaccharide, or any combination thereof. In some embodiments, the analyte may comprise a polysaccharide. In some cases, the polysaccharide can be starch, glycogen, galactogen, inulin, arabinoxylan, cellulose, chitin, pectin, or any combination thereof.

[0208] In certain embodiments, analyte can comprise non-nucleic acid based polymer analyte.In certain embodiments, a part of non-nucleic acid based polymer analyte can comprise nucleic acid molecule.In some cases, the part of described non-nucleic acid based polymer analyte can be 0% to about 100% of described non-nucleic acid based polymer analyte.In some cases, part non-nucleic acid based 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% non-nucleic acid based polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte can be 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%, or up to about 0% of the non-nucleic acid polymer analyte. In some 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.

[0209] In certain embodiments, a part for non-nucleic acid based polymer analyte can include oligosaccharide molecules.In some cases, the part of described non-nucleic acid polymer analyte can be 0% to about 100% of described non-nucleic acid polymer analyte.In some cases, part 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% non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte can be 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%, or up to about 0% of the non-nucleic acid polymer analyte. In some 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.

[0210] The analyte may include a contour length. The analyte may include a linear length. The linear length may be the length of the analyte in the unfolded state. The analyte in the unfolded state may or may not contain secondary structural elements. Secondary structural elements may include α-helices, β-helices, coils, or β-sheets. The helices may be left-handed or right-handed. The analyte in the unfolded state may be fully unfolded or partially unfolded (e.g., an intermediate state of unfolding). The contour length may be the length of the polymer analyte when its two ends are fully extended relative to each other. In some embodiments, the analyte may include a structured portion, an unstructured portion, a denatured portion, a partially denatured portion, or a combination thereof. In some embodiments, the analyte may include about 1 to about 6 ends. In some embodiments, the analyte may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or at least about 6 ends or more. In some embodiments, the analyte may include up to about 6 ends, up to about 5 ends, up to about 4 ends, up to about 3 ends, up to about 2 ends, up to about 1 end, or less. In some embodiments, the analyte can include about 1 end, about 2 ends, about 3 ends, about 4 ends, about 5 ends, or 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.

[0211] The analyte may comprise a repeating unit. The unit may comprise a peptide unit, a carbohydrate unit, a lipid unit, a nucleotide unit, a water-soluble plastic monomer, or a combination thereof. The analyte may comprise a polypeptide, a polysaccharide, a nucleic acid, a water-soluble plastic, or a combination thereof. In some embodiments, the analyte may comprise a charge. The charge may be positive or negative. The charge may be evenly or unevenly distributed over the analyte. In some embodiments, the charge may be derived from an amino acid residue. The amino acid residue may be natural or mutated. In some cases, the mutated amino acid residue may comprise one or more additional chemical groups compared to a natural amino acid. In some embodiments, the analyte may comprise a peptide. The peptide may be composed of a polypeptide or a protein. The protein may be a full-length protein or a truncated protein. A truncated protein may be a protein that is shorter than when the protein was originally formed. For example, a protein may be shortened due to cleavage (e.g., by a peptidase) or degradation (e.g., due to acidic or alkaline conditions). The protein may comprise a sequence that is a natural protein sequence or a modified protein sequence. The sequence may be modified by mutation, deletion, or insertion of a sequence. A sequence may be a combination of multiple sequences. For example, a first native sequence can be appended to or inserted into a second native sequence to form a third sequence that is a combination of the first and second sequences.

[0212] In some embodiments, the analyte comprises a protein. In some embodiments, the analyte comprises a peptide or polypeptide (e.g., a protein). In some embodiments, a peptide, polypeptide, or protein can be targeted. The target analyte can be a target peptide, target polypeptide, or target protein. The target analyte can be an analyte that is bound to a translocase. The analyte can be bound to a translocase to form a complex (e.g., a translocase-analyte complex).

[0213] In some embodiments, the analyte and the translocase can be located on the cis side of the membrane. In some cases, the analyte and the translocase can form a complex on the cis side of the membrane. In some embodiments, the analyte and the translocase can form a complex in a nanopore system.

[0214] In some embodiments, the analyte and translocase can be located on the trans side of the membrane. In some cases, the analyte and translocase can form a complex on the trans side of the membrane. In some embodiments, the analyte can be in contact with the translocase outside the nanopore system. In some cases, the analyte and translocase can form a complex in a container separate from the nanopore system. In some cases, the analyte and translocase complex can be added to the nanopore system.

[0215] In some embodiments, the analyte can be a target analyte. In some cases, the target analyte can be a protein. In some cases, the target analyte can be a polypeptide. In some cases, the target analyte can be a peptide. In some cases, the target analyte can be a nucleic acid molecule. In some cases, the nucleic acid molecule can be an RNA molecule. In some cases, the nucleic acid molecule can be a DNA molecule. In some cases, the target analyte can be a polysaccharide. Non-limiting examples of polysaccharides include cellulose, chitin, pullulan, chitosan, dextran, hemi-glycan, galactose, lentinan, amylose, amylopectin, starch, hemicellulose, alginic acid, glycosaminoglycans, gellan gum, carrageenan, glycogen, pectin, dextran, inulin, homopolysaccharides, fucoidan, and polydextrose. In some cases, the target analyte can be an oligosaccharide. The non-limiting examples of oligosaccharides include lactose, maltose, sucrose, cellobiose, black triose, maltose, maltose, maltose, ketose, black tetraose, maltose, lychee sugar, mold erythralose, sesame sugar, stachyose, pentasaccharide, oligofructose, galacto-oligosaccharide, hexaose and heptasaccharide. In certain embodiments, the target analyte can be a glycosylated substance. In some cases, glycosylation can be an oligosaccharide that is N-connected. In some cases, glycosylation can be an oligosaccharide that is O-connected.

[0216] In some embodiments, electroosmotic flow (also known as electroosmotic force, EOF) acts across the membrane in a cis-to-trans direction or a trans-to-cis direction. Electroosmotic flow can be a flow generated by a net flow of ions moving along a surface induced by an applied potential (e.g., an applied voltage potential). For example, a charged surface can form a static layer composed of mobile ions with opposite charges. Under the action of an applied potential, the charged mobile ions may be induced to move in the direction of higher potential, or in the direction of lower potential if negative. The flow of charged ions creates resistance to the surrounding solvent (e.g., water) molecules, thereby generating a net flow that exerts a force on the surrounding charged and neutral molecules. For example, in a negatively charged nanopore cavity, electroosmotic flow can be caused by a net flow of positive ions in a cis-to-trans direction (e.g., due to the lower potential on the trans side) causing the surrounding water to flow from cis to trans and exert a force on the surrounding molecules. The amount of ion flux and the corresponding magnitude of the electroosmotic flux can be affected by parameters including the ion concentration difference across the membrane, the potential difference, the net charge of the nanopore lumen, the geometry of the nanopore lumen, or any combination thereof.

[0217] In some embodiments, electroosmotic flow can be a flow caused by one or more constrictions present in a nanopore channel. For example, a constriction in a nanopore can affect the flow of certain ions (e.g., larger hydrated ions) more than other ions (e.g., smaller hydrated ions). In some embodiments, electroosmotic flow can be a flow resulting from a net flow of mobile ions along a surface induced by an applied potential and one or more constrictions present in a nanopore channel.

[0218] In some embodiments, electroosmotic flow can be generated or modified by the difference between the solution on the cis side of the membrane and the solution on the trans side of the membrane. In some cases, the solution on the cis side of the membrane can be a first solution. In some cases, the solution on the trans side of the membrane can be a second solution. This difference can be a difference in the concentration of a molecule, including ions, electrolytes, or permeate.

[0219] In some embodiments, the difference between the solutions can be salt asymmetry or ionic asymmetry, where one side of the membrane (e.g., the cis side) contains a different concentration of ions than the other side (e.g., the trans side). Ionic asymmetry can affect the ionic current across the membrane, as described by the Goldman-Hodgkin-Katz equation.

[0220]

[0221] where ionic current (I(S)) is the ionic species S across the membrane as a function of applied potential (Vm): (S) is the membrane permeability of ionic species S, z s is the valence state of the ion, F is the Faraday constant, R is the gas constant, T is the temperature, [S] 顺式and [S] 反式 are the cis and trans concentrations of an ion species, S, respectively. Since the difference in cis and trans ion concentrations affects ion flux, the combined flux of different ion species affects the electroosmotic force when ions flow in different directions across the membrane. This can be used to enhance or weaken the electroosmotic force by minimizing or maximizing the contribution of the ionic current of species S to the net ion flux by varying the ionic concentrations between the cis and trans forms.

[0222] The difference in molecular concentrations on both sides of the membrane can change the electroosmotic flux by providing a competitive or auxiliary osmotic flux. The concentration difference across the membrane can produce an osmotic gradient, in which the solvent (e.g., water) can 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 water flow in the same or different direction as the electroosmotic force. For example, the high ion concentration on the cis side relative to the trans side can produce an osmotic gradient that competes with the cis-to-trans electroosmotic force because the osmotic gradient can drive water to flow from the trans-to-cis direction. Ion concentrations may support cis-to-trans electroosmotic flow even if they also provide an osmotic gradient.

[0223] In certain embodiments, EOF can be produced by the asymmetric salt distribution between the cis side and the trans side of the membrane.In some cases, the concentration of one or more salts of the membrane cis side may be different from the concentration of one or more salts of the membrane trans side.In some cases, the concentration of one or more salts of the membrane cis side may be higher than the concentration of one or more salts of the membrane trans side.In some cases, the concentration of one or more salts of the membrane cis side may be lower than the concentration of one or more salts of the membrane trans side.In some cases, the concentration of one or more salts of the membrane trans side may be higher than the concentration of one or more salts of the membrane cis side.In some cases, the concentration of one or more salts of the membrane trans side may be lower than the concentration of one or more salts of the membrane cis side.

[0224] 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 nanomolar. 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, to about 100 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 the 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, or at most about 250 nM. nM, up to about 150 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 45 nM, up to about 40 nM, up to about 35 nM, up to about 30 nM, up to about 25 nM, up to about 20 nM, up to about 15 nM, up to about 10 nM, up to about 5 nM, up to 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 105 nM, about 106 nM, about 107 nM, about 108 nM, about 109 nM, about 110 nM, about 111 nM, about 112 nM, about 113 nM, about 114 nM, about 115 nM, about 116 nM, about 117 nM, about 118 nM, about 119 nM, about 120 nM, about 121 nM, about 122 nM, about 123 nM, about 124 nM, about 125 nM, about 126 nM, about 127 nM, about 128 nM, about 129 nM, about 130 nM, about 131 nM, about 132 nM, about 133 nM, about 134 nM 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.

[0225] In some embodiments, the salt, ion, osmotic agent or electrolyte concentration 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 salt, ion, osmotic agent or electrolyte concentration of one 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.

[0226] In certain embodiments, the salt, ion, osmotic agent, cis side or electrolyte concentration can range from about 0.01 M to 5 M. In certain embodiments, the salt, ion, osmotic agent, cis side or electrolyte concentration can range from about 0.01 M to 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to 0.5 M, about 0.1 M to about 1M, about 0.1M to about 1.5M, about 0.1M to about 2M, about 0.1M to about 2.5M, about 0.1M to about 3M, about 0.1M to about 3.5M, about 0.1M to about 4M, about 0.1m to 4.5M, about 0.1M to about 5M, about 0.5m to 1M, about 0.5M to about 1.5M, about 0.5M to about 2M, about 0.5M to about 2.5M, about 0.5M to about 3M, about 0.5M to about 3.5M, about 0.5M to about 4M, about 0.5M to about 4.5M, about 0. .5M to about 5M, about 1 meter to 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2M, about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3 .5M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M or about 4.5M to about 5M.

[0227] In certain embodiments, the salt, ion, osmotic agent or electrolyte concentration 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, about 5 M.

[0228] 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 nanomolar. 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, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more salts on the 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, to at least about 100 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 the 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. M, up to about 150 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 45 nM, up to about 40 nM, up to about 35 nM, up to about 30 nM, up to about 25 nM, up to about 20 nM, up to about 15 nM, up to about 10 nM, up to about 5 nM, up to 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 105 nM, about 106 nM, about 107 nM, about 108 nM, about 109 nM, about 110 nM, about 111 nM, about 112 nM, about 113 nM, about 114 nM, about 115 nM, about 116 nM, about 117 nM, about 118 nM, about 119 nM, about 120 nM, about 121 nM, about 122 nM, about 123 nM, about 124 nM, about 125 nM, about 126 nM, about 127 nM, about 128 nM, about 129 nM, about 130 nM, about 131 nM, about 132 nM, about 133 nM, about 134 nM 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, about 1,000 nM.

[0229] In some embodiments, the salt, ion, osmotic agent or electrolyte concentration 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 of a salt, the concentration of ions, osmotic agents, or electrolytes on the trans side can be at most about 5 M, at most about 4.5, at most about 4 M, at most about 3.5, 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.

[0230] In certain embodiments, the salt, ion, osmotic agent or electrolyte concentration on the trans side can be from about 0.01 M to about 5 M. In certain embodiments, the salt, ion, osmotic agent or electrolyte concentration on the cis side can be from about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to about 0.5 M, about 0.1 M to about about 1M, about 0.1M to about 1.5M, about 0.1M to about 2M, about 0.1M to about 2.5M, about 0.1M to about 3M, 0.1M to about 3.5M, about 0.1M to about 4M, about 0.1M to about 4.5M, about 0.1M to about 5M, about 0.5M to about 1M, about 0.5 meters to 1.5M, about 0.5M to about 2M, about 0.5M to about 2.5M, about 0.5M to about 3M, about 0.5M to about 3.5M, about 0.5M to about 4M, about 0.5M to about 4.5M, about 0. .5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2M, about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3.5M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M or about 4.5M to 5M.

[0231] In certain embodiments, the salt, ion, osmotic agent or electrolyte concentration 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, about 5 M.

[0232] In some embodiments, the difference in salt, ion, or electrolyte concentration between the cis and trans forms 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, at least about 0.50, 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 3.5 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 difference in salt, ion, or electrolyte concentration between the cis and trans forms may 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.

[0233] In some embodiments, the concentration of the salt, ion, or electrolyte that differs between the cis and trans forms can range from about 0.01 M to about 5 M. In some embodiments, the concentration of the salt, ion, or electrolyte that differs between the cis and trans forms can range from about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to about 0.5 M, about 0.1 M to about 1M, about 0.1M to about 1.5M, about 0.1M to about 2M, about 0.1M to about 2.5M, about 0.1M to about 3M, about 0.1M to about 3.5M, about 0.1M to about 4M, about 0.1M to about 4.5M, about 0.1M to about 5M, about 0.5M to about 1M, about 0.5M to about 1.5M, about 0.5M to about 2M, about 0.5M to about 2.5M, about 0.5 meters to about 3M, about 0.5M to about 3.5M, about 0.5M to about 4M, about 0.5M to about 4.5M, about 0.5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2M, about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3.5M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M or about 4.5M to about 5M.

[0234] In some embodiments, the different salt, ion or electrolyte concentrations between the cis and trans forms 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, about 5 M.

[0235] In some embodiments, the one or more salts can include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, tetraaminocopper sulfate, zinc chloride monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, potassium glutamate, sodium ferrocyanide, sodium ferrocyanide, potassium ferrocyanide, potassium ferrocyanide, or any combination thereof.

[0236] In some embodiments, the one or more salts on the cis side of the membrane may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc chloride monohydrate, sodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof. In some embodiments, the one or more salts on the trans side of the membrane may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc chloride monohydrate, sodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof.

[0237] In certain embodiments, the one or more salts on the cis side of the film can be identical with the one or more salts on the trans side of the film. In certain embodiments, the one or more salts on the cis side of the film can be salts of the same type as the trans side of the film. In certain embodiments, the one or more salts on the cis side of the film can be different from the one or more salts on ...

[0238] In certain embodiments, one or more salts can comprise about one salt to about ten salts. In some cases, one or more salts can comprise at least about one salt, at least about two salts, at least about three salts, at least about four salts, at least about five salts, at least about six salts, at least about seven salts, at least about eight salts, at least about nine salts, at least about ten salts or more than ten salts. In some cases, one or more salts can only account for about ten salts, at most about nine salts, at most about eight salts, at most about seven salts, at most about six salts, at most about five salts, at most about four salts, at most about three salts, at most about two salts, at most about a salt or less than a salt. In some cases, one or more salts can comprise one salt, about two salts, about three salts, about four salts, about five salts, about six salts, about seven salts, about eight salts, about nine salts or about ten salts.

[0239] In certain embodiments, the one or more salts on the cis side membrane can be salts 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 existing on the cis side and trans side of the membrane can exist with identical concentrations. In some cases, the same type of salts existing on the cis side and trans side of the membrane can exist with different concentrations.

[0240] In certain embodiments, the one or more salts on the cis side membrane can be salt types that are different from the one or more salts on the trans side of the membrane. In certain embodiments, the dissimilar salts present in the cis side of the membrane and the trans side can exist with identical concentrations. In some cases, dissimilar salts can exist with different concentrations on the cis side of the membrane and the trans side.

[0241] 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% higher than the concentration of one or more salts on the trans side of the membrane. %, 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 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 510%, about 510% to about 520%, about 520% to about 530%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 600%, about 610% to about 620%, about 630% to about 640%, about 650% to about 660%, about 670% to about 680%, about 690% to about 700%, about 710% to about 720%, about 50% 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%.

[0242] In some cases, the concentration of the 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 201%, at least about 210%, at least about 211%, at least about 212%, at least about 213%, at least about 214%, at least about 215%, at least about 216%, at least about 217%, at least about 218%, at least about 220%, at least about 221%, at least about 222%, at least about 223%, at least about 224%, at least about 225%, at least about 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 360%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, 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.

[0243] In some cases, the concentration of the one or more salts on the cis side of the membrane is 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 242%, at most about 263%, at most about 274%, at most about 275%, at most about 286%, at most about 297%, at most about 308%, at most about 319%, at most about 320%, at most about 331%, at most about 340%, at most about 351%, at most about 361%, at most about 370%, at most about 380%, at most about 390%, at most about 390%, at most about 3 %, 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%.

[0244] In some cases, the concentration of the one or more salts on the cis side of the membrane can be greater than 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 201%, 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%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 0%, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0245] In some embodiments, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75% lower than the concentration of one or more salts on the trans side of the membrane. , 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 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 510%, about 510% to about 520%, about 520% to about 530%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 600%, about 610% to about 620%, about 630% to about 640%, about 650% to about 660%, about 670% to about 680%, about 690% to about 700%, about 710% to about 720%, about 50% 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%.

[0246] In some cases, the concentration of the 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%, 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 30%, 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.

[0247] In some cases, the concentration of the 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 240%, 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%.

[0248] In some cases, the concentration of the 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 201%, 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 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about %, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0249] In some embodiments, the concentration of the one or more salts on the trans side of the membrane can be about 0.1% to about 500% higher than the concentration of the one or more salts on the cis side of the membrane. In some cases, the concentration of the 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% higher than the concentration of the one or more salts on the cis side of the membrane. %, 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 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 510%, about 510% to about 520%, about 520% to about 530%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 600%, about 610% to about 620%, about 630% to about 640%, about 650% to about 660%, about 670% to about 680%, about 690% to about 700%, about 710% to about 720%, about 50% 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%.

[0250] In some cases, the concentration of the 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 201%, 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 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 360%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, 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.

[0251] In some cases, the concentration of the 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 2410%, up to about 2620%, up to about 2640%, up to about 2650%, up to about 2700%, up to about 2750%, up to about 27600%, up to about 27700%, up to about 27800%, up to about 27900%, up to about 28000%, up to about 29900%, up to about 30100%, up to about 31100%, up to about 32100%, up to about 33000%, up to about 34000%, up to about 35000%, up to about 36000 %, 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%.

[0252] In some cases, the concentration of the 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 500%, about 550%, about %, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0253] In some embodiments, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75% lower than the concentration of one or more salts on the trans side of the membrane. , 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 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 510%, about 510% to about 520%, about 520% to about 530%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 600%, about 610% to about 620%, about 630% to about 640%, about 650% to about 660%, about 670% to about 680%, about 690% to about 700%, about 710% to about 720%, about 50% 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%.

[0254] In some cases, the concentration of the 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%, 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 30%, 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.

[0255] In some cases, the concentration of the 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 2410%, up to about 2620%, up to about 2640%, up to about 2650%, up to about 2700%, up to about 2710%, up to about 2720%, up to about 2730%, up to about 2740%, up to about 2750%, up to about 2760%, up to about 2770%, up to about 2780%, up to about 2790%, up to about 2800%, up to about 2810%, up to about 2830%, up to about 2840%, up to about 2860%, up to about 2870%, up to about 2880%, up to about 2890%, up to about 2910%, up to about 2920%, up to about 2930%, up to about 2940%, up to about 2950%, up to about 2960%, up to about 2970%, up to about %, 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%.

[0256] In some cases, the concentration of the 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 201%, 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 500%, about 550%, about %, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0257] In some embodiments, EOF can be produced by an asymmetric salt distribution. Asymmetric salt distribution can occur when the concentration of one or more salts on the cis side of the membrane is greater than about 1% or less than the concentration of one or more salts on the trans side of the membrane.

[0258] Alternatively, EOF can be produced by a symmetrical salt distribution between the cis and trans sides of the membrane. A symmetrical salt distribution may occur when the concentration of one or more salts on the cis side of the membrane is the same as the concentration of one or more salts on the trans side of the membrane. In some embodiments, the concentration of one or more salts on the cis side of the membrane may be the same as the concentration of one or more salts on the trans side of the membrane.

[0259] In some embodiments, EOF can be produced by an asymmetric ion distribution between the cis side and the trans side of the membrane. Asymmetric ion distribution may occur when the concentration of one or more ions on the cis side of the membrane is greater than about 1% 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 about 1% or less than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be higher than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be lower than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the trans side of the membrane may be higher than the concentration of one or more ions on the cis side of the membrane. In some cases, the concentration of one or more ions on the trans side of the membrane may be lower than the concentration of one or more ions on the cis side of the membrane.

[0260] In some cases, the concentration of one or more ions on the cis side of the membrane may 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 may be between about 1 nM and about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more ions on the cis side of the membrane may 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, to about 1,000 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 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, or at most about 350 nM. nM, up to about 150 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 45 nM, up to about 40 nM, up to about 35 nM, up to about 30 nM, up to about 25 nM, up to about 20 nM, up to about 15 nM, up to about 10 nM, up to about 5 nM, up to about 1 nM or less than 1 nM.In some cases, the concentration of salt in the cis 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, about 1,000 nM.

[0261] 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 nanomolar. 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, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more ions on the 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 10 ... 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 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, or at most about 350 nM. nM, up to about 150 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 45 nM, up to about 40 nM, up to about 35 nM, up to about 30 nM, up to about 25 nM, up to about 20 nM, up to about 15 nM, up to about 10 nM, up to about 5 nM, up to 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 90 nM, about 95 nM, about 96 nM, about 97 nM, about 98 nM, about 99 nM, about 100 nM, about 101 nM, about 102 nM, about 103 nM, about 104 nM, about 105 nM, about 106 nM, about 107 nM, about 108 nM, about 109 nM, about 110 nM, about 111 nM, about 112 nM, about 113 nM, about 114 nM, about 115 nM, about 116 nM, about 117 nM, about 118 nM, about 119 nM, about 120 nM, about 121 nM, about 122 nM, about 123 nM, about 124 nM 5nM, about 100nM, about 150nM, about 200nM, about 250nM, about 300nM, about 350nM, about 400nM, about 450nM, about 500nM, about 550nM, about 600nM, about 650nM, about 700nM, about 750nM, about 800nM, about 850nM, about 900nM, about 950nM, about 1,000nM.

[0262] In some embodiments, the one or more ions may include chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, 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.

[0263] In some embodiments, the one or more ions on the cis side of the membrane can include chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, 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.

[0264] In some embodiments, the one or more ions on the trans side of the membrane may include chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, 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.

[0265] In some embodiments, the one or more ions on the cis side of the membrane may be the same type of ion as the one or more ions on the trans side of the membrane. In some embodiments, the one or more ions on the cis side of the membrane may be a different type of ion than the one or more ions on the trans side of the membrane.

[0266] In some embodiments, one or more ions may comprise about one ion to about ten ions. In some cases, one or more ions may comprise at least about one ion, at least about two ions, at least about three ions, at least about four ions, at least about five ions, at least about six ions, at least about seven ions, at least about eight ions, at least about nine ions, at least about ten ions or more than ten ions. In some cases, one or more ions may comprise 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 may comprise about 1 ion, about 2 ions, about 3 ions, about 4 ions, about 5 ions, about 6 ions, about 7 ions, about 8 ions, about 9 ions or about 10 ions.

[0267] In some embodiments, one or more ions on the cis side of the membrane may be present at the same concentration as one or more ions on the trans side of the membrane. In some cases, one or more ions on the cis side of the membrane may be present at a different concentration than one or more ions on the trans side of the membrane.

[0268] In some embodiments, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 500% greater than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, or about 75% to about 76%. 5%, 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 360% to about 370%, about 380% to about 390%, about 400% to about 410%, about 420% to about 430%, about 440% to about 450%, about 460% to about 470%, about 480% to about 490%, about 500% to about 510%, about 510% to about 520%, about 520% to about 530%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 600%, about 610% to about 620%, about 630% to about 640%, about 650% to about 660%, about 670% to about 680%, about 690% to about 700%, about 50% 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%.

[0269] 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%, 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 30%, 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.

[0270] 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 240%, up to about 250%, 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%.

[0271] 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 201%, 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%, about 500%, about 510%, about 511%, about 520%, about 530%, about 540%, about 550%, about 0%, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0272] In some embodiments, the concentration of one or more ions on the cis side of the membrane can be about 0.1% to about 500% lower than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane 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% lower than the concentration of one or more ions on the trans side of the membrane. %, 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 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 370% to about 380%, about 390% to about 400%, about 410% to about 420%, about 430% to about 440%, about 450% to about 460%, about 470% to about 480%, about 490% to about 500%, about 510% to about 520%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 590%, about 600% to about 610%, about 620% to about 630%, about 640% to about 650%, about 660% to about 670%, about 680% to about 690%, about 700% to about 710%, about % 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%, between about 460% to about 470%, about 470% to about 480%, about 480% to about 490%, or about 490% to about 500%.

[0273] 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%, 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 30%, 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.

[0274] 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 242%, up to about 264%, up to about 275%, up to about 286%, up to about 297%, up to about 308%, up to about 311%, up to about 320%, up to about 330%, up to about 340%, up to about 350%, up to about 360%, up to about 370%, up to about 380%, up to about 390%, up to about 390%, up to about 390%, up to about 3 %, 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%.

[0275] In some cases, the concentration of one or more ions on the cis side of the membrane may 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 201%, 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%, about 500%, about 510%, about 511%, about 520%, about 530%, about 540%, about 550%, about 0%, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0276] 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% greater 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 76%, about 77% to about 78%, about 79% to about 80%, about 81% to about 82%, about 83% to about 84%, about 85% to about 86%, about 87% to about 88%, about 89% to about 90%, about 91% to about 92%, about 93% to about 94%, about 95% to about 96%, about 97% to about 98%, about 99% to about 100%, about 100% to about 101%, about 100% to about 102%, about 100% to about 103%, about 100% to about 104%, about 100% to about 105%, about 100% to about 106%, about 100% to about 107%, about 100% to about 108%, about 100% to about 109%, about 110% to about 111%, about 111% to about 112%, about 5% 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 370% to about 380%, about 390% to about 400%, about 410% to about 420%, about 430% to about 440%, about 450% to about 460%, about 470% to about 480%, about 490% to about 500%, about 510% to about 520%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 590%, about 600% to about 610%, about 620% to about 630%, about 640% to about 650%, about 660% to about 670%, about 680% to about 690%, about 700% to about 710%, about 0% 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%.

[0277] 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%, 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 30%, 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.

[0278] 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 240%, up to about 250%, 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%.

[0279] 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 201%, 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%, about 500%, about 510%, about 511%, about 520%, about 530%, about 530%, about 540%, about 550%, about 0%, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0280] 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% lower than the concentration of one or more ions on the cis side of the membrane. In some cases, the concentration of one or more ions between 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 20 0% 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%.

[0281] 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%, 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 30%, 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.

[0282] In some cases, the concentration of one or more ions on the trans side of the membrane may 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 240%, up to about 250%, 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%.

[0283] In some cases, the concentration of one or more ions on the trans side of the membrane may 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 201%, 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%, about 500%, about 510%, about 511%, about 520%, about 530%, about 530%, about 540%, about 550%, about 0%, 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 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, about 500%.

[0284] Alternatively, EOF can be produced by a symmetrical ion distribution between the cis and trans sides of the membrane. A symmetrical ion distribution can occur when the concentration of one or more ions on the cis side of the membrane is the same as the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane is the same as the concentration of one or more ions on the trans side of the membrane.

[0285] In some embodiments, EOF can be produced by an asymmetric concentration of one or more salts and an asymmetric concentration of one or more ions between the cis and trans sides of the membrane.

[0286] In some embodiments, the electroosmotic force can act in the same direction as the electrophoretic force or in a direction opposite to the electrophoretic force. In some embodiments, the electroosmotic force can be greater than about 100 Å. In some embodiments, the electroosmotic force can be less than the electrophoretic force.

[0287] In some embodiments, the cis-to-trans EOF may 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 may 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 may include a total ionic current. In some cases, the net ionic current may include a flow rate that is less than the total flow of all ions in the nanopore system. In some cases, the net ionic current may include a flow rate that is less than the flow rate of all ions in a particular direction in the nanopore system. In some cases, the particular direction may be from the cis side of the membrane to the trans side of the membrane. In some cases, the particular direction may be from the trans side of the membrane to the cis side of the membrane. In some cases, the total ionic current may include the total flow rate of all ions in the nanopore system. In some cases, the total flow rate of all ions in the nanopore system may be from the cis side of the membrane to the trans side of the membrane. In some cases, the total flow rate of all ions in the nanopore system may be from the trans side of the membrane to the cis side of the membrane. In some cases, the total ionic current may include the total flow rate of all ions in a particular direction in the nanopore system. In some cases, the particular direction may 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.

[0288] In some embodiments, the net ionic current can comprise between about 0.001% and about 100% of the total ionic current. In some cases, the net ionic current can comprise between about 0.001% and about 0.01%, about 0.01% and about 0.1%, about 0.1% and about 1%, about 1% and about 10%, or about 10% and about 100% of the total ionic current. In some cases, the net ionic current can comprise 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 may 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% of the total ionic current, 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 about 0.001%. In some cases, the net ionic current may comprise about 0.001%, about 0.005%, about 0.01%, about 0.05%, 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.

[0289] In some embodiments, the cis-to-trans EOF is derived from the ratio of the net ionic current cis-to-trans to the total ionic current, also referred to as the cis-to-trans relative net current, 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, greater than about 0.99, or more. In certain embodiments, the cis-to-trans EOF is derived from the net ionic current cis-to-trans over the total ionic current, also referred to as the cis-to-trans relative net current, 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.

[0290] In some embodiments, the absolute relative net electroosmotic current exceeds the applied voltage (I relV ), greater than about 0.01, greater than about 0.02, greater than about 0.03, greater than about 0.04, greater than about 0.05, greater than about 0.06, greater than about 0.07, greater than about 0.08, greater than about 0.09, greater than about 0.10, greater than about 0.15, 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, or greater than about 1 pA / mV. In some embodiments, the absolute relative net electroosmotic current is greater than about 0.01, greater than about 0.02, greater than about 0.03, greater than about 0.04, greater than about 0.05, greater than about 0.06, greater than about 0.07, greater than about 0.08, greater than about 0.09, greater than about 0.10, greater than about 0.15, 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, or greater than about 1 pA / mV. relV ), less than about 0.01, less than about 0.02, less than about 0.03, less than about 0.04, less than about 0.05, less than about 0.06, less than about 0.07, less than about 0.08, less than about 0.09, less than about 0.10, less than about 0.15, 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, or less than about 1 pA / mV.

[0291] In some embodiments, the system may include a translocase. The translocase may include a molecular motor (e.g., an unfoldase). In some embodiments, the molecular motor may move the analyte via the translocase. In some cases, the translocase may move the analyte through the translocase and into the nanopore. In some cases, the translocase may move the analyte into the nanopore via the translocase and through the nanopore channel. The molecular motor may be NTP-driven, ATP-driven, or neither. The translocase may include an unfoldase. The unfoldase may be an AAA+ enzyme. The translocase may include a molecular motor (e.g., an unfoldase). A translocase may include ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, (ClpY), LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, functional homologs, homologs, or analogs, or any combination thereof. The translocase may bind to the analyte or form a complex with the analyte (e.g., a translocase-analyte complex). The transposase can drive the analyte through the transposase. The transposase can translocate the analyte through the nanopore. Driving the analyte through the transposase can destroy (e.g., unfold) the quaternary, tertiary or secondary structure of the analyte (e.g., protein). The destruction of the quaternary, tertiary or secondary structure helps the analyte to translocate through the nanopore. The transposase can form a complex at the N-terminus or C-terminus of the analyte (e.g., peptide, protein). The transposase can form a transposase-analyte complex with the analyte on the cis side of the fluid chamber. The transposase-analyte complex can be formed outside the fluid chamber or inside the fluid chamber (e.g., on the cis side of the fluid chamber). Before being added to the cis side of the fluid chamber, a transposase-analyte complex can be formed (e.g., by preloading with a preload solution). The transposase can control the rate of translocation. The translocation rate of the transposase can be regulated. Regulation can be accomplished by changing the energy source concentration of the transposase (e.g., NTP, ATP).

[0292] In some embodiments, a transposase may be capable of moving an analyte. In some embodiments, a transposase may not be able to separate the strands of a double-stranded nucleic acid. In some cases, a transposase may not be a helicase. In some embodiments, a transposase may not be able to replicate nucleic acids. In some cases, a transposase may not be a nucleic acid polymerase. In some cases, a transposase may not be a DNA polymerase or an RNA polymerase. In some embodiments, a transposase may not participate in nucleic acid replication. In some embodiments, a transposase may not be able to separate an analyte. In some embodiments, a transposition may not be a topoisomerase.

[0293] In some embodiments, the translocase can be coupled to the nanopore. The translocase can be covalently coupled (e.g., genetic fusion) or non-covalently coupled (e.g., via a recognition element).

[0294] In some embodiments, the translocase may not be coupled to the nanopore. In some cases, the translocase may not be coupled to the opening of the nanopore. In some cases, the translocase may not be coupled to the membrane near the nanopore.

[0295] In some embodiments, the translocase may not bind to the nanopore. In some cases, the translocase may not bind to the opening of the nanopore. In some cases, the translocase may not bind to the membrane near the nanopore.

[0296] In some embodiments, the electroosmotic force can capture a translocase-analyte complex (e.g., a translocase-analyte complex). Capture can be the result of pulling a portion of the analyte that is not in the translocase of the translocase-analyte complex into the nanopore. Capturing a portion of the analyte by the nanopore channel can cause a portion of the analyte of the transposition-analyte complex to be translocated through the nanopore because the analyte can be further pulled into the nanopore channel by the electroosmotic force. This translocation can occur in the opposite direction of the electrophoretic force or simultaneously with the electrophoretic force. The translocation of the analyte portion can bring the translocase of the transposition-analyte complex close to the nanopore channel because a portion of the translocated analyte is close to the portion of the analyte in the translocase. If the electroosmotic force acts in the cis-to-trans direction, the translocase can be brought to the vicinity of the nanopore on the cis side of the nanopore channel; if the electroosmotic force acts in the trans-to-cis direction, the translocase can be brought to the trans side of the nanopore channel. The electroosmotic force can hold the translocase of the translocase-analyte complex near the nanopore of the nanopore channel, for example by continuing to attract the analyte through the nanopore and transferring the electroosmotic force to the attached translocase. The vicinity of the nanopore channel can be near the opening of the nanopore channel, also referred to as the "top" of the nanopore, or the portion of the nanopore that is not within the membrane.

[0297] In some embodiments, the EOF can retain the translocase at the top of the nanopore in the absence of analyte. In some cases, a portion of the translocase can be captured in the nanopore. In some cases, a portion of the translocase can be captured in the nanopore due to electroosmotic forces. In some cases, a portion of the translocase can be captured in the nanopore due to electrophoretic forces. In some cases, a portion of the translocase can be captured in the nanopore due to both electroosmotic and electrophoretic forces. In some cases, the portion of the translocase captured in the nanopore can be a charged linker or a peptide extension of the translocase.

[0298] When maintained adjacent to the nanopore channel, the transposase of the transposase-analyte complex can be oriented so that the channel of the transposase is adjacent to the channel of the nanopore. This orientation can be provided by the analyte pulled into the nanopore by the electroosmotic force, which can align the connection point of the analyte with the transposase (e.g., transposase channel) and the transposase channel adjacent to the nanopore channel. In some embodiments, the transposase can control the rate of analyte translocation. The translocation rate can be the result of the transposase acting as a molecular motor on the analyte. In some embodiments, the translocation rate can be from about 0.1 amino acid / second (aa / s) to about 1,000 amino acid / second. 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 least 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 certain 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.

[0299] The translocation orientation can be such that the feed direction of the translocase is aligned with the channel of the nanopore. The feed direction can be cis-to-trans or trans-to-cis. There may or may not be a gap between the lumen of the translocase channel and the lumen of the nanopore channel. The translocase can be held such that it feeds the analyte through the nanopore in the direction of the electroosmotic force or it can be held such that it pulls the analyte through the nanopore in the direction of the electroosmotic force.

[0300] The translocase can transport the analyte into the nanopore in the direction of the electroosmotic force, causing it to translocate faster or slower than an analyte that is translocated by electroosmotic force alone.

[0301] In some embodiments, the rate of translocation of an analyte through a nanopore with a translocase can be faster than the rate of translocation of the analyte through a nanopore without a translocase. In some cases, the rate of translocation of an analyte through a nanopore with a translocase can be about 0.1% to about 500% faster than the rate of translocation of the analyte through a nanopore without a translocase. In some cases, the rate of translocation of an analyte through a nanopore with a translocase 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 75% to about 80%, about 85% to about 90%, about 90% to about 100%, about 100% to about 15 ... 0% 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%.

[0302] In some cases, the rate of translocation of an analyte through a nanopore with a translocase 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%, 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 201%, at least about 210%, at least about 211%, at least about 212%, at least about 213%, at least about 214%, at least about 215%, at least about 216%, at least about 217%, at least about 218%, at least about 220%, at least about 221%, at least about 222%, at least about 223%, at least about 224%, at least about 225%, at least about 30%, 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.In some cases, the rate of translocation of an analyte through a nanopore with a translocase 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 420%, up to about 410%, up to about 400%, up to about 420%, up to about 430%, up to about 440%, up to about 450%, up to about 460%, up to about 470%, up to about 480%, up to about 490%, up to about 500%, up to about 510%, up to about 520%, up to about 530%, up to about 540%, up to about 550%, up to about 560%, up to about 570%, up to about 580%, up to about 590%, up to about 600%, up to about 610%, up to about 620%, up to about 630%, up to about 640%, up to about 650%, up to about 660%, up to about 670%, up to about 680%, up to about 690%, up to about 700%, up to about 710%, up to about 720%, up to about 730%, up to about 740%, up to about 750%, up to about 760%, up to about 770%, up to about 780%, up to about 790%, up to about 800%, up to about 8 %, up to about 30%, 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%. In some cases, the rate of translocation of an analyte through a nanopore with a translocase 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 201%, 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%, about 500%, about 510%, about 511%, about 520%, about 530%, about 530%, about 540%, about 550%, about %, about 70%, 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 380%, about 390%, about 400%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490%, or about 500%.

[0303] In some embodiments, the translocation rate of an analyte through a nanopore with a translocase can be slower than the translocation rate of an analyte through a nanopore without a translocase. In some cases, the translocation rate of an analyte through a nanopore with a translocase can be about 0.1% to about 500% slower than the translocation rate of an analyte through a nanopore without a translocase. In some cases, the translocation rate of an analyte through a nanopore with a translocase 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 75% to about 80%, about 85% to about 90%, about 90% to about 100%, about 100% to about 15 ... 0% 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%.

[0304] In some cases, the rate of translocation of an analyte through a nanopore with a translocase 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%, 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 30%, 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.In some cases, the rate of translocation of an analyte through a nanopore with a translocase 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%, or at most about 370%. The rate at which the analyte is translocated passes through the nanopore translocase is 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 rate at which the analyte is translocated passes through the nanopore translocase. In some cases, the rate of translocation of an analyte through a nanopore with a translocase 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 201%, 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%, about 500%, about 510%, about 511%, about 520%, about 530%, about 531%, about 540%, about 550%, about %, about 70%, 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 380%, about 390%, about 400%, about 420%, about 430%, about 440%, about 450%, about 450%, about 460%, about 470%, about 480%, about 480%, about 490% or about 500%.

[0305] In some embodiments, the primary force for translocation of an analyte through a nanopore can be an EOF. In some embodiments, translocation of an analyte through a nanopore can occur using an EOF. In some cases, translocation of an analyte through a nanopore can occur without a translocation enzyme. In some cases, translocation of an analyte through a nanopore can occur without a translocation enzyme using an EOF.

[0306] In some embodiments, translocation of the analyte through the nanopore may not occur in the absence of an EOF. In some cases, translocation of the analyte through the nanopore may not occur in the presence of a translocase. In some cases, translocation of the analyte through the nanopore may not occur in the absence of an EOF and a translocase.

[0307] The transposase can be retained on the cis or trans side of the nanopore without being coupled to the nanopore. Electroosmotic force can keep the transposase near the nanopore without the need for additional coupling to the nanopore channel. Electroosmotic force can keep the transposase near the nanopore, thereby allowing the nanopore to be coupled to the transposase. When the analyte is translocated through the nanopore, the transposase can be retained near the nanopore channel. After the analyte is completely translocated through the nanopore, the transposase can continue to remain near the nanopore or the transposase can be released from the position near the nanopore. The released transposase can then form a transposition-analyte complex with another analyte. In some embodiments, the transposase may not be coupled to the nanopore. In some cases, the transposase may not be coupled to the nanopore. In some cases, the transposase may not be coupled to the membrane near the nanopore.

[0308] Alternatively, in some embodiments, the translocase can be coupled to the cis side or the trans side of the nanopore. In some cases, the translocase can be coupled to the nanopore via a covalent bond. In some cases, the covalent bond is a polar covalent bond. In some cases, the covalent bond is a non-polar covalent bond. In some cases, the translocase can be coupled to the nanopore via a non-covalent bond. In some cases, the non-covalent bond can include electrostatic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, or any combination thereof. In some cases, the translocase can be coupled to the nanopore via a linker. In some cases, the linker can include (GGGGS)3, (GGGGS) n 、(GGGGS) n , (Gly)8, (Gly)6, (EAAAK)3, (EAAAK) n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG, A(EAAAK)4, ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro) n , disulfide bonds, cysteine bonds, or any combination thereof. In some embodiments, the linker may comprise any combination of amino acids. In some cases, the amino acids may be standard amino acids. In some cases, typical amino acids may include alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or any combination thereof. In some cases, the amino acids may be unnatural amino acids. In some cases, unnatural amino acids may include hydroproline, β-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, pyroglutamic acid, naphthylalanine, Abu, DAB, methionine sulfoxide, methionine sulfone, α-amino-n-butyric acid, norvaline, isoleucine, t-leucine, α-amino-n-heptanoic acid, piperic acid, isothreonine, homocysteine, homoserine, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, β- Alanine, β-amino-n-butyric acid, β-amino-isobutyric acid, β-amino-isobutyric acid, α,β-alanine, β-amino-n-butyric acid, β-amino-isobutyric acid, γ-amino-butyric acid, α-amino-isobutyric acid, isovaline, sarcosine, N-ethylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, N-methyl-β-alanine, N-ethyl-β-alanine, isoserine, α-hydroxy-γ-amino-butyric acid, or any combination thereof. In some cases, the linker can comprise any combination of standard amino acids and unnatural amino acids. In some cases, the linker can be ethylene glycol. In some cases, the linker can be polyethylene glycol. In some cases, the linker can be biotin. In some cases, the linker can be streptavidin. In some cases, the linker can be a cysteine linker. In some cases, the linker can be formed using Spycatcher, Halo-tag, Snap-tag, or other bioconjugation methods. In some cases, the linker can be formed by click chemistry. In some cases, a linker is attached to the unnatural amino acid.

[0309] In some embodiments, the method includes providing a system. 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 include a first fluid solution. The trans-side tank includes a second fluid solution. In some embodiments, the solution or the solution on the cis side or trans side of the fluid chamber can be configured to have a fixed pH. The pH of the solution is greater than about 1, greater than about 2, greater than about 3, greater than about 3.8, greater than about 4, greater than about 4.5, greater than about 6, greater than about 7, greater than about 8, greater than about 9, greater than about 10, greater than about 10.5, greater than about 11, and a pH greater than about 12, greater than about 13, or greater than about 14 can be used. The pH of the solution or solutions that can be used can be less than about 1, less than about 2, less than about 3, less than about 3.8, less than about 4, less than about 4.5, less than about 6, less than about 7, less than about 8, less than about 9, less than about 10, less than about 10.5, less than about 11, less than about 12, less than about 13, or less than about 14.

[0310] In some embodiments, the first solution and the second solution can be different solutions. In some embodiments, the first solution and the second solution can be different solutions and can have different concentrations of one or more types of ions. In some embodiments, the first solution and the second solution can be different solutions and can have different concentrations of one or more types of salts. In some embodiments, the first solution and the second solution can be different solutions and can have different concentrations of one or more types of salts and different concentrations of one or more types of ions.

[0311] Alternatively, in some embodiments, the first solution and the second solution can be different solutions and can have the same concentration of one or more salts. In some embodiments, the first solution and the second solution can be different solutions and can have the same concentration of one or more types of ions. In some embodiments, the first solution and the second solution can be different solutions and can have the same concentration of one or more ions and the same concentration of one or more salts.

[0312] In some embodiments, the first solution and the second solution can be the same solution. In some embodiments, the first solution and the second solution can be the same solution and can have different concentrations of one or more types of ions. In some embodiments, the first solution and the second solution can be the same solution and can have different concentrations of one or more salts. In some embodiments, the first solution and the second solution can be the same solution and can have different concentrations of one or more ions and different concentrations of one or more salts.

[0313] Alternatively, in some embodiments, the first solution and the second solution can be the same solution and can have the same concentration of one or more salts. In some embodiments, the first solution and the second solution can be the same solution and can have the same concentration of one or more types of ions. In some embodiments, the first solution and the second solution can be the same solution and can have the same concentration of one or more ions and the same concentration of one or more salts.

[0314] The fluid solution can be configured to provide electroosmotic flow, also known as electroosmotic force. In some embodiments, electroosmotic flow can be generated by having an asymmetric distribution of one or more salts between the cis side and the trans side of the membrane. In some embodiments, electroosmotic flow can be generated by having an asymmetric distribution of one or more ions between the cis side and the trans side of the membrane. In some embodiments, electroosmotic flow can be generated by having an asymmetric distribution of one or more ions and one or more salts between the cis side and the trans side of the membrane. The electroosmotic force can act across the membrane. In some embodiments, the membrane includes a nanopore. In some embodiments, a pair of electrodes is provided. The pair of electrodes can be configured with one electrode located on the cis side of the fluid chamber and the other electrode located on the trans side of the fluid chamber. In some embodiments, the pair of electrodes can be configured to provide an applied voltage. The applied voltage can pass through the membrane. The applied voltage will generate an electrophoretic force. In some embodiments, the pair of electrodes can be configured to provide an electrophoretic force across the membrane. This pair of electrodes can be used to measure a signal.

[0315] In some embodiments, the applied membrane voltage can be at least about 1, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, 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, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 600, at least about 700, at least about 800-, at least about 900-, or at least about 1,000 mV. The applied voltage can be at most about 1, at most about 5, at most about 10, at most about 20, at most about 30, at most about 40, at most about 50, at most about 60, at most about 70, at most about 80, at most about 90, at most about 100, at most about 150, at most about 200, at most about 250, at most about 300, at most about 350, at most about 400, at most about 450, at most about 500, at most about 600, at most about 700, at most about 800, at most about 900, or at most about 1,000 mV. In some embodiments, the voltage is negative cis to trans. In some embodiments, the voltage is positive cis to trans.

[0316] In some embodiments, translocation of the analyte through the nanopore occurs in a cis-to-trans direction. In some embodiments, translocation of the analyte through the nanopore occurs in a trans-to-cis direction. In some embodiments, translocation of the analyte through the nanopore occurs in the direction of the electroosmotic force (EOF). In some embodiments, translocation of the analyte through the nanopore occurs in a direction opposite to the electrophoretic force (EPF). In some embodiments, translocation of the analyte through the nanopore occurs in a direction opposite to the EOF direction and the EPF direction.

[0317] Alternatively, in some embodiments, the EPF can be greater than the EOF. In some embodiments, the EOF can be greater than about the EPF. In some cases, the EOF is 0.1% to 500% greater than the EPF. In some cases, the EOF 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 ... 5%, 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 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%. 0%, 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%.

[0318] In some cases, the 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%, 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 30%, 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%, at least about 510%, at least about 520%, at least about 530%, at least about 540%, at least about 550%, at least about 560%, at least about 570%, at least about 580%, at least about 590%, at least about 600%, at least about 610%, at least about 620%, at least about 630%, at least about 640%, at least about 650%, at least about 660%, at least about 670%, at least about 680%, at least about 690%, at least about 700%, at least about 710%, at least about 720%, at least about 730%, at least about 740%, at least about 750%, at least about 760%, at least about 770%, at least about 780%, at least about 790%, at least about 800%, at least about

[0319] In some cases, the EOF 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 240%, 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 1%, up to about 0.5%, up to about 0.1%, or 0.1% or less.

[0320] 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% greater than the EPF. , 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%.

[0321] In some embodiments, translocation of the analyte through the nanopore occurs in the EOF direction. In some embodiments, translocation of the analyte through the nanopore occurs in the EPF direction. In some embodiments, translocation of the analyte through the nanopore occurs in both the EOF and EPF directions.

[0322] Alternatively, in some embodiments, translocation of the analyte through the nanopore occurs in the direction of the EPF. In some embodiments, translocation of the analyte through the nanopore occurs in a direction opposite to the EOF. In some embodiments, translocation of the analyte through the nanopore occurs in a direction opposite to the EPF and the EOF.

[0323] In some embodiments, the EOF can be greater than the EPF. In some 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 ... 5%, 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 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%. 0%, 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%.

[0324] In some cases, the EPF can be greater than the EOF by 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%, 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 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%.

[0325] In some cases, the EPF 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 2 %, 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%.

[0326] In some 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% greater than the EOF. , 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%.

[0327] In some embodiments, a signal is measured. The signal may comprise an electrical signal. The signal may be related to or caused by translocation of the analyte. The signal may comprise an ionic current or a change in ionic current. The signal may comprise a measurement of a change in current between nanopore states. The states of the nanopore may comprise an open channel, analyte captured by the nanopore, or a passage of a polymer through the nanopore from a captured state. In some embodiments, measuring the signal may comprise comparing signals during different states of the nanopore.

[0328] In some embodiments, an electrophoretic force is provided. In some embodiments, the method comprises transporting an analyte through the nanopore. The translocation may be assisted by electroosmotic force, electrophoretic force, or a combination thereof. The translocation may be opposed by electroosmotic force, electrophoretic force, or a combination thereof. In some embodiments, the analyte is in a pre-denatured state prior to translocation. In some embodiments, the method comprises measuring a signal. The signal may be caused or influenced by the translocation of the analyte. In some embodiments, one or more analytes are translocated. The signal of one or more translocated analytes may be measured. In some embodiments, multiple analytes may be measured. The signal or signals from multiple analytes may be used to characterize them. In some embodiments, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 8, at least about 9, at least about 20, at least about 30, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 1,500, at least about 2,000, or more. About 2,000, at least about 2,500, at least about 3,500, at least about 4,000, at least about 4,500, at least about 5,000, at least about 5,500, at least about 6,000, at least about 6,500, at least about 7,000, at least about 7,500, at least about 8,000, at least about 8,500, at least about 9,000, at least about 9,500, or at least about 10,000 analytes can be characterized. In some embodiments, 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 20, at most about 30, at most about 50, at most about 100, at most about 200, at most about 300, at most about 400, at most about 500, at most about 600, at most about 700, at most about 800, at most about 900, at most about 1,000, at most about 1,500, at most about 2 About 2,000, up to about 2,500, up to about 3,500, up to about 4,000, up to about 4,500, up to about 5,000, up to about 5,500, up to about 6,000, up to about 6,500, up to about 7,000, up to about 7,500, up to about 8,000, up to about 8,500, up to about 9,000, up to about 950, up to about 10,000 analytes can be characterized.

[0329] In some embodiments, the solutions on the cis side and the trans side of the fluid chamber are configured to generate an electromagnetic force. The electromagnetic force can be generated due to the difference in solute concentration between the solution on the cis side and the solution on the cis side. The solute can be an ion or a permeant. These ions or permeant can flow through the membrane through the nanopores. These ions can be high-mobility ions or low-mobility ions.

[0330] In some embodiments, the electrophoretic force can act in a cis-to-trans or trans-to-cis direction. The electrophoretic force can be in the same direction as the electroosmotic force or in the opposite direction. The electrophoretic force can have a greater or lesser effect on the analyte than the electroosmotic force. The electrophoretic force can assist or oppose the translocation of the analyte.

[0331] In some embodiments, the highly mobile ions may comprise less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, or less than about 95% of the salt content that flows through the nanopore from one side of the membrane. In some embodiments, highly mobile ions may comprise about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, greater than about 8%, greater than about 9%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% of the salt content that flows through the nanopore.

[0332] In some embodiments, low mobility ions may comprise less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, or less than about 95% of the salt content that flows through the nanopore from one side of the membrane. In some embodiments, low mobility ions may comprise about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, greater than about 8%, greater than about 9%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% of the salt content that flows through the nanopore.

[0333] In some embodiments, the salt, ion, osmotic agent or electrolyte concentration on the cis side is greater than about 0.01, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.25, 1.50, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, or 5 M. In some embodiments, the difference in salt ion or electrolyte concentration between the cis and trans sides is greater than about 0.01, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.25, 1.50, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, or about 5 M.

[0334] In some embodiments, the concentration of the solute is greater on the cis side than on the trans side. In some embodiments, the concentration of the solute is greater on the trans side than on the cis side.

[0335] In some embodiments, the analyte is an unmodified analyte. In some embodiments, the analyte is a label-free or label-free analyte. In some embodiments, the analyte includes a main branch structure. The main branch structure may include a molecule conjugated or coupled to the analyte. The conjugation or coupling of the molecule to the analyte can improve the performance characteristics of the provided analyte characterization method. The performance characteristics may include read length, throughput, processing speed, sequence accuracy, sequence coverage, or a combination thereof. The main branch structure may include a label, a barcode, or a combination thereof. In some embodiments, the main branch structure can be configured to modify the characteristics of the analyte. The main branch structure can be configured to modify an analyte bound to a translocase, an analyte bound to an unfolding enzyme, an analyte bound to a membrane, an analyte captured by a nanopore, or a combination thereof. In some embodiments, the main branch structure can be configured to couple the analyte to a translocation. In some cases, the main branch structure can be configured to bind the analyte to a translocase. In some cases, the main branch structure can be bound to the analyte via a covalent bond. In some instances, the main branch structure can be configured to bind the analyte via a non-covalent bond. In some cases, the main branch structure can be configured to bind the analyte via a linker. In some embodiments, the main branch structure can be configured to couple the analyte to the unfolded product. In some embodiments, the main branch structure may not be configured to couple to the nanopore. In some cases, the main branch structure may not be coupled to the nanopore. In some cases, the main branch structure may not be bound to the nanopore. In some cases, the main branch structure may not be configured to assist the nanopore in capturing the analyte.

[0336] In some embodiments, the main branch structure may include nucleic acids. In some cases, the nucleic acids may include DNA, RNA, locked nucleic acids (LNA), peptide nucleic acids (PNA), bridged nucleic acids (BNA), glycol nucleic acids (GNA), sucrose nucleic acids (TNA), hexanol nucleic acids (HNA), or any combination thereof. In some embodiments, the main branch structure may include one or more peptides or proteins. In some embodiments, the main branch structure may include nucleic acids, proteins, polypeptides, or any combination thereof.

[0337] In some embodiments, the main branch structure may exist in a 5' to 3' direction. In some embodiments, the main branch structure may exist in an N-terminal to C-terminal orientation.

[0338] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, blocking motif, interception motif, recognition motif, and capture motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, blocking motif, interception motif, recognition motif, and capture motif.

[0339] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, blocking motif, interception motif, capture motif, and recognition motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, blocking motif, interception motif, capture motif, and recognition motif.

[0340] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, blocking motif, recognition motif, interception motif, and capture motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, blocking motif, interception motif, capture motif, and recognition motif.

[0341] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, blocking motif, recognition motif, capture motif, and interception motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, blocking motif, interception motif, capture motif, and recognition motif.

[0342] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, blocking motif, capture motif, interception motif, and recognition motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, blocking motif, capture motif, interception motif, and recognition motif.

[0343] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, blocking motif, capture motif, recognition motif, and interception motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, blocking motif, capture motif, recognition motif, and interception motif.

[0344] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, interception motif, retardation motif, recognition motif, and capture motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, interception motif, retardation motif, recognition motif, and capture motif.

[0345] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, interception motif, retardation motif, capture motif, and recognition motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, interception motif, retardation motif, capture motif, and recognition motif.

[0346] In some embodiments, the main branch structure may include the following 5' to 3' orientation: coupling motif, interception motif, recognition motif, retardation motif, and capture motif. In some embodiments, the main branch structure may include the following N-terminal to C-terminal orientation: coupling motif, interception motif, recognition motif, retardation motif, and capture motif.

[0347] In s...

Claims

1. A method comprising: (a) Provide: (i) A nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side and a trans side; (b) contacting a complex comprising a non-nucleic acid-based polymer analyte and a translocase with the cis side of the nanopore; and (c) translocating the non-nucleic acid-based polymer analyte to the trans side of the fluidic channel using the cis-side to trans-side electroosmotic force, wherein the cis-side to trans-side electroosmotic force maintains the complex translocated from the cis-side entrance of the nanopore channel. 2 . The method according to claim 1 , further comprising contacting the non-nucleic acid-based polymer analyte with a translocase to form a complex before (c).

3. The method of claim 2, wherein the complex described herein is produced on the cis side of the fluid chamber.

4. The method according to any one of claims 1 to 3, wherein the electroosmotic force from the cis side to the trans side comprises a net ionic current from the cis side to the trans side.

5. The method according to any one of claims 1 to 4, wherein the electroosmotic force from the cis side to the trans side is regulated by pH value, salt type, salt concentration, transmembrane osmotic pressure of the membrane, modification of the nanopore, or any combination thereof. The method of claim 5 , wherein the modification of the nanopore comprises modification of the charge of the nanopore. 7 . The method according to claim 5 , wherein the electroosmotic force from the cis side to the trans side is regulated by an asymmetric salt distribution between the cis side and the trans side of the fluid chamber.

8. The method of any one of claims 1-7, wherein the complex is formed in a solution on the cis side of the fluid chamber.

9. The method of any one of claims 1-8, wherein the complex is formed prior to contacting the complex with the cis side of a nanopore.

10. The method of any one of claims 1-9, wherein the translocase comprises an adenosine triphosphate (ATP) driven unfoldase.

11. The method of any one of claims 1-10, wherein the unfolding translocase comprises a nucleotide triphosphate (NTP) driven unfolding enzyme.

12. The method of claim 11, wherein the translocase comprises an ATPase associated with various cell-active (AAA+) enzymes.

13. The method of claim 12, wherein the AAA+ enzyme is selected from caseinolytic mitochondrial matrix peptidase partner subunit X (ClpX), caseinolytic mitochondrial matrix peptidase partner subunit A (ClpA), proteasome-activating nucleotidase (PAN), LON, valosin-containing protein-like ATPase (VAT) of pyrogenic acidophilus, AMA, 854, membrane-bound AAA (MBA), archaeal ubiquitin-like modifying protein (SAMP), caseinolytic mitochondrial matrix peptidase partner subunit C (ClpC), caseinolytic mitochondrial matrix peptidase partner subunit E (ClpE), HsIU, caseinolytic mitochondrial matrix peptidase partner subunit Y (ClpY), LonA, LonB, FtsH, Mpa, Cdc48-like protein (Cpa) of actinomycetes, Msp1, SecA and functional homologs, homologs or analogs thereof.

14. The method of any one of claims 1-13, wherein the system further comprises a pair of electrodes.

15. The method of claim 14, wherein the electrode pair is configured to provide an applied voltage to generate an electrophoretic force. The method of claim 15 , wherein the applied voltage is a negative voltage of the trans side. The method of claim 15 , wherein the applied voltage is a positive voltage of the trans side.

18. The method of any one of claims 15-17, wherein the amplitude of the applied voltage is less than 300 millivolts (mV).

19. The method of any one of claims 15-18, wherein the magnitude of the applied voltage is greater than about 20 mV.

20. The method of any one of claims 15-19, wherein the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

21. The method according to any one of claims 1 to 20, wherein the non-nucleic acid-based polymer analyte comprises a main branch structure located at the N-terminus or the C-terminus.

22. The method of claim 21, wherein the main branch structure is configured to couple one or more translocations to the non-nucleic acid-based polymer analyte.

23. A method according to claim 21 or claim 22, wherein the main branch structure is configured to retard one or more transpositions.

24. The method according to any one of claims 21 to 23, wherein the main branch structure comprises a recognition motif.

25. The method according to any one of claims 21-24, wherein the main branch structure further comprises a capture motif, a blocking motif, an interception motif or a combination thereof.

26. A system comprising: Fluid chamber; and a membrane comprising a nanopore that separates the fluid chamber into a cis side comprising the first solution and a trans side comprising the second solution; The first solution and the second solution are configured to generate an electroosmotic force, wherein the electroosmotic force is configured to couple a translocase of a complex at the cis-side entrance of the nanopore channel, wherein the complex includes a non-nucleic acid-based polymer analyte and the translocase.

27. The system of claim 26, further comprising a translocase.

28. The system of claim 27, wherein the translocase comprises an ATP-driven unfoldase.

29. The system of claim 27, wherein the translocase comprises an NTP-driven unfoldase.

30. The system of claim 29, wherein the translocase comprises an AAA+ enzyme.

31. The system of claim 30, wherein the AAA+ enzyme is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Mspl, SecA, and functional homologs, orthologs, or analogs thereof.

32. The system of any one of claims 26-31, wherein the translocase is configured to sequentially translocate the non-nucleic acid based polymer analyte through the nanopore.

33. The system of any one of claims 26-32, wherein the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of a solute.

34. The system of claim 33, wherein the solute comprises an ion or an osmolyte.

35. The system of claim 33, wherein the difference between the first concentration of solute and the second concentration of solute is configured to generate an electro-osmotic force.

36. The system of any one of claims 26-35, wherein the electroosmotic force comprises a net ionic current flowing from the cis side to the trans side.

37. The system of any one of claims 26-36, wherein the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the membrane of the system, modification of the nanopore, or any combination thereof.

38. The system of any one of claims 26-37, wherein the electroosmotic force is modulated by charge modification of the nanopore.

39. The system of any one of claims 26-38, wherein the electroosmotic force is regulated by an asymmetric salt distribution between the cis and trans sides of the membrane.

40. The system of any one of claims 26-39, further comprising a pair of electrodes.

41. The system of claim 40, wherein a first electrode of the pair of electrodes is disposed on the cis side of the membrane and a second electrode of the pair of electrodes is disposed on the trans side of the membrane.

42. The system of claim 40, wherein the pair of electrodes is configured to detect a signal during translocation of a non-nucleic acid based polymer analyte.

43. The system of claim 42, wherein the signal is associated with a characteristic of the non-nucleic acid based polymer analyte.

44. The system of any one of claims 40-43, wherein the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force.

45. The system of claim 44, wherein the applied voltage is a negative voltage on the trans side.

46. The system of claim 44, wherein the applied voltage is a positive voltage on the trans side.

47. The system of any one of claims 44-46, wherein the amplitude of the applied voltage is less than 300 mV.

48. The system of any one of claims 44-47, wherein the magnitude of the applied voltage is greater than about 20 mV.

49. The system of any one of claims 44-48, wherein the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

50. The system of any one of claims 44-49, wherein the signal comprises an ionic current or a change therein.

51. A method comprising: (a) Provide: (i) A nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side and a trans side; (ii) a non-nucleic acid-based polymer analyte, wherein the non-nucleic acid-based polymer analyte is coupled to a main branch structure, and the main branch structure comprises a blocking motif, an intercepting motif, a coupling motif, or a combination thereof; and (iii) a translocase; and (b) translocating the non-nucleic acid-based polymer analyte from the cis side to the trans side of the fluid chamber.

52. The method of claim 51, wherein the main branch structure comprises a nucleic acid.

53. A method according to claim 51 or claim 52, wherein the main branch structure comprises a peptide.

54. The method of any one of claims 51-53, wherein the main branch structure comprises a nucleic acid and a peptide.

55. The method of any one of claims 51-54, wherein the blocking motif is configured to disrupt the interaction of the translocase with the non-nucleic acid based polymer analyte.

56. The method of any one of claims 51-55, wherein the blocking motif comprises an amino acid sequence.

57. The method of claim 56, wherein the amino acid sequence comprises n repeats of (Gly)n, (Ser-Gly)n, (Gly-Ser)n, (Alanine-Ser)n, (Val)n, (Alanine-Ser)n, (Alanine-Ser)n, (Valine-Ser)n, or (Ser-Val)n.

58. The method of claim 57, wherein n is greater than about 2, 3, 6, 9, 12, 15, 18, or 21.

59. The method of any one of claims 51-58, wherein the blocking motif comprises a non-amino acid chemical region.

60. The method of claim 59, wherein the non-amino acid chemical region comprises polyethylene glycol.

61. The method of any one of claims 51-60, wherein the interception motif is configured to prevent the translocase from translocating the non-nucleic acid-based polymer analyte through the interception motif.

62. The method of claim 61, wherein the interceptor motif is configured to prevent the translocase from translocating the non-nucleic acid-based polymer analyte through the main branch structure.

63. The method of claim 61, wherein the interceptor motif is configured to prevent the translocase from translocating the non-nucleic acid-based polymer analyte through the nanopore.

64. The method of any one of claims 51-63, wherein the intercepting motif comprises steric hindrance.

65. The method of claim 64, wherein the steric hindrance comprises one or more bulky amino acids.

66. The method of claim 65, wherein the one or more bulky amino acids comprise histidine, phenylalanine, tyrosine, or tryptophan.

67. The method of claim 65 or 66, wherein the steric hindrance comprises at least about one bulky amino acid.

68. The method of claim 65 or claim 66, wherein the steric hindrance comprises at least about five bulky amino acids.

69. The method of claim 64, wherein the steric hindrance comprises at least about a portion of an unfolding-resistant protein.

70. The method of claim 69, wherein the anti-unfolding protein comprises maltose binding protein, titin, dihydrofolate reductase, a nuclease, or a combination thereof.

71. The method of claim 69 or claim 70, wherein The unfolding-resistant protein comprises disulfide bonds.

72. The method of claim 64, wherein the steric hindrance comprises a large tethering molecule.

73. The method of claim 72, wherein the large tethering molecule comprises a carbohydrate, a polycyclic molecule, a branched dextran, biotin, streptavidin, a nanobody, an antibody, or a small antigenic element.

74. The method of any one of claims 51-73, wherein the coupling motif is configured to couple the main-branch structure to the non-nucleic acid-based polymer analyte.

75. The method of claim 74, wherein the non-nucleic acid based polymer analyte comprises a peptide.

76. The method of claim 75, wherein the coupling motif is attached to the C-terminus of the peptide.

77. The method of claim 75, wherein the coupling motif is attached to the N-terminus of the peptide.

78. The method of any one of claims 75-77, wherein the coupling motif comprises an enzyme having peptide ligase activity.

79. The method of any one of claims 51-78, wherein the coupling motif comprises a chemical group.

80. The method of claim 79, wherein the chemical group comprises maleimide, iodoacetamide, 2-thiopyridine, 3-arylpropionitrile, NHS-ester, isocyanate, isothiocyanate, benzoyl fluoride, diazonium salt, or PTAD.

81. The method of any one of claims 51-80, wherein the coupling motif comprises an enzyme coupling region.

82. The method of claim 81, wherein the enzyme coupling region links the coupling motif to an enzyme.

83. The method of claim 82, wherein the enzyme comprises a peptidase, a holoacylase, or a sortase.

84. The method of any one of claims 51-83, wherein the coupling motif of the main branch structure is coupled to the non-nucleic acid based polymer analyte via a bond.

85. The method of any one of claims 51-84, wherein the coupling motif of the main-branch structure is coupled to the non-nucleic acid-based polymer analyte via a linker.

86. The method according to any one of claims 51 to 85, wherein the main branch structure further comprises: (i) a recognition motif; or (ii) Capture motif.

87. The method of claim 86, wherein the capture motif comprises a polycationic tag.

88. The method of claim 87, wherein the polycationic tag comprises n repeats of (Ser-Gly-Arg)n, (Ser-Arg)n, (Arg)n.

89. The method of claim 86, wherein the capture motif comprises a polyanionic tag.

90. The method of claim 89, wherein the polyanionic tag comprises n repeats of (Ser-Gly-Asp)n, (Ser-Asp)n, (Asp)n.

91. The method of claim 86, wherein the recognition motif comprises a portion of ssrA, a prokaryotic ubiquitin-like protein, SulA, a peroxisomal membrane protein (Pex15), or a combination thereof.

92. The method of claim 86, wherein the sequence of the recognition motif comprises one or more of SEQ ID NOs: 201-206.

93. The method according to any one of claims 51-92, wherein the main branch structure is connected to the C-terminus or N-terminus of the non-nucleic acid-based polymer analyte.

94. The method of claim 93, wherein the non-nucleic acid based polymer analyte comprises a polypeptide.

95. The method of claim 94, wherein the main branch structure is coupled to the N-terminus of the polypeptide.

96. The method of claim 94, wherein the main branch structure is coupled to the C-terminus of the polypeptide.

97. The method of any one of claims 51-96, wherein the non-nucleic acid based polymer analyte comprises another main branch structure.

98. The method of claim 97, wherein the main branch structure and the another main branch structure are configured to translocate the non-nucleic acid-based polymer analyte through the nanopore in a C-terminal to N-terminal direction or an N-terminal to C-terminal direction.

99. The method of any one of claims 51-98, wherein the non-nucleic acid based polymer analyte is translocated using electroosmotic forces.

100. The method according to claim 99, further comprising: (c) providing an electrophoretic force, the electrophoretic force acting in a direction opposite to the electroosmotic force.

101. The method of claim 100, wherein the electroosmotic force pushes the non-nucleic acid-based polymer analyte through the nanopore against the electrophoretic force.

102. The method of claim 99, wherein the electroosmotic force comprises a net ionic current from the cis side to the trans side.

103. The method of any one of claims 99-102, wherein the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the nanopore system, modification of the nanopore, or any combination thereof.

104. The method of any one of claims 99-103, wherein the electroosmotic force is modulated by modification of the nanopore charge.

105. The method of any one of claims 99-104, wherein the electroosmotic force is modulated by an asymmetric salt distribution between the cis and trans sides of the membrane.

106. The method of any one of claims 51-105, wherein the nanopore system further comprises a pair of electrodes.

107. The method of claim 106, wherein the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force.

108. The method of claim 107, wherein the applied voltage is a negative voltage on the trans side.

109. The method of claim 107, wherein the applied voltage is a positive voltage on the reverse side.

110. The method of any one of claims 107-109, wherein the amplitude of the applied voltage is less than 300 mV.

111. The method of any one of claims 107-110, wherein the magnitude of the applied voltage is greater than about 20 mV.

112. The method of any one of claims 107-111, wherein the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

113. The method of any one of claims 51-112, wherein the non-nucleic acid based polymer analyte is translocated using a translocase.

114. The method of claim 113, wherein the translocase comprises an ATP-driven unfoldase.

115. The method of claim 113, wherein the translocase comprises an NTP-driven unfoldase.

116. The method of claim 115, wherein the translocase comprises an AAA+ enzyme.

117. The method of claim 116, wherein the AAA+ enzyme is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, homologs, or analogs thereof.

118. A system comprising: Fluid chamber; a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte; translocase; and A main branch structure comprising at least about one of a blocking motif, an intercepting motif, or a coupling motif, or a combination thereof, wherein the main branch structure is configured to couple to the non-nucleic acid-based polymer analyte.

119. A system comprising: Fluid chamber; a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte; and A controller operatively coupled to the fluid chamber and the nanopore, wherein during or after translocation of a non-nucleic acid-based polymer analyte coupled to a main branch structure through the nanopore using a translocase, the controller is configured to detect one or more signals associated with at least about one feature of the main branch structure and one or more signals associated with at least about one feature of the non-nucleic acid-based polymer analyte, wherein the main branch structure includes at least about one of a blocking motif, an intercepting motif, or a coupling motif, or a combination thereof.

120. The system of claim 119, wherein the controller is further configured to detect, using a pair of electrodes, one or more signals associated with at least about one feature of the main branch structure and one or more signals associated with at least about one feature of the non-nucleic acid-based polymer analyte.

121. A system according to claim 119 or claim 120, wherein the controller is further configured to separate one or more signals associated with at least about one feature of the main branch structure from one or more signals associated with at least about one feature of the non-nucleic acid-based polymer analyte.

122. The system of any one of claims 118-121, wherein the main branch structure comprises one or more nucleic acid molecules.

123. The system of any one of claims 118-122, wherein the main branch structure comprises one or more peptides.

124. The system of any one of claims 118-123, wherein the main branch structure comprises one or more nucleic acid molecules and one or more peptides.

125. The system of any one of claims 118-124, wherein the blocking motif is configured to disrupt the interaction of the translocase with the non-nucleic acid based polymer analyte.

126. The system of any one of claims 118-125, wherein the blocking motif comprises an amino acid sequence.

127. The system of claim 126, wherein the amino acid sequence comprises n repeats of (Gly)n, (Serine-Gly)n, (Glycine-Ser)n, (Alanine-Ser)n, (Alanine)n, (Valine)n, (Alanine-Ser)n, (Serine-Alanine)n, (Valine-Ser)n, or (Serine-Valine)n.

128. The system of claim 127, wherein n is greater than approximately 2, 3, 6, 9, 12, 15, 18, or 21.

129. The system of any one of claims 118-128, wherein the blocking motif comprises a non-amino acid chemical region.

130. The system of claim 129, wherein the region of non-amino acid chemistry comprises polyethylene glycol.

131. The system of any one of claims 118-130, wherein the interception motif is configured to prevent a translocase from translocating the non-nucleic acid-based polymer analyte through the interception motif.

132. The system of claim 131, wherein the interceptor motif is configured to prevent the translocase from translocating the non-nucleic acid-based polymer analyte through the main branch structure.

133. The system of claim 131, wherein the interceptor motif is configured to prevent the translocase from translocating the non-nucleic acid-based polymer analyte through the nanopore.

134. The system of any one of claims 118-133, wherein the intercepting motif comprises steric hindrance.

135. The system of claim 134, wherein the steric hindrance comprises one or more bulky amino acids.

136. The system of claim 135, wherein the one or more bulky amino acids comprise histidine, phenylalanine, tyrosine, or tryptophan.

137. The system of claim 135, wherein the steric hindrance comprises at least about one bulky amino acid.

138. The system of claim 135, wherein the steric hindrance comprises at least about five bulky amino acids.

139. The system of any one of claims 134-138, wherein the steric hindrance comprises at least about a portion of a protein that resists unfolding.

140. The system of claim 139, wherein the anti-unfolding protein comprises maltose binding protein, titin, dihydrofolate reductase, a nuclease, or a combination thereof.

141. The system of claim 139, wherein the unfolding-resistant protein comprises a disulfide bond.

142. The system of any one of claims 134-141, wherein the steric hindrance comprises a large tethering molecule.

143. The system of claim 142, wherein the large tethering molecule comprises a carbohydrate, a polycyclic molecule, a branched dextran, biotin, streptavidin, a nanobody, an antibody, or a small antigenic element.

144. The system of any one of claims 118-143, wherein the coupling motif is configured to couple the main branch structure to the non-nucleic acid based polymer analyte.

145. The system of claim 144, wherein the non-nucleic acid based polymer analyte comprises a peptide.

146. The system of claim 145, wherein the coupling motif is attached to the C-terminus of the peptide.

147. The system of claim 145, wherein the coupling motif is attached to the N-terminus of the peptide.

148. The system of any one of claims 145-147, wherein the coupling motif comprises an enzyme having peptide ligase activity.

149. The system of any one of claims 118-148, wherein the coupling motif comprises a chemical group.

150. The system of claim 149, wherein the chemical group comprises maleimide, iodoacetamide, 2-thiopyridine, 3-arylpropionitrile, NHS-ester, isocyanate, isothiocyanate, benzoyl fluoride, diazonium salt, or PTAD.

151. The system of any one of claims 118-150, wherein the coupling motif comprises an enzyme coupling region.

152. The system of claim 151, wherein the enzyme coupling region connects the coupling motif to the enzyme.

153. The system of claim 152, wherein the enzyme comprises a peptidase, a holoenzyme, or a sortase.

154. The system of any one of claims 118-153, wherein the coupling motif of the main branch structure is coupled to the non-nucleic acid based polymer analyte via a bond.

155. The system of any one of claims 118-154, wherein the coupling motif of the main branch structure is coupled to the non-nucleic acid based polymer analyte via a linker.

156. The system of any one of claims 118-155, wherein the main branch structure further comprises: (i) a recognition motif; or (ii) Capture motif.

157. The system of claim 156, wherein the capture motif comprises a polycationic tag.

158. The system of claim 157, wherein the polycationic tag comprises n repeats of (Ser-Gly-Arg)n, (Ser-Arg)n, (Arg)n, and wherein the capture motif comprises a polyanionic tag.

159. The system of claim 156, wherein the capture motif comprises a polyanionic tag.

160. The system of claim 159, wherein the polyanionic tag comprises n repeats of (Ser-Gly-Asp)n, (Ser-Asp)n, (Asp)n.

161. The system of any one of claims 156-160, wherein the recognition motif comprises a portion of ssrA, a prokaryotic ubiquitin-like protein, SulA, a peroxisomal membrane protein (Pex15), or a combination thereof.

162. The system of any one of claims 156-161, wherein the sequence of the recognition motif comprises one or more of SEQ ID NOs: 201-206.

163. The system of any one of claims 118-161, wherein the main branch structure is attached to the C-terminus or N-terminus of the non-nucleic acid-based polymer analyte.

164. The system of claim 163, wherein the non-nucleic acid-based polymer analyte comprises a polypeptide, wherein the main branch structure is added to the N-terminus of the polypeptide.

165. The system of claim 163, wherein the non-nucleic acid based polymer analyte comprises a polypeptide, wherein a main branch structure is added to the C-terminus of the polypeptide.

166. The system of any one of claims 118-165, wherein the non-nucleic acid based polymer analyte comprises a second main branch structure.

167. The system of any one of claims 118-166, wherein the first solution and the second solution are configured to generate an electro-osmotic force across the membrane.

168. The system of claim 167, wherein the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of a solute.

169. The system of claim 168, wherein the solute comprises an ion or an osmolyte.

170. The system of claim 169, wherein the difference between the first concentration of solute and the second concentration of solute is configured to generate an electro-osmotic force.

171. The system of any one of claims 167-170, wherein the electro-osmotic force comprises a net ionic current flowing from the cis side of the membrane to the trans side of the membrane.

172. The system of any one of claims 167-171, wherein the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of a membrane of the system, modification of nanopores, or any combination thereof.

173. The system of any one of claims 167-172, wherein the electroosmotic force is modulated by charge modification of the nanopore.

174. The system of any one of claims 167-173, wherein the electro-osmotic force is regulated by an asymmetric salt distribution between the cis side of the membrane and the trans side of the membrane.

175. The system of any one of claims 167-174, further comprising a pair of electrodes comprising a first electrode and a second electrode.

176. The system of claim 175, 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.

177. A system according to claim 175 or claim 176, wherein the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force across the membrane in a direction opposite to the electro-osmotic force.

178. The system of any one of claims 175-177, wherein the electroosmotic force is strong enough to translocate the non-nucleic acid based polymer analyte through the nanopore against the electrophoretic force.

179. The system of claim 177 or claim 178, wherein the applied voltage is a negative voltage on the trans side.

180. The system of claim 177 or claim 178, wherein the applied voltage is a positive voltage of the trans side and the trans side; and (ii) a non-nucleic acid polymer analyte; and (b) translocating the non-nucleic acid-based polymer analyte from the cis side to the trans side of the fluid chamber; in, The nanopore includes an adaptor protein, wherein at least about a portion of the adaptor protein is located within a channel of the nanopore.

181. The method of claim 180, wherein the adaptor protein is configured to modify the geometry of the channel of the nanopore.

182. The method of claim 181, wherein the adaptor protein is configured to constrict the channel of the nanopore.

183. The method of any one of claims 180-182, wherein the adaptor protein is configured to modify the charge of the channel of the nanopore.

184. The method of claim 183, wherein the adaptor protein is configured to modify the channel of the nanopore to have a positive net charge.

185. The method of claim 183, wherein the adaptor protein is configured to modify the channel of the nanopore to have a negative net charge.

186. The method of any one of claims 180-185, wherein the adaptor protein comprises a protein adaptor protein or a chemical adaptor protein.

187. The method of claim 186, wherein the protein adaptor protein comprises a CsgF subunit, a CsgF subunit truncation, or a CsgF subunit homolog.

188. The method of claim 186, wherein the chemical adaptor comprises a cyclodextrin, a cucurbituril, a crown ether, a calixarene, a porphyrin, a cyclosporine, a cyclem, or 1,4,8,11-tetraazacyclotetradecane.

189. The method of any one of claims 180-188, wherein the adaptor protein is coupled to the channel of the nanopore.

190. The method of claim 189, wherein the adaptor protein is coupled to the channel of the nanopore via a covalent bond.

191. The method of claim 189, wherein the adaptor protein is coupled to the channel of the nanopore via a non-covalent bond.

192. The method of claim 189, wherein the adaptor protein is coupled to the channel of the nanopore via a linker.

193. The method of any one of claims 180-192, wherein the nanopore system comprises a cis-side to trans-side electroosmotic force resulting from a net ionic current cis-side to trans-side.

194. The method according to claim 193, further comprising: An electrophoretic force is provided that acts in a direction opposite to the cis-side electroosmotic force.

195. The method of claim 194, wherein the electroosmotic force from the cis side to the trans side is strong enough to push the non-nucleic acid based polymer analyte through the nanopore against the electrophoretic force.

196. The method of any one of claims 180-195, wherein the non-nucleic acid based polymer analyte is translocated through the nanopore using electroosmotic forces.

197. The method according to claim 196, further comprising: An electrophoretic force is provided that acts in a direction opposite to the electroosmotic force.

198. The method of claim 197, wherein the electroosmotic force propels the non-nucleic acid based polymer analyte through the nanopore against the electrophoretic force.

199. The method of any one of claims 196-198, wherein the electroosmotic force comprises a net ionic current from the cis side to the trans side.

200. The method of any one of claims 196-199, wherein the electroosmotic force is modulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the nanopore system, modification of the nanopore, or any combination thereof.

201. The method of any one of claims 196-200, wherein the electroosmotic force is modulated by modifying the charge of the nanopore.

202. The method of any one of claims 196-201, wherein the electro-osmotic force is modulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

203. The method of any one of claims 180-202, wherein the nanopore system further comprises a pair of electrodes.

204. The method of claim 203, wherein the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force.

205. The method of claim 204, wherein the applied voltage is a negative voltage on the trans side.

206. The method of claim 204, wherein the applied voltage is a positive voltage on the reverse side.

207. The method of any one of claims 204-206, wherein the amplitude of the applied voltage is less than 300 mV.

208. The method of any one of claims 204-207, wherein the amplitude of the applied voltage is greater than about 20 mV.

209. The method of any one of claims 201-208, wherein the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

210. The method of any one of claims 180-209, wherein the non-nucleic acid based polymer analyte is translocated using a translocase.

211. The method of claim 210, wherein the translocase comprises an ATP-driven unfoldase.

212. The method of claim 210, wherein the translocase comprises an NTP-driven unfolding enzyme.

213. The method of claim 212, wherein the translocase comprises an AAA+ enzyme.

214. The method of claim 213, wherein the AAA+ enzyme is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, orthologs, or analogs thereof.

215. A system comprising: (a) Fluid chamber; and (b) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, wherein the first solution and the second solution are configured to translocate a non-nucleic acid-based polymer analyte; Wherein, the nanopore comprises an adaptor protein within the channel of the nanopore.

216. The system of claim 215, wherein the adaptor protein comprises a protein adaptor protein or a chemical adaptor protein.

217. The system of claim 216, wherein the protein adaptor protein comprises a CsgF subunit, a CsgF subunit truncation, or a CsgF subunit homolog.

218. The system of claim 216, wherein the chemical adaptor protein comprises a cyclodextrin, a cucurbituril, a crown ether, a calixarene, a porphyrin, a cyclosporine, a cyclem, or a 1,4,8,11-tetraazacyclotetradecane.

219. The system of any one of claims 215-218, wherein the adaptor protein is coupled to the channel of the nanopore.

220. The system of claim 219, wherein the adaptor protein is coupled to the channel of the nanopore via a covalent bond.

221. The system of claim 219, wherein the adaptor protein is coupled to the channel of the nanopore via a non-covalent bond.

222. The system of claim 219, wherein the adaptor protein is coupled to the channel of the nanopore via a linker.

223. The system of any one of claims 215-222, wherein the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of a solute.

224. The system of claim 223, wherein the solute comprises an ion or an osmolyte.

225. The system of claim 223, wherein the difference between the first concentration of solute and the second concentration of solute is configured to generate an electroosmotic force.

226. The system of any one of claims 215-225, wherein the first solution and the second solution are configured to generate an electro-osmotic force across a membrane.

227. The system of claim 226, wherein the electro-osmotic force is generated by a net ionic current flowing from the cis side to the trans side of the membrane.

228. The system of claim 226 or claim 227, further comprising an electrophoretic force acting in a direction opposite to the electroosmotic force, wherein the electroosmotic force is sufficiently strong to propel the non-nucleic acid based polymer analyte through the nanopore against the electrophoretic force.

229. The system of any one of claims 226-228, wherein the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of a membrane of the system, modification of nanopores, or any combination thereof.

230. The system of any one of claims 226-229, wherein the electroosmotic force is modulated by modifying the charge of the nanopore.

231. The system of any one of claims 226-230, wherein the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

232. The system of any one of claims 215-231, further comprising a pair of electrodes.

233. A system according to claim 232, wherein the first electrode of the pair of electrodes is disposed on the cis side and the second electrode of the pair of electrodes is disposed on the trans side of the membrane.

234. The system of claim 232 or claim 233, wherein the pair of electrodes is configured to detect a signal during translocation of the non-nucleic acid based polymer analyte.

235. The system of claim 234, wherein the signal is associated with a characteristic of the non-nucleic acid based polymer analyte.

236. The system of any one of claims 232-235, wherein the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force.

237. The system of claim 236, wherein the applied voltage is a negative voltage on the trans side.

238. The system of claim 236, wherein the applied voltage is a positive voltage on the reverse side.

239. The system of any one of claims 236-238, wherein the amplitude of the applied voltage is less than 300 mV.

240. The system of any one of claims 236-239, wherein the magnitude of the applied voltage is greater than about 20 mV.

241. The system of any one of claims 236-240, wherein the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

242. A method comprising: (a) Provide: (i) A nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a cis side and a trans side; (b) adding a combined solution to the cis side of the fluid chamber, wherein the combined solution comprises a non-nucleic acid-based polymer analyte and a preload solution; and (c) translocating the non-nucleic acid-based polymer analyte from the cis side to the trans side of the fluid chamber.

243. The method of claim 242, wherein prior to (b), further comprising combining the sample comprising the non-nucleic acid-based polymer analyte with a preload solution.

244. The method of claim 242 or claim 243, wherein the preload solution comprises a translocase.

245. The method of claim 244, wherein the non-nucleic acid based polymer analyte is translocated using the translocase.

246. The method of claim 245, wherein the translocase comprises an ATP-driven unfoldase.

247. The method of claim 245, wherein the translocase comprises an NTP-driven unfolding enzyme.

248. The method of claim 257, wherein the translocase comprises an AAA+ enzyme.

249. The method of claim 248, wherein the AAA+ enzyme is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, orthologs, or analogs thereof.

250. The method of any one of claims 245-249, wherein the sample and the preload solution are combined to form a non-nucleic acid based polymer analyte-translocase complex.

251. The method of any one of claims 245-250, wherein the sample and the preload solution are combined to form a non-nucleic acid based polymer analyte-backbone structure complex.

252. The method of any one of claims 242-251, wherein the preload solution comprises a main branch structure.

253. The method of any one of claims 242-252, wherein the preload solution comprises a chemical that enhances binding of the non-nucleic acid based polymer analyte to a component of the preload solution.

254. The method of claim 253, wherein the binding of the non-nucleic acid based polymer analyte to components of the loading solution is higher than the binding of the non-nucleic acid based polymer analyte to components in the fluid chamber.

255. The method of any one of claims 242-253, wherein the preload solution comprises one or more cofactors.

256. The method of claim 255, wherein the one or more cofactors comprise NTP, M 2+ , NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, polysucrose, manganese iron, cobalt, copper, penicillamine, trientine, ethylenediaminetetraacetic acid.

257. The method of any one of claims 242-256, wherein the non-nucleic acid based polymer analyte is translocated using electroosmotic forces.

258. The method of claim 257, wherein the translocation comprises providing an electrophoretic force acting in a direction opposite to the electroosmotic force.

259. The method of claim 258, wherein the electroosmotic force pushes the non-nucleic acid based polymer analyte through the nanopore against the electrophoretic force.

260. The method of any one of claims 257-259, wherein the electroosmotic force comprises a net ionic current from the cis side to the trans side.

261. The method of any one of claims 257-260, wherein the electroosmotic force is modulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the nanopore system, modification of the nanopore, or any combination thereof.

262. The method of any one of claims 257-261, wherein the electroosmotic force is modulated by modifying the charge of the nanopore.

263. The method of any one of claims 257-262, wherein the electro-osmotic force is modulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

264. The method of any one of claims 242-263, wherein the nanopore system further comprises a pair of electrodes.

265. The method of claim 264, wherein the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force.

266. A method according to claim 265, wherein the applied voltage is a negative voltage on the trans side.

267. A method according to claim 265, wherein the applied voltage is a positive voltage on the reverse side.

268. A method according to any one of claims 265-267, wherein the amplitude of the applied voltage is less than 300mV.

269. The method of any one of claims 265-268, wherein the amplitude of the applied voltage is greater than about 20 mV.

270. The method of any one of claims 262-269, wherein the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

271. A system comprising: Fluid chamber; and a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into (i) a cis side comprising a first solution and (ii) a trans side comprising 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; and A preloading solution is configured to interact with the non-nucleic acid based polymer analyte.

272. The system of claim 271, wherein the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of a solute.

273. The system of claim 272, wherein the solute comprises an ion or an osmolyte.

274. The system of claim 272, wherein the difference between the first concentration of solute and the second concentration of solute is configured to generate an electroosmotic force.

275. The system of any one of claims 271-274, wherein the first solution and the second solution are configured to generate an electro-osmotic force across the membrane.

276. The system of claim 275, wherein the electro-osmotic force is generated by a net ionic current flowing from the cis side to the trans side of the membrane.

277. The system of claim 275 or 276, wherein the electroosmotic force is regulated by pH, salt type, salt concentration, transmembrane osmotic pressure of the membrane of the system, modification of nanopores, or any combination thereof.

278. The system of any one of claims 275-277, wherein the electroosmotic force is modulated by modifying the charge of the nanopore.

279. The system of any one of claims 275-278, wherein the electro-osmotic force is regulated by an asymmetric salt distribution between the cis side and the trans side of the membrane.

280. The system of any one of claims 275-279, further comprising a pair of electrodes disposed on the cis side and the trans side of the membrane, wherein the pair of electrodes are configured to provide an applied voltage to generate an electrophoretic force across the membrane in a direction opposite to the electroosmotic flow.

281. The system of claim 280, wherein the applied voltage is a negative voltage on the trans side.

282. The system of claim 280, wherein the applied voltage is a positive voltage on the reverse side.

283. The system of any one of claims 280-282, wherein the amplitude of the applied voltage is less than 300 mV.

284. The system of any one of claims 280-283, wherein the amplitude of the applied voltage is greater than about 20 mV.

285. The system of any one of claims 280-284, wherein the absolute relative net electroosmotic current at the applied voltage is greater than about 0.10 pA / mV.

286. The system of any one of claims 271-285, wherein the preload solution comprises one or more cofactors.

287. The system of claim 286, wherein the one or more cofactors include NTP, M 2+ , NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, polysucrose, manganese iron, cobalt, copper, penicillamine, calcium edetate, and ethylenediaminetetraacetic acid.

288. The system of any one of claims 271-287, wherein the preload solution comprises a translocase.

289. The system of claim 288, wherein the translocase comprises an ATP-driven unfolding enzyme.

290. The system of claim 288, wherein the translocase comprises an NTP-driven unfolding enzyme.

291. The system of claim 290, wherein the translocase comprises an AAA+ enzyme.

292. The system of claim 291, wherein the AAA+ enzyme is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Mspl, and SecA.

293. The system of any one of claims 271-292, wherein the preload solution comprises a main branch structure.

294. The system of any one of claims 271-293, wherein the preload solution comprises a chemical that enhances binding of the non-nucleic acid based polymer analyte to components of the preload solution relative to binding in solution on the cis side of the fluid chamber.

295. The method of any one of claims 1-25, 51-117, 180-214, or 242-270, wherein the nanopore has an ion selectivity P greater than about 2.

0. (+) / P (-) .

296. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295, wherein the nanopore has an ion selectivity P of less than 0.

50. (+) / P (-) .

297. The method of any one of claims 1-25, 51-117, 180-214, 242-270, 295 or 296, wherein the non-nucleic acid based polymer analyte is an unmodified (unlabeled) non-nucleic acid based polymer analyte.

298. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-297, wherein the termini of the non-nucleic acid-based polymer analyte lack a three-dimensional structure.

299. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-298, wherein at least about a portion of the non-nucleic acid based polymer analyte is denatured.

300. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-299, wherein the non-nucleic acid-based polymer analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof.

301. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-300, wherein the non-nucleic acid-based polymer analyte comprises a polypeptide, a polysaccharide, or a water-soluble plastic.

302. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-301, wherein the non-nucleic acid based polymer analyte comprises a polypeptide.

303. The method of claim 302, wherein the polypeptide comprises at least about 30 peptide units.

304. The method of claim 303, wherein at least 30 of the peptide units comprise positively charged residues.

305. The method of claim 303, wherein the at least about 30 peptide units comprise negatively charged residues.

306. The method of claim 303, wherein the at least about 30 peptide units comprise positively charged residues and negatively charged residues.

307. The method of any one of claims 303-306, wherein the polypeptide is in a denatured state.

308. The method of any one of claims 303-306, wherein the polypeptide is provided in a folded state.

309. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-308 further comprising measuring a signal generated by translocating a non-nucleic acid based polymer analyte through the nanopore.

310. The method of claim 309, wherein the measuring comprises: Signals are measured for: (i) an open channel of the nanopore; (ii) capture of the non-nucleic acid based polymer analyte by the nanopore; or (iii) passage of the non-nucleic acid based polymer analyte through the nanopore.

311. A system according to claim 310, wherein the measuring includes detecting the difference between states (i), (ii) and (iii).

312. The method of claim 309, wherein the signal comprises an ionic current, a change in an ionic current, or a derivative thereof.

313. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-312, wherein the nanopore comprises a biological nanopore.

314. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-313, wherein the nanopore comprises an internal pore constriction of from about 0.5 nM to about 2 nM.

315. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-314, wherein the nanopore comprises an α-helical oligomeric pore structure.

316. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-315, wherein the nanopore comprises a β-barrel oligomeric pore structure.

317. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-316, wherein the nanopore comprises a recombinant nanopore.

318. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-317, wherein the nanopore comprises a protein selected from the group consisting of hemolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, phage-derived portal protein, modified variants thereof, or ion-selective mutants thereof.

319. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-318, wherein the nanopore comprises a biological nanopore.

320. The method of claim 319, wherein the biological nanopore is modified to restrict the passage of one or more ions through the nanopore.

321. The method of claim 320, wherein the biological nanopore restricts passage of one or more ions through the nanopore by modifying the charge of the nanopore's channel.

322. The method of claim 320, wherein the net charge is negative.

323. The method of claim 320, wherein the net charge is positive.

324. The method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-323, wherein the nanopore is a mutant CytK nanopore.

325. The method of claim 324, wherein the mutant CytK comprises one or more amino acid substitutions.

326. The method of claim 325, wherein the one or more amino acid substitutions comprise K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof.

327. The method of claim 324, wherein the one or more amino acid substitutions comprise K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof.

328. The method of claim 324, 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.

329. The method of claim 324, wherein the mutant CytK nanopore comprises one or more combinations of the following amino acid substitutions: (i) S120D, G122D or K155D; (ii) S120D is used in combination with K128F / K128D; (iii) Q122D or S151D; (iv) K128D or K128F; (v) S120D, K115D, Q122D; (vi) K128F, S120D, G122D; and (vii)K128F, S120D, G122D, K155D.

330. The system of any one of claims 26-50, 118-180, 219-244, or 273-296, wherein the nanopore has an ion selectivity P greater than about 2.

0. (+) / P (-) .

331. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330, wherein the nanopore has an ion selectivity P of less than 0.

50. (+) / P (-) .

332. The system of any one of claims 26-50, 118-180, 215-241, 271-294, 330, or 331, wherein the non-nucleic acid based polymer analyte is an unmodified (unlabeled) non-nucleic acid based polymer analyte.

333. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-332, wherein the termini of the non-nucleic acid based polymer analyte lack a three-dimensional structure.

334. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-333, wherein at least about a portion of the non-nucleic acid based polymer analyte is denatured.

335. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-334, wherein the non-nucleic acid-based polymer analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof.

336. The system of any one of claims 26-50, 118-180, 215-241, 271-264, or 330-335, wherein the non-nucleic acid-based polymer analyte comprises a polypeptide, a polysaccharide, or a water-soluble plastic.

337. The system of any of claims 26-50, 118-180, 215-241, 271-294, or 330-336, wherein the non-nucleic acid-based polymer analyte comprises a polypeptide.

338. The system of claim 337, wherein the polypeptide comprises at least about 30 peptide units.

339. The system of claim 337, wherein the at least about 30 peptide units comprise positively charged residues.

340. The system of claim 337, wherein the at least about 30 peptide units comprise negatively charged residues.

341. The system of claim 337, wherein the at least about 30 peptide units comprise positively charged residues and negatively charged residues.

342. The system of any one of claims 337-341, wherein the polypeptide is in a denatured state.

343. The system of any one of claims 337-341, wherein the polypeptide is provided in a folded state.

344. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-343, further comprising measuring a signal generated by translocating the non-nucleic acid based polymer analyte through the nanopore.

345. The system of claim 344, wherein the measuring comprises: Signals are measured for: (i) an open channel of the nanopore; (ii) capture of the non-nucleic acid based polymer analyte by the nanopore; or (iii) passage of the non-nucleic acid based polymer analyte through the nanopore.

346. A system according to claim 345, wherein measuring includes detecting differences between states (i), (ii) and (iii).

347. A system according to claim 344, wherein the signal comprises an ionic current, a change in ionic current, or a derivative thereof.

348. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-347, wherein the nanopore comprises a biological nanopore.

349. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-348, wherein the nanopore comprises an internal pore constriction from about 0.5 nm to about 2 nm.

350. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-349, wherein the nanopore comprises an α-helical oligomeric pore structure.

351. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-350, wherein the nanopore comprises a β-barrel oligomeric pore structure.

352. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-351, wherein the nanopore comprises a recombinant nanopore.

353. The system of any one of claims 26-50, 118-180, 245-241, 271-294, or 330-352, wherein the nanopore comprises the following protein: hemolysin (Aer), cytolysin K (CytK), MspA, α-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, phage-derived portal protein, a modified variant thereof, or an ion-selective mutant thereof.

354. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-353, wherein the nanopore comprises a biological nanopore.

355. The system of claim 354, wherein the biological nanopore is modified to restrict the passage of one or more ions through the nanopore.

356. The system of claim 355, wherein the biological nanopore restricts passage of one or more ions through the channel of the nanopore by modifying the charge of the channel of the nanopore.

357. The system of claim 355, wherein the net charge is negative.

358. The system of claim 355, wherein the net charge is positive.

359. The system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-358, wherein the nanopore is a mutant CytK nanopore.

360. The system of claim 359, wherein the mutant CytK comprises one or more amino acid substitutions.

361. The system of claim 360, wherein the one or more amino acid substitutions comprise K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof.

362. The system of claim 359, wherein the one or more amino acid substitutions comprise K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof.

363. The system of claim 359, 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.

364. The system of claim 359, wherein the mutant CytK nanopore comprises one or more combinations of the following amino acid substitutions: (i) S120D, G122D or K155D; (ii) Combination of S120D and K128F / K128D; (iii) Q122D or S151D; (iv) K128D or K128F; (v) S120D, K115D, Q122D; (vi) K128F, S120D, G122D; and (vii)K128F, S120D, G122D, K155D.

365. An apparatus comprising an array of systems comprising a system according to any one of claims 26-50, 118-180, 214-241, 271-294 or 330-364.

366. Use of the method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-329 to characterize at least about one structural feature of the non-nucleic acid based polymer analyte.

367. Use of the method of any one of claims 1-25, 51-117, 180-214, 242-270, or 295-329 for analyzing the amino acid sequence or amino composition of one or more non-nucleic acid based polymer analytes at the single molecule level.

368. Use of the system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-363 to characterize at least about one structural feature of the non-nucleic acid based polymer analyte.

369. Use of the system of any one of claims 26-50, 118-180, 215-241, 271-294, or 330-363 for analyzing the amino acid sequence or amino composition of one or more of said non-nucleic acid based polymer analytes at the single molecule level.

370. A method for translocating a target protein through a nanopore, said nanopore being contained in a membrane separating a fluidic chamber of said nanopore system into a cis side and a trans side, comprising: (a) preparing a protein translocase solution, optionally in the presence of NTPs, to capture and form a complex with the target protein to be translocated; (b) contacting the translocase-target protein complex with the cis side of the nanopore and allowing the target protein to translocate to the trans side; The nanopore system has a cis-to-trans electroosmotic force (EOF), generating a cis-to-trans net ionic current, whereby the target protein is captured in the nanopore, and the protein translocase at the top of the nanopore controls the translocation.

371. The method of claim 370, wherein the nanopore system has a cis-to-trans EOF determined by the ratio of the net ionic current cis-to-trans to the total ionic current (I rel ) is greater than 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, most preferably greater than 0.35 or less than -0.

35.

372. A method according to claim 370 or claim 371, wherein the cis-to-trans EOF is set by adjusting the pH, type and / or concentration and / or osmotic pressure of salts passing through the nanopore system membrane, by modifying (e.g., genetic engineering) the nanopore charge, or any combination thereof, preferably by modifying the nanopore and / or asymmetric salt distribution between the cis side and the trans side of the fluid chamber.

373. The method of any one of claims 370-372, wherein the translocase-target protein complex is formed in solution on the cis side of the fluid chamber.

374. The method of any one of claims 370-373, wherein the translocase-target protein complex is formed in a separate step in solution prior to adding the complex to the cis side of the fluid chamber to contact the nanopore.

375. The method of any one of claims 370-374, wherein the target protein comprises a backbone structure at its N- and / or C-terminus to allow preloading and optionally arrest translocation of one or more proteins.

376. A method according to claim 375, wherein the main branch structure includes (i) a recognition motif for the protein translocase, and more preferably further includes one or more of the following elements: (ii) a capture motif; (iii) a blocking motif; (iv) an interception motif.

377. A nanopore system for transporting a target protein through a nanopore, comprising: (a) A membrane having a nanopore therein, the membrane separating a chamber into a cis side and a trans side, wherein a target protein is added to the cis side and translocated to the trans side through the nanopore; (b) a target protein captured on the cis side of the chamber by a protein translocase that can sequentially bind to and translocate the target protein through the nanopore; (c) means for providing a voltage difference between the cis side and the trans side of the membrane; wherein the nanopore system has a cis-to-trans electroosmotic force (EOF), the electroosmotic force is generated by a cis-to-trans net ionic current, whereby the target protein is captured at the nanopore, and the translocase controls the translocation at the nanopore, preferably wherein the nanopore system has a cis-to-trans EOF, the electroosmotic force is generated by a cis-to-trans net ionic current, the total ionic current of the net ionic 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.

378. A nanopore system according to claim 377, further comprising a method for measuring a signal based on an ionic current passing through the nanopore during translocation, wherein the measurement method detects a change in the signal that is characteristic of the protein as it translocates.

379. The method or nanopore system of any one of claims 370-378, wherein the nanopore system has ion selectivity. (+) / P (-) Greater than 2.0 or less than 0.5, preferably greater than 2.5 or less than 0.4, most preferably greater than 3.0 or less than 0.

33.

380. The method or nanopore system of any one of claims 370-379, wherein the nanopore is a biological nanopore, preferably having an internal pore constriction in the range of 0.5-2 nM, more preferably wherein the nanopore is an α-helical or β-barrel oligomeric pore forming toxin or porin.

381. The method or nanopore system of claim 380, wherein the nanopore is selected from the group consisting of Aerolysin (Aer), Cytolysin K (CytK), MspA, α-Hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, phage-derived portal proteins, and modified variants thereof, preferably wherein the nanopore is modified to have a net charge in the lumen-facing region of >21, preferably >28, more preferably >35, most preferably wherein the net charge is negative.

382. The method or nanopore system of claim 380, wherein the nanopore is a mutant CytK nanopore comprising one or more amino acid substitutions selected from the group consisting of K128D, K128F, K115D, S120D, Q122D and S151D, preferably comprising one of the following combinations of amino acid substitutions: S120D, G122D and / or K155D; a combination of S120D and K128F / K128D, preferably further comprising Q122D or S151D; K128D / K128F, S120D, K115D, Q122D; K128F, S120D, G122D, optionally in combination with K155D.

383. The method or nanopore system of any one of claims 370-382, wherein the protein translocase is an NTP-driven unfolding enzyme, preferably an AAA+ enzyme.

384. The method or nanopore system of claim 383, wherein the AAA+ enzyme is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, (ClpY), LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, homologs, or analogs thereof.

385. The method or nanopore system of any one of claims 370-384, wherein the nanopore system has ion selectivity. (+) / P (-) Greater than about 2.0, preferably greater than 2.5, more preferably greater than 3.0, with a negative applied voltage on the trans side.

386. The method or nanopore system of claim 385, wherein the system comprises a cation selective (mutant) nanopore.

387. An analytical device comprising a nanopore system array according to any one of claims 377-386.

388. Use of a nanopore system or device as described in any one of claims 377-387 for characterizing at least about one structural feature of a target protein, preferably for analyzing the amino acid sequence or amino composition of one or more target proteins at the single molecule level.

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