Nanopore-based analysis of proteins

Abstract

This disclosure relates to systems and methods for the analysis of proteins, and more specifically to nanopore systems, devices and methods for the analysis and sequencing of single-molecule proteins. A method is provided for dislocating a target protein through nanopores, wherein the nanopores are contained in a membrane that separates the fluid chamber of the nanopore system into a cis side and a trans side, and the method comprises (a) enabling a protein translocase in solution to capture the target protein to be dislocated and form a complex with it, and (b) bringing the translocase-target protein complex into contact with the cis side of the nanopore to enable the dislocation of the target protein to the trans side, wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) resulting from a large net ionic current flow from cis to trans compared to the total ionic current flow, and as a result the target protein is captured within the nanopores using a translocase that controls the dislocation located at the top of the nanopore.

Description

[Technical Field]

[0001] Embedding by reference This application claims the benefits of European application No. EP22204590.8, filed on 28 October 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Determining the characteristics of an analyte is a crucial aspect of scientific research. These characteristics may be important for further scientific research or clinical applications. [Overview of the project]

[0003] In one embodiment, the present disclosure provides a method comprising: (a) a nanopore system comprising a fluid chamber and a membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side and a trans side; (b) contacting a complex comprising a non-nucleoside polymer analyte and a trans-locase with the cis side of the nanopores; and (c) dislocating the non-nucleoside polymer analyte to the trans side of the fluid channel using electroosmotic force from the cis side to the trans side, wherein the electroosmotic force from the cis side to the trans side dislocates the trans-locase of the complex to the cis side inlet of the channel of the nanopores.

[0004] In some embodiments, prior to (c), the method further comprises contacting a non-nucleic acid polymer analyte with a translocase to form a complex. In some embodiments, the complex is formed on the cis side of the fluid chamber. In some embodiments, the electroosmotic force from the cis side to the trans side includes a net flow of ionic current from the cis side to the trans side. In some embodiments, the electroosmotic force from the cis side to the trans side is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane, modification of the nanopores, or any combination thereof. In some embodiments, modification of the nanopores includes modification of the charge of the nanopores. In some embodiments, the electroosmotic force from the cis side to the trans side is regulated by an asymmetric salt distribution between the cis and trans sides of the fluid chamber. In some embodiments, the complex is formed in the solution on the cis side of the fluid chamber. In some embodiments, the complex is formed before contacting the complex with the cis side of the nanopores.

[0005] In some embodiments, the translocase includes an adenosine triphosphate (ATP)-driven unfoldase. In some embodiments, the translocase includes an nucleotide triphosphate (NTP)-driven unfoldase. In some embodiments, the translocase includes ATPases associated with various cell-active (AAA+) enzymes. In some embodiments, the AAA+ enzymes include ATP-dependent Clp protease ATP-binding subunit ClpX (ClpX) and ClpX-like proteases, ATP-dependent Clp protease ATP-binding subunit ClpA (ClpA), proteasome-activated nucleotidase (PAN), LON protease, VCP-like ATPase (VAT), AMA, 854, membrane-bound AAA (MBA), small-molecule archaeal ubiquitin-like modified protein (SAMP), ATP-dependent Clp protease ATP-binding subunit ClpC (ClpC), ATP-dependent Clp protease ATP-binding subunit ClpE (ClpE), and ATP-dependent proteases. The following are selected from the group consisting of the ATPase subunit HslU (HsIU), casein-degrading mitochondrial matrix peptidase chaperone subunits Y (ClpY), LonA, LonB, ATP-dependent zinc metalloproteinase FtsH (FtsH), proteasome-associated ATPase (Mpa), cell division cycle protein 48 (also called Cdc48, p97, and VCP) and actinomycete Cdc48-like protein (Cpa), outer mitochondrial transmembrane helix translocase (Msp1), protein translocase subunit SecA (SecA), and their functional homologs, orthologs, or paralogs.

[0006] In some embodiments, the system further includes a pair of electrodes. In some embodiments, the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side.

[0007] In some embodiments, the magnitude of the applied voltage is less than 300 millivolts (mV). In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the absolute relative net electroosmotic current with respect to the applied voltage is greater than approximately 0.10 picoamperes / millivolts (pA / mV).

[0008] In some embodiments, the non-nucleoside polymer analyte includes a leader construct at its N-terminus or C-terminus. In some embodiments, the leader construct is configured to couple one or more translocases to the non-nucleoside polymer analyte. In some embodiments, the leader construct is configured to stall one or more translocases. In some embodiments, the leader construct includes a recognition motif. In some embodiments, the leader construct further includes a capture motif, a stall motif, a blocking motif, or a combination thereof.

[0009] In another embodiment, the present disclosure provides a system comprising a fluid chamber and a membrane having nanopores that separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, wherein the first and second solutions are configured to generate electroosmotic force, which is configured to couple the translocase of a complex to the cis-side inlet of a channel in the nanopore, and the complex comprises a non-nucleic acid polymer analyte and a translocase.

[0010] In some embodiments, the system further comprises a translocase. In some embodiments, the translocase comprises an ATP-driven unfoldase. In some embodiments, the translocase comprises an NTP-driven unfoldase. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, 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 their functional homologs, orthologues, and paralogs.

[0011] In some embodiments, the translocase is configured to rearrange non-nucleic acid polymer analytes through nanopores in a sequential order. In some embodiments, the first solution contains a first concentration of solute, and the second solution contains a second concentration of solute. In some embodiments, the solute contains ions or osmolite. In some embodiments, the difference between the first and second concentrations of solute is configured to generate electroosmosis. In some embodiments, the electroosmosis includes a net ionic current flow from the cis side to the trans side. In some embodiments, the electroosmosis is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the system, modification of nanopores, or any combination thereof. In some embodiments, the electroosmosis is regulated by modification of the charge of the nanopores. In some embodiments, the electroosmosis is regulated by an asymmetric salt distribution between the cis and trans sides of the membrane.

[0012] In some embodiments, the system further includes a pair of electrodes. In some embodiments, the first electrode of the pair of electrodes is positioned on the cis side, and the second electrode of the pair of electrodes is positioned on the transform side of the membrane. In some embodiments, the pair of electrodes is configured to detect a signal during dislocation of a non-nucleoside polymer analyte. In some embodiments, the signal is associated with a feature of the non-nucleoside polymer analyte. In some embodiments, the pair of electrodes is configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the net electroosmotic current absolute relative to the applied voltage is greater than about 0.10 pA / mV. In some embodiments, the signal includes an ionic current or a change thereof.

[0013] In another embodiment, the Disclosure provides a nanopore system comprising a nanopore system comprising a fluid chamber and a membrane comprising nanopores, wherein the membrane separates the fluid chamber into a cis side and a trans side; a non-nucleic acid polymer analyte, wherein the non-nucleic acid polymer analyte is coupled to a leader construct comprising a stall motif, a block motif, a coupling motif, or a combination thereof; and a translocase; and a method comprising dislocating the non-nucleic acid polymer analyte from the cis side to the trans side of the fluid chamber.

[0014] In some embodiments, the leader construct comprises nucleic acids. In some embodiments, the leader construct comprises peptides. In some embodiments, the leader construct comprises nucleic acids and peptides. In some embodiments, the stall motif is configured to disrupt the interaction between the translocase and the non-nucleoside polymer analyte. In some embodiments, the stall 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)n, (valine)n, (alanine-serine)n, (serine-alanine)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 stall motif comprises a non-amino acid chemistry region. In some embodiments, the non-amino acid chemistry region comprises polyethylene glycol.

[0015] In some embodiments, the block motif is configured to prevent the translocase from rearranging the non-nucleoside polymer analyte beyond the block motif. In some embodiments, the block motif is configured to prevent the translocase from rearranging the non-nucleoside polymer analyte beyond the leader construct. In some embodiments, the block motif is configured to prevent the translocase from rearranging the non-nucleoside polymer analyte through nanopores. In some embodiments, the block motif includes steric hindrance. In some embodiments, the steric hindrance includes one or more bulky amino acids. In some embodiments, the one or more bulky amino acids include histidine, phenylalanine, tyrosine, or tryptophan. In some embodiments, the steric hindrance includes at least one bulky amino acid. In some embodiments, the steric hindrance includes at least five bulky amino acids. In some embodiments, the steric hindrance includes at least a portion of a stretch-resistant protein. In some embodiments, the stretch-resistant protein includes a maltose-binding protein, titin, dihydrofolate reductase, burnase, or a combination thereof. In some embodiments, the deficiency-resistant protein includes a disulfide bond. In some embodiments, the steric hindrance includes a large binding molecule. In some embodiments, the large binding molecule includes a carbohydrate, a polycyclic molecule, a branched dextran, biotin, streptavidin, a nanobody, an antibody, or a small antigenic element.

[0016] In some embodiments, the coupling motif is configured to couple a leader construct to a non-nucleoside polymer analyte. In some embodiments, the non-nucleoside polymer analyte includes a peptide. In some embodiments, the coupling motif attaches to the C-terminus of the peptide. In some embodiments, the coupling motif attaches to the N-terminus of the peptide. In some embodiments, the coupling motif includes a recognition sequence that can be recognized by an enzyme having peptide ligase activity. In some cases, the enzyme can interact with the recognition sequence within the coupling motif. In some embodiments, the coupling motif includes a chemical group. In some embodiments, the chemical group includes maleimide, iodoacetamide, 2-thiopyridine, 3-arylpropioronitrile, NHS ester, isocyanate, isothiocyanate, benzoyl fluoride, diazonium salt, or PTAD. In some embodiments, the coupling motif includes an enzyme coupling region. In some embodiments, the enzyme coupling region causes the coupling motif to attach to an enzyme. In some cases, the enzyme can interact with the coupling motif.

[0017] In some embodiments, the enzyme includes peptiligase, omniligase, or saltase. In some embodiments, the coupling motif of the leader construct is coupled to a non-nucleoside polymer analyte via a covalent bond. In some embodiments, the coupling motif of the leader construct is coupled to a non-nucleoside polymer analyte via a linker.

[0018] In some embodiments, the reader construct further includes at least one of a recognition motif or a capture motif.

[0019] In some embodiments, the capture motif includes a polycation tag. In some embodiments, the polycation tag includes n repeats of (serine-glycine-arginine)n, (serine-arginine)n, (arginine)n. In some cases, the polycation tag may include serine, glycine, arginine, lysine, or any combination thereof. In some embodiments, the capture motif includes a polyanion tag. In some embodiments, the polyanion tag includes n repeats of (serine-glycine-aspartic acid)n, (serine-aspartic acid)n, (aspartic acid)n, (serine-glycine-glutamic acid)n, (serine-glutamic acid)n, or (glutamic acid)n. In some cases, the polyanion tag may include serine, glycine, aspartic acid, glutamic acid, or any combination thereof. In some embodiments, the recognition motif includes ssrA, a prokaryotic ubiquitin-like protein, SulA, a peroxisome membrane protein (Pex15), or a portion of a combination thereof. In some embodiments, the sequence of the recognition motif includes one or more of sequence numbers 101-206.

[0020] In some embodiments, the leader construct is attached to the C-terminus or N-terminus of a non-nucleoside polymer analyte. In some embodiments, the non-nucleoside polymer analyte comprises a polypeptide. In some embodiments, the leader construct is coupled to the N-terminus of the polypeptide. In some embodiments, the leader construct is coupled to the C-terminus of the polypeptide. In some embodiments, the non-nucleoside polymer analyte comprises another leader construct. In some embodiments, the leader construct and the other leader construct are configured to displace the non-nucleoside polymer analyte through nanopores in the C-terminus to N-terminus direction, N-terminus to C-terminus direction, or C-terminus to N-terminus direction and N-terminus to C-terminus direction. In some embodiments, the non-nucleoside polymer analyte is displaced using electroosmosis.

[0021] In some embodiments, the method further includes providing electrophoretic force acting in the opposite direction to electroosmosis. In some embodiments, electroosmosis pushes non-nucleic acid polymer analytes through nanopores against electrophoretic force. In some embodiments, electroosmosis includes a net ionic current flow from the cis side to the trans side. In some embodiments, electroosmosis is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the nanopore system, modification of the nanopores, or any combination thereof. In some embodiments, electroosmosis is regulated by modification of the charge of the nanopores. In some cases, the charge of the nanopores may be modified at the cis inlet of the channel. In some cases, the charge of the nanopores may be modified at the trans inlet of the channel. In some cases, the charge of the nanopores may be modified within the central channel of the nanopores. In some embodiments, electroosmosis is regulated by an asymmetric salt distribution between the cis and trans sides of the membrane.

[0022] In some embodiments, the nanopore system further includes a pair of electrodes. In some embodiments, the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the net electroosmotic current absolute relative to the applied voltage is greater than about 0.10 pA / mV.

[0023] In some embodiments, non-nucleoside polymer analytes are rearranged using a translocase. In some embodiments, the translocase comprises an ATP-driven unfoldase. In some embodiments, the translocase comprises an NTP-driven unfoldase. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, 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 their functional homologs, orthologues, and paralogs.

[0024] In another embodiment, the present disclosure provides a system comprising: a fluid chamber; a membrane comprising nanopores, wherein the membrane is configured to separate the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, the first and second solutions being configured to rearrange a non-nucleic acid polymer analyte; and a leader construct comprising a translocase and at least one of a stall motif, a block motif, or a coupling motif, or a combination thereof, wherein the leader construct is configured to couple to a non-nucleic acid polymer analyte.

[0025] In another embodiment, the Disclosure provides a system comprising: a fluid chamber; a membrane comprising nanopores, the membrane being configured to separate the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, the first solution and the second solution being configured to displace a non-nucleoside polymer analyte; and a controller operably coupled to the fluid chamber and the nanopores, the controller being configured to detect one or more signals related to at least one feature of a leader construct and one or more signals related to at least one feature of a non-nucleoside polymer analyte during or after the displacement of a non-nucleoside polymer analyte coupled to a leader construct through the nanopores using a translocase, the leader construct comprising at least one of a stall motif, a block motif, or a coupling motif, or a combination thereof.

[0026] In some embodiments, the controller is further configured to use a pair of electrodes to detect one or more signals associated with at least one feature of the leader construct and one or more signals associated with at least one feature of the non-nucleoside polymer analyte. In some embodiments, the controller is further configured to separate one or more signals associated with at least one feature of the leader construct from one or more signals associated with at least one feature of the non-nucleoside polymer analyte.

[0027] In some embodiments, the leader construct comprises one or more nucleic acid molecules. In some embodiments, the leader construct comprises one or more peptides. In some embodiments, the leader construct comprises one or more nucleic acid molecules and one or more peptides. In some embodiments, the stall motif is configured to disrupt the interaction between the translocase and the non-nucleoside polymer analyte. In some embodiments, the stall motif comprises a sequence of amino acids. In some embodiments, the amino acid sequence comprises n repeats of (glycine)n, (serine-glycine)n, (glycine-serine)n, (alanine)n, (valine)n, (alanine-serine)n, (serine-alanine)n, (valine-serine)n, or (serine-valine)n. In some embodiments, n may be about 1 to about 50. In some embodiments, n is approximately 1 to 2, approximately 1 to 3, approximately 1 to 4, approximately 1 to 5, approximately 1 to 10, approximately 1 to 15, approximately 1 to 20, approximately 1 to 25, approximately 1 to 30, approximately 1 to 40, approximately 1 to 50, approximately 2 to 3, approximately 2 to 4, approximately 2 to 5, approximately 2 to 10, approximately 2 to 15, approximately 2 to 20, approximately 2 to 25, approximately 2 to 30, approximately 2 to 40, approximately 2 to 50, approximately 3 to 4, approximately 3 to 5, approximately 3 to 10, approximately 3 to 15, approximately 3 to 20, approximately 3 to 25, approximately 3 to 30, approximately 3 to 40, approximately 3 to 50, approximately 4 to 5, approximately 4 to 10, approximately 4 to 15, approximately 4 to 20, approximately 4 to 25, approximately 4-30, approximately 4-40, approximately 4-50, approximately 5-10, approximately 5-15, approximately 5-20, approximately 5-25, approximately 5-30, approximately 5-40, approximately 5-50, approximately 10-15, approximately 10-20, approximately 10-25, approximately 10-30, approximately 10-40, approximately 10-50, approximately 15-20, approximately 15-25, approximately 15-30, approximately 15-40, approximately 15-50, approximately 20-25, approximately 20-30, approximately 20-40, approximately 20-50, approximately 25-30, approximately 25-40, approximately 25-50, approximately 30-40, approximately 30-50, or approximately 40-50.

[0028] In some embodiments, n may 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 stall motif includes a non-amino acid chemical region. In some embodiments, the non-amino acid chemical region includes polyethylene glycol.

[0029] In some embodiments, the block motif is configured to prevent the translocase from rearranging the non-nucleoside polymer analyte beyond the block motif. In some embodiments, the block motif is configured to prevent the translocase from rearranging the non-nucleoside polymer analyte beyond the leader construct. In some embodiments, the block motif is configured to prevent the translocase from rearranging the non-nucleoside polymer analyte through nanopores. In some embodiments, the block motif includes steric hindrance. In some embodiments, the steric hindrance includes one or more bulky amino acids. In some embodiments, the one or more bulky amino acids include histidine, phenylalanine, tyrosine, or tryptophan. In some embodiments, the steric hindrance includes at least one bulky amino acid. In some embodiments, the steric hindrance includes at least five bulky amino acids. In some embodiments, the steric hindrance includes at least a portion of a unfolding-resistant protein. In some cases, the unfolding-resistant protein may include any folded protein. In some cases, a stretch-resistant protein may contain an alpha-helix, beta-strand, beta-turn, helix-hairpin-helix motif, or any combination thereof. In some cases, a stretch-resistant protein may contain one or more stretch-resistant domains. In some cases, one or more stretch-resistant domains may contain an alpha-helix, beta-strand, beta-turn, helix-hairpin-helix motif, or any combination thereof. In some cases, a stretch-resistant protein may have about 1 to about 10 stretch-resistant domains. In some cases, a stretch-resistant protein may have at least about 1 stretch-resistant domain, at least about 2 stretch-resistant domains, at least about 3 stretch-resistant domains, at least about 4 stretch-resistant domains, at least about 5 stretch-resistant domains, at least about 6 stretch-resistant domains, at least about 7 stretch-resistant domains, at least about 8 stretch-resistant domains, at least about 9 stretch-resistant domains, at least about 10 stretch-resistant domains, or more than 10 stretch-resistant domains.In some cases, the expansion-resistant protein may have up to approximately 10 expansion-resistant domains, up to approximately 9 expansion-resistant domains, up to approximately 8 expansion-resistant domains, up to approximately 7 expansion-resistant domains, up to approximately 6 expansion-resistant domains, up to approximately 5 expansion-resistant domains, up to approximately 4 expansion-resistant domains, up to approximately 3 expansion-resistant domains, up to approximately 2 expansion-resistant domains, up to approximately 1 expansion-resistant domain, or less than 1 expansion-resistant domain. In some cases, the expansion-resistant protein may have approximately 1 expansion-resistant domain, approximately 2 expansion-resistant domains, approximately 3 expansion-resistant domains, approximately 4 expansion-resistant domains, approximately 5 expansion-resistant domains, approximately 6 expansion-resistant domains, approximately 7 expansion-resistant domains, approximately 8 expansion-resistant domains, approximately 9 expansion-resistant domains, or approximately 10 expansion-resistant domains. In some embodiments, the expansion-resistant protein comprises a maltose-binding protein, titin, dihydrofolate reductase, burnase, or a combination thereof. In some embodiments, the expansion-resistant protein comprises a disulfide bond. In some embodiments, the steric hindrance includes a large binding molecule. In some embodiments, the large binding molecule includes a carbohydrate, a polycyclic molecule, a branched dextran, biotin, streptavidin, a nanobody, an antibody, or a small antigenic element.

[0030] In some embodiments, the coupling motif is configured to couple a leader construct to a non-nucleoside polymer analyte. In some embodiments, the non-nucleoside polymer analyte includes a peptide. In some embodiments, the coupling motif attaches to the C-terminus of the peptide. In some embodiments, the coupling motif attaches to the N-terminus of the peptide. In some embodiments, the coupling motif includes an enzyme having peptide ligase activity. In some embodiments, the coupling motif includes a recognition sequence that can be recognized by the enzyme having peptide ligase activity. In some cases, the enzyme can interact with the coupling motif. In some embodiments, the coupling motif includes a chemical group. In some embodiments, the chemical group includes maleimide, iodoacetamide, 2-thiopyridine, 3-arylpropioronitrile, NHS ester, isocyanate, isothiocyanate, benzoyl fluoride, diazonium salt, or PTAD. In some embodiments, the coupling motif includes an enzyme coupling region. In some embodiments, the enzyme coupling region causes the coupling motif to attach to the enzyme. In some embodiments, the enzyme includes peptiligase, omniligase, buterase, tripsiligase, peptideamidase, asparaginyl endopeptidase, or saltase. In some embodiments, the coupling motif of the leader construct is coupled to a non-nucleoside polymer analyte via binding. In some embodiments, the coupling motif of the leader construct is coupled to a non-nucleoside polymer analyte via a linker.

[0031] In some embodiments, the leader construct further comprises at least one of a recognition motif or a capture motif. In some embodiments, the capture motif comprises a polycation tag. In some embodiments, the polycation tag comprises n repeats of (serine-glycine-arginine)n, (serine-arginine)n, (arginine)n, and the capture motif comprises a polyanion tag. In some embodiments, the capture motif comprises a polyanion tag. In some embodiments, the polyanion tag comprises n repeats of (serine-glycine-aspartic acid)n, (serine-aspartic acid)n, (aspartic acid)n. In some embodiments, the recognition motif comprises a portion of ssrA, a prokaryotic ubiquitin-like protein, SulA, a peroxisome membrane protein (Pex15), or a combination thereof. In some embodiments, the sequence of the recognition motif comprises one or more of sequence numbers 201-206.

[0032] In some embodiments, the leader construct is attached to the C-terminus or N-terminus of a non-nucleic acid polymer analyte. In some embodiments, the non-nucleic acid polymer analyte comprises a polypeptide, and the leader construct is attached to the N-terminus of the polypeptide. In some embodiments, the non-nucleic acid polymer analyte comprises a polypeptide, and the leader construct is attached to the C-terminus of the polypeptide. In some embodiments, the non-nucleic acid polymer analyte comprises a second leader construct.

[0033] In some embodiments, a first solution and a second solution are configured to generate electroosmotic force across the membrane. In some embodiments, the first solution contains a first concentration of solute, and the second solution contains a second concentration of solute. In some embodiments, the solute contains ions or osmolite. In some embodiments, the difference between the first and second concentrations of solute is configured to generate electroosmotic force. In some embodiments, the electroosmotic force includes a net ionic current flow from the cis side to the transform side of the membrane. In some embodiments, the electroosmotic force is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the system, modification of nanopores, or any combination thereof. In some embodiments, the electroosmotic force is regulated by modification of the charge of the nanopores. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis and transform sides of the membrane.

[0034] In some embodiments, the system further includes a pair of electrodes comprising a first electrode and a second electrode. In some embodiments, the first electrode is located on the cis side of the fluid chamber, and the second electrode is located on the transformer side of the fluid chamber. In some embodiments, the pair of electrodes are configured to provide an applied voltage to generate an electrophoretic force across the membrane in the opposite direction to the electroosmotic force. In some embodiments, the electroosmotic force is strong enough to dislocate non-nucleic acid polymer analytes through nanopores against the electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the net electroosmotic current absolute relative to the applied voltage is greater than about 0.10 pA / mV.

[0035] In another embodiment, the present disclosure provides a method comprising: (1) a nanopore system, wherein the nanopore system includes a fluid chamber; and (2) a membrane comprising nanopores, wherein the membrane separates the fluid chamber into a cis side and a trans side; and a non-nucleic acid polymer analyte; and dislocating the non-nucleic acid polymer analyte from the cis side to the trans side of the fluid chamber, wherein the nanopores include an adapter, and at least a portion of the adapter is located within the channel of the nanopores.

[0036] In some embodiments, the adapter is configured to alter the geometry of the channels in the nanopore. In some embodiments, the adapter is configured to constrict the channels in the nanopore. In some embodiments, the adapter is configured to alter the charge of the channels in the nanopore. In some embodiments, the adapter is configured to alter the channels or a portion thereof in the nanopore to have a net positive charge. In some embodiments, the adapter is configured to alter the channels or a portion thereof in the nanopore to have a net negative charge. In some embodiments, the adapter includes a proteinaceous adapter or a chemical adapter. In some embodiments, the proteinaceous adapter includes a CsgF subunit, a CsgF subunit cleavage, or a CsgF subunit homolog, paralog, or ortholog. In some embodiments, the chemical adapter includes a cyclodextrin, cucurbituryl, crown ether, calixsalen, porphyrin, cyclosporine, cyclomethicone, or cyclomethicone. In some embodiments, the adapter is coupled to the channels in the nanopore. In some embodiments, the adapter is coupled to the channels in the nanopore via a covalent bond. In some embodiments, the adapter is coupled to the channels of the nanopores via non-covalent bonds. In some embodiments, the adapter is coupled to the channels of the nanopores via linkers. In some embodiments, the nanopore system includes a cis-to-transformer electroosmotic force resulting from a net ionic current flow from the cis side to the transformer side.

[0037] In some embodiments, the method further includes providing an electrophoretic force acting in the opposite direction to the electroosmotic force from the cis side to the trans side. In some embodiments, the electroosmotic force from the cis side to the trans side is strong enough to push the non-nucleic acid polymer analyte through the nanopores against the electrophoretic force. In some embodiments, the non-nucleic acid polymer analyte is displaced through the nanopores using the electroosmotic force.

[0038] In some embodiments, the method further includes providing an electrophoretic force acting in the opposite direction to the electroosmotic force. In some embodiments, the electroosmotic force pushes the non-nucleic acid polymer analyte through the nanopores against the electrophoretic force. In some embodiments, the electroosmotic force includes a net ionic current flow from the cis side to the transform side. In some embodiments, the electroosmotic force is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the nanopore system, modification of the nanopores, or any combination thereof. In some embodiments, the electroosmotic force is regulated by modification of the charge of the nanopores. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis and transform sides of the membrane.

[0039] In some embodiments, the nanopore system further includes a pair of electrodes. In some embodiments, the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the net electroosmotic current absolute relative to the applied voltage is greater than about 0.10 pA / mV.

[0040] In some embodiments, non-nucleoside polymer analytes are rearranged using a translocase. In some embodiments, the translocase comprises an ATP-driven unfoldase. In some embodiments, the translocase comprises an NTP-driven unfoldase. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, 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 their functional homologs, orthologues, and paralogs.

[0041] In another embodiment, the present disclosure provides a system comprising a fluid chamber and a membrane including nanopores, wherein the membrane is configured to separate the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, the first solution and the second solution being configured to displace a non-nucleic acid polymer analyte, and the nanopores include adapters within the channels of the nanopores.

[0042] In some embodiments, the adapter includes a proteinaceous adapter or a chemical adapter. In some embodiments, the proteinaceous adapter includes a CsgF subunit, a CsgF subunit cleavage, or a CsgF subunit homolog, paralog, or ortholog. In some embodiments, the chemical adapter includes a cyclodextrin, cucurbituryl, crown ether, calixsalen, porphyrin, cyclosporine, cyclam, or cyclam. In some embodiments, the adapter is coupled to a channel in a nanopore. In some embodiments, the adapter is coupled to a channel in a nanopore via a covalent bond. In some embodiments, the adapter is coupled to a channel in a nanopore via a non-covalent bond. In some embodiments, the adapter is coupled to a channel in a nanopore via a linker.

[0043] In some embodiments, the first solution contains a first concentration of the solute, and the second solution contains a second concentration of the solute. In some embodiments, the solute contains ions or osmolite. In some embodiments, the difference between the first and second concentrations of the solute is configured to generate electroosmotic force. In some embodiments, the first and second solutions are configured to generate electroosmotic force across the membrane. In some embodiments, the electroosmotic force arises from a net ionic current flow from the cis side to the transform side of the membrane.

[0044] In some embodiments, the system further includes an electrophoretic force acting in the opposite direction to the electroosmotic force, and the electroosmotic force is strong enough to push non-nucleic acid polymer analytes through the nanopores against the electrophoretic force. In some embodiments, the electroosmotic force is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the system, modification of the nanopores, or any combination thereof. In some embodiments, the electroosmotic force is regulated by modification of the charge of the nanopores. In some embodiments, the electroosmotic force is regulated by an asymmetric salt distribution between the cis and trans sides of the membrane.

[0045] In some embodiments, the system further includes a pair of electrodes. In some embodiments, the first electrode of the pair of electrodes is positioned on the cis side, and the second electrode of the pair of electrodes is positioned on the transform side of the membrane. In some embodiments, the pair of electrodes is configured to detect a signal during dislocation of a non-nucleoside polymer analyte. In some embodiments, the signal is associated with a feature of the non-nucleoside polymer analyte. In some embodiments, the pair of electrodes is configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the absolute relative net electroosmotic current with respect to the applied voltage is greater than approximately 0.10 pA / mV.

[0046] In another embodiment, the present disclosure provides a method comprising: (a) a nanopore system comprising a fluid chamber and a membrane comprising nanopores, 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 polymer analyte and a pre-loading solution; and (c) dislocating the non-nucleic acid polymer analyte from the cis side to the trans side of the fluid chamber.

[0047] In some embodiments, the process further includes combining the sample containing the non-nucleoside polymer analyte with a preloading solution prior to (b). In some embodiments, the preloading solution contains a translocase. In some embodiments, the non-nucleoside polymer analyte is rearranged using the translocase. In some embodiments, the translocase contains an ATP-driven unfoldase. In some embodiments, the translocase contains an NTP-driven unfoldase. In some embodiments, the translocase contains an AAA+ enzyme. In some embodiments, 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 their functional homologs, orthologues, and paralogs.

[0048] In some embodiments, the sample and preloading solution are combined to form a non-nucleoside polymer analyte-translocase complex. In some embodiments, the sample and preloading solution are combined to form a non-nucleoside polymer analyte-leader construct complex. In some embodiments, the preloading solution includes a leader construct. In some embodiments, the preloading solution includes a chemical that enhances the binding of the non-nucleoside polymer analyte to the components of the preloading solution. In some embodiments, the binding of the non-nucleoside polymer analyte to the components of the preloading solution is higher than the binding of the non-nucleoside polymer analyte to the components in the fluid chamber. In some embodiments, the preloading solution includes one or more cofactors. In some embodiments, one or more cofactors are NTP, M 2+ It contains NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, Ficol, iron manganese, cobalt, copper, penicillamine, trientine, calcium sodium edetate, or ethylenediaminetetraacetic acid.

[0049] In some embodiments, non-nucleic acid polymer analytes are displaced using electroosmosis. In some embodiments, displacement involves providing electrophoretic force acting in the opposite direction to the electroosmosis. In some embodiments, the electroosmosis pushes the non-nucleic acid polymer analyte through nanopores against the electrophoretic force. In some embodiments, the electroosmosis involves a flow of net ionic current from the cis side to the transform side.

[0050] In some embodiments, electroosmosis is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the nanopore system, modification of the nanopores, or any combination thereof. In some embodiments, electroosmosis is regulated by modification of the charge of the nanopores. In some embodiments, electroosmosis is regulated by an asymmetric salt distribution between the cis and transform sides of the membrane.

[0051] In some embodiments, the nanopore system further includes a pair of electrodes. In some embodiments, the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 20 mV. In some embodiments, the net electroosmotic current absolute relative to the applied voltage is greater than about 0.10 pA / mV.

[0052] In another embodiment, the Disclosure provides a system comprising a fluid chamber, a membrane comprising nanopores, the membrane being configured to separate the fluid chamber into a cis side comprising a first solution and a trans side comprising a second solution, the first and second solutions being configured to displace a non-nucleic acid polymer analyte across the nanopores, and a preloading solution configured to interact with the non-nucleic acid polymer analyte.

[0053] In some embodiments, the first solution contains a first concentration of solute, and the second solution contains a second concentration of solute. In some embodiments, the solute contains ions or osmolite. In some embodiments, the difference between the first and second concentrations of solute is configured to generate electroosmosis. In some embodiments, the first and second solutions are configured to generate electroosmosis across the membrane. In some embodiments, electroosmosis arises from a net ionic current flow from the cis side to the transform side of the membrane. In some embodiments, electroosmosis is regulated by pH, type of salt, salt concentration, osmotic pressure across the membrane of the system, modification of nanopores, or any combination thereof. In some embodiments, electroosmosis is regulated by modification of the charge of the nanopores. In some embodiments, electroosmosis is regulated by an asymmetric salt distribution between the cis and transform sides of the membrane.

[0054] In some embodiments, the system further includes a pair of electrodes positioned on the cis and transformer sides of the membrane, the pair of electrodes configured to provide an applied voltage to generate electrophoretic force across the membrane in the direction opposite to the electroosmotic flow. In some embodiments, the applied voltage is a negative voltage relative to the transformer side. In some embodiments, the applied voltage is a positive voltage relative to the transformer side. In some embodiments, the magnitude of the applied voltage is less than 300 mV. In some embodiments, the magnitude of the applied voltage is greater than 300 mV. In some embodiments, the net electroosmotic current absolute relative to the applied voltage is greater than about 0.10 pA / mV. In some embodiments, the preloading solution contains one or more cofactors. In some embodiments, the one or more cofactors are divalent metal ions, NTPs, M 2+ It contains NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, Ficol, iron manganese, cobalt, copper, penicillamine, trientine, sodium calcium edetate, glycine betaine, or ethylenediaminetetraacetic acid.

[0055] In some embodiments, the preloading solution comprises a translocase. In some embodiments, the translocase comprises an ATP-driven unfoldase. In some embodiments, the translocase comprises an NTP-driven unfoldase. In some embodiments, the translocase comprises an AAA+ enzyme. In some embodiments, 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, or their functional homologs, orthologues, or paralogs.

[0056] In some embodiments, the preloading solution includes a leader construct. In some embodiments, the preloading solution includes a chemical that enhances the binding of the non-nucleoside polymer analyte to the components of the preloading solution compared to binding in the solution on the cis side of the fluid chamber. In some embodiments, the nanopores have an ion selectivity P(+) / P(-) greater than 2.0. In some embodiments, the nanopores have an ion selectivity P(+) / P(-) less than 0.50. In some embodiments, the non-nucleoside polymer analyte is an unmodified (label-free) non-nucleoside polymer analyte.

[0057] In some embodiments, the ends of the non-nucleoside polymer analytes lack a three-dimensional structure. In some embodiments, at least a portion of the non-nucleoside polymer analytes is denatured. In some embodiments, the non-nucleoside polymer analytes comprise peptide units, sugar units, water-soluble plastic monomers, or any combination thereof. In some embodiments, the non-nucleoside polymer analytes comprise polypeptides, polysaccharides, or water-soluble plastics. In some embodiments, the non-nucleoside polymer analytes comprise polypeptides. In some embodiments, the polypeptide comprises at least 30 peptide units. In some embodiments, at least 30 peptide units comprise positively charged residues. In some embodiments, at least 30 peptide units comprise negatively charged residues. In some embodiments, at least 30 peptide units comprise both positively charged 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.

[0058] In some embodiments, the method further includes measuring a signal generated by dislocation of a non-nucleoside polymer analyte through nanopores. In some embodiments, the measurement includes measuring a signal about (a) the open channel of the nanopore, (b) the capture of the non-nucleoside polymer analyte by the nanopore, or (c) the passage of the non-nucleoside polymer analyte through the nanopore. In some embodiments, the measurement includes detecting the difference between states (a), (b), and (c).

[0059] In some embodiments, the signal includes an ionic current, a change in ionic current, or a derivative thereof. In some embodiments, the nanopore includes an internal pore constriction of about 0.5 nm to about 2 nanometers (nm). In some embodiments, the internal pore constriction is about 1 nm to about 2 nanometers (nm). In some embodiments, the nanopore includes an alpha-helical oligomeric pore structure. In some embodiments, the nanopore includes a beta-barrel oligomeric pore structure. In some embodiments, the nanopore includes recombinant nanopores. In some embodiments, the nanopore includes proteins such as erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, their modified variants, or their ion-selective variants.

[0060] In some embodiments, the nanopores include biological nanopores. In some embodiments, the biological nanopores are modified to restrict the passage of one or more ions through the channels of the nanopores. In some embodiments, the biological nanopores restrict the passage of one or more ions through the channels of the nanopores by modifying the charge of the channels of the nanopores. In some embodiments, the net charge is negative. In some embodiments, the net charge is positive.

[0061] In some embodiments, the nanopores are mutant CytK nanopores. In some embodiments, the mutant CytK includes one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions include K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof. In some embodiments, one or more amino acid substitutions include K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof. In some embodiments, the mutant CytK nanopores include (a) K128D and K155D, (b) K128D, K155D, and T116D, (c) T147D or S151D, (d) K128D, K155D, and S120D, (e) Q122D, T147D, or S155D, (f) K128D, K155D, Q145D, and S151D, and (g) one of the amino acid substitution combinations of those combinations. In some embodiments, the mutant CytK nanopores include one or more of the following amino acid substitution combinations: (a) S120D, G122D, or K155D; (b) S120D in combination with K128F / K128D; (c) Q122D or S151D; (d) K128D or K128F; (e) S120D, K115D, and Q122D; (f) K128F, S120D, and G122D; (g) K128F, S120D, G122D, and K155D; and combinations thereof.

[0062] In some embodiments, the nanopores have an ion selectivity P(+) / P(-) greater than 2.0. In some embodiments, the nanopores have an ion selectivity P(+) / P(-) less than 0.50. In some embodiments, the non-nucleoside polymer analyte is an unmodified (unlabeled) non-nucleoside polymer analyte. In some embodiments, the ends of the non-nucleoside polymer analyte lack a three-dimensional structure. In some embodiments, at least a portion of the non-nucleoside polymer analyte is denatured. In some embodiments, the non-nucleoside polymer analyte comprises peptide units, sugar units, water-soluble plastic monomers, or any combination thereof. In some embodiments, the non-nucleoside polymer analyte comprises polypeptides, polysaccharides, or water-soluble plastics. In some embodiments, the non-nucleoside polymer analyte comprises polypeptides. In some embodiments, the polypeptide comprises at least 30 peptide units. In some embodiments, at least 30 peptide units contain positively charged residues. In some embodiments, at least 30 peptide units contain negatively charged residues. In some embodiments, at least 30 peptide units contain both positively charged 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.

[0063] In some embodiments, the system further includes measuring signals generated by dislocation of non-nucleoside polymer analytes through nanopores. In some embodiments, the measurement includes measuring signals about (a) the open channels of the nanopores, (b) the capture of non-nucleoside polymer analytes by the nanopores, or (c) the passage of non-nucleoside polymer analytes through the nanopores. In some embodiments, the measurement includes detecting the difference between states (a), (b), and (c).

[0064] In some embodiments, the signal includes an ionic current, a change in ionic current, or a derivative thereof. In some embodiments, the nanopore includes an internal pore constriction of about 0.5 nm to about 2 nm. In some embodiments, the internal pore constriction is about 1 nm to about 2 nm. In some embodiments, the nanopore includes an alpha-helical oligomer pore structure. In some embodiments, the nanopore includes a beta-barrel oligomer pore structure. In some embodiments, the nanopore includes recombinant nanopores. In some embodiments, the nanopore includes proteins such as erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, their modified variants, or their ion-selective variants.

[0065] In some embodiments, the nanopores include biological nanopores. In some embodiments, the biological nanopores are modified to restrict the passage of one or more ions through the channels of the nanopores. In some embodiments, the biological nanopores restrict the passage of one or more ions through the channels of the nanopores by modifying the charge of the channels of the nanopores. In some embodiments, the net charge is negative. In some embodiments, the net charge is positive.

[0066] In some embodiments, the nanopores are mutant CytK nanopores. In some embodiments, the mutant CytK includes one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions include K128D, K128F, K115D, S120D, Q122D, S151D, or any combination thereof. In some embodiments, one or more amino acid substitutions include K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, or any combination thereof. In some embodiments, the mutant CytK nanopores include (a) K128D and K155D, (b) K128D, K155D, and T116D, (c) T147D or S151D, (d) K128D, K155D, and S120D, (e) Q122D, T147D, or S155D, (f) K128D, K155D, Q145D, and S151D, and (g) one of the amino acid substitution combinations of those combinations. In some embodiments, the mutant CytK nanopores include one or more of the following amino acid substitution combinations: (a) S120D, G122D, or K155D; (b) S120D in combination with K128F / K128D; (c) Q122D or S151D; (d) K128D or K128F; (e) S120D, K115D, and Q122D; (f) K128F, S120D, and G122D; (g) K128F, S120D, G122D, and K155D; and combinations thereof.

[0067] In another aspect, the disclosure provides a device including an array of systems including the systems disclosed herein.

[0068] In another aspect, the disclosure provides the use of any of the methods, kits, or devices disclosed herein for characterizing at least one structural feature of a non-nucleic acid polymer analyte.

[0069] In another aspect, the 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 polymer analytes at the single-molecule level.

[0070] In another aspect, the disclosure provides the use of any of the systems disclosed herein for characterizing at least one structural feature of a non-nucleic acid polymer analyte.

[0071] In another aspect, the present disclosure provides the use of any of the systems disclosed herein for analyzing the amino acid sequence or amino composition of one or more non-nucleic acid polymer analytes at the single-molecule level.

[0072] Another aspect of the present disclosure provides a method for dislocating a target protein through nanopores, wherein the nanopores are contained within a membrane separating the fluid chamber of the nanopore system into cis and trans sides, and the method comprises (a) enabling a protein translocase in solution to capture the target protein to be dislocated and form a complex with it, optionally in the presence of an NTP, and (b) bringing the translocase-target protein complex into contact with the cis side of the nanopores to enable the dislocation of the target protein to the trans side, wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) resulting from a net ionic current flow from cis to trans, and as a result, the target protein is captured within the nanopores using a dislocation-controlling protein translocase located at the top of the nanopores.

[0073] In some embodiments, the nanopore system allows for a net ion current flow from cis to transformer (I) that exceeds the total ion current flow of greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, and greater than 0.35 or less than -0.35. rel ) has an EOF from cis to trans resulting from.

[0074] In some embodiments of any one of the prior embodiments, the cis-to-trans EOF is regulated by modifying the charge of the nanopores (e.g., genetically engineered) or any combination thereof, by adjusting the pH, type and / or concentration of salts and / or osmotic pressure across the membrane of the nanopore system, for example, by modifying the nanopores and / or by creating an asymmetric salt distribution between the cis and trans sides of the chamber.

[0075] In some embodiments of any one of the prior embodiments, the translocase-target protein complex is formed in the solution on the cis side of the fluid chamber.

[0076] In some embodiments of any one of the prior embodiments, the translocase-target protein complex is formed in solution during a separate step prior to adding the complex to the cis side of the fluid chamber and bringing it into contact with the nanopores.

[0077] In some embodiments of any one of the prior embodiments, the target protein includes a leader construct at its N-terminus and / or C-terminus, which allows preloading and optionally stalling one or more protein translocases. In some embodiments, the leader construct includes (i) a recognition motif for the protein translocase, and further includes one or more elements of (ii) a capture motif, (iii) a stall motif, and (iv) a block motif.

[0078] Another aspect of the present disclosure is a nanopore system for displacing a target protein through nanopores, comprising: (a) a membrane having nanopores therein, the membrane separating a chamber into a cis side and a trans side, the target protein being added to the cis side and displacing through the nanopores to the trans side; (b) a target protein captured by a protein translocase on the cis side of the chamber, the target protein binding to the target protein and capable of displacing the target protein through the nanopores in a continuous sequence; and (c) a voltage difference between the cis side and the trans side of the membrane. The present invention provides a nanopore system comprising a mechanism for providing a nanopore system having a cis-to-trans electroosmotic flow (EOF) resulting from a net cis-to-trans ionic current flow, thereby trapping target proteins within the nanopores using translocases that control dislocations located at the top of the nanopores, and the nanopore system having a cis-to-trans EOF resulting from a net cis-to-trans ionic current flow exceeding a total ionic current flow of 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.

[0079] In some embodiments, the system further includes a method for measuring a signal based on ionic currents flowing through nanopores during the rearrangement period, the measuring method detecting changes in the signal that reflect the characteristics of the protein as it is rearranged.

[0080] In some embodiments of any one of the prior embodiments, the nanopore system has an ion selectivity P greater than 2.0 or less than 0.5, greater than 2.5 or less than 0.4, greater than 3.0 or less than 0.33. (+) / P (-) It holds.

[0081] In some embodiments of any one of the prior embodiments, the nanopores are biological nanopores having internal pore constriction in the range of 0.5 to 2 nm, and the nanopores are alpha-helical or beta-barrel oligomeric pore-forming toxins or porins. In some embodiments, the nanopores are selected from the group consisting of erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragasceatoxin C (FraC), lysenin, phage-derived portal proteins, and modified variants thereof, and the nanopores are modified to have a net charge in a region facing the lumen greater than 21, greater than 28, or greater than 35, and the net charge is negative. In some embodiments, the nanopores are selected from the group consisting of erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, phage-derived portal proteins, and modified variants thereof, and the nanopores are modified to have a net charge in more than 21, more than 28, or more than 35 lumen-facing regions, and the net charge is positive. In some embodiments, any amino acid residue in the lumen-facing region of the nanopore may be mutated. In some cases, the mutated amino acid residue may be mutated to a negatively charged amino acid. In some cases, the mutated amino acid residue may be mutated to a positively charged amino acid. In some cases, the mutated amino acid residue may be mutated to a neutrally charged amino acid.

[0082] In some embodiments, the nanopores can be oligomers. In some cases, the oligomeric nanopores can comprise one or more subunits. In some cases, each of the one or more subunits can comprise about 20 to about 40 charges in the region facing the lumen of the subunit. In some cases, each of the one or more subunits can comprise 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 region facing the lumen of the subunit. In some cases, each subunit of one or more subunits may contain up to approximately 40 charges, up to approximately 39 charges, up to approximately 38 charges, up to approximately 37 charges, up to approximately 36 charges, up to approximately 35 charges, up to approximately 34 charges, up to approximately 33 charges, up to approximately 32 charges, up to approximately 31 charges, up to approximately 30 charges, up to approximately 29 charges, up to approximately 28 charges, up to approximately 27 charges, up to approximately 26 charges, up to approximately 25 charges, up to approximately 24 charges, up to approximately 23 charges, up to approximately 22 charges, up to approximately 21 charges, up to approximately 20 charges, or less than 20 charges within the region facing the lumen of the subunit. In some cases, each subunit of one or more subunits may contain approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 charges within the region facing the lumen of the subunit.

[0083] In some embodiments, the nanopores are mutant CytK nanopores comprising one or more amino acid substitutions selected from the group consisting of K128D, K128F, K115D, S120D, Q122D, and S151D, further comprising S120D, G122D and / or K155D; S120D, Q122D, or S151D in combination with K128F / K128D; K128D / K128F, S120D, K115D, and Q122D; and one of the amino acid substitution combinations of K128F, S120D, and G122D in combination with K155D, optionally.

[0084] In some embodiments of any one of the prior embodiments, the protein translocase is an AAA+ enzyme, which is an unfoldase driven by an NTP. In some embodiments, 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 their functional homologs, orthologues, or paralogs.

[0085] In some embodiments of any one of the prior embodiments, the nanopore system has an ion selectivity P greater than 2.0, greater than 2.5, and greater than 3.0. (+) / P (-) The system has a negative voltage applied to the transformer side. In some embodiments, the system includes cation-selective (mutant) nanopores.

[0086] One aspect of this disclosure provides an analytical device including an array of nanopore systems according to any one of the prior embodiments.

[0087] One aspect of the present disclosure provides the use of a nanopore system or device according to any one of the prior embodiments for analyzing the amino acid sequence or amino composition of one or more target proteins at the single-molecule level in order to characterize at least one structural feature of a target protein.

[0088] Another aspect of this disclosure provides a non-temporary computer-readable medium containing machine-executable code that, when executed by one or more computer processors, implements any of the methods described herein or elsewhere.

[0089] Another aspect of this disclosure provides a system comprising one or more computer processors and computer memory coupled thereto, the computer memory containing machine-executable code that, when executed by the one or more computer processors, implements any of the methods described herein or elsewhere.

[0090] Additional aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As realized, other different embodiments of the present disclosure are possible, and some of its details are modifiable in various obvious ways without departing from the present disclosure. Accordingly, the drawings and description should be considered illustrative and not limiting in nature.

[0091] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. To the extent that any publications and patents or patent applications incorporated by reference conflict with any disclosures contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material. [Modes for carrying out the invention]

[0092] This disclosure relates to a system and method for analyzing non-nucleic acid polymer analytes (e.g., analytes) using nanopore-based sensors. This disclosure provides nanopore systems, devices and methods for the analysis and sequencing of single-molecule non-nucleic acid polymer analytes (e.g., single-molecule proteins).

[0093] Various studies have demonstrated both free and motor-controlled migration of polypeptides (e.g., proteins deployed during or before rearrangement through narrow nanopores) (typically less than 2 nm in diameter). However, unlike polynucleotides with a fixed negative charge that can be electrophoretically drawn into nanopores by an electric field from an applied voltage, capturing and controlling the migration of peptides of diverse compositions remains a challenge. This is because diverse compositions result in a range of electrical and structural properties (e.g., positive, negative, neutral, hydrophilic, hydrophobic, aromatic mixtures) that prevent simple capture under electrophoretic conditions and rearrangement in the deployed state.

[0094] Due to their complex composition, it was previously impossible to push / supply analytes into the pores from the cis side in their natural form (e.g., without attaching to or conjugating to a DNA reader, or without adding other (e.g., polyanion) tags to create electrophoretic capture motifs). Because diverse charges, depending on the charge and applied voltage, can sometimes attract and sometimes release the deployed peptides from the nanopores, electrophoretic mechanisms alone cannot transpose the diverse repertoire of complex peptides through the nanopores. Indeed, previous studies have shown the transposition of either very short peptides with contour lengths shorter than the length of the nanopore channels, or very carefully selected (model) analytes, whose charge, structure, or added electrophoretic tags favor electrophoretic capture and transposition through the nanopores. However, this by no means represents the broad amino acid composition of proteins found in nature. For example, see Motone et al. (iScience 24, September 24, 2021), which outlines recent approaches using various techniques aimed at driving protein chains and peptides through nanopores. It states that nanopore protein sequencing remains a challenging and unexplored field.

[0095] This disclosure provides a novel approach that may be simple and / or a robust mechanism for supplying non-nucleic acid polymer analytes (e.g., full-length proteins) through nanopores (e.g., for sequencing and / or characterizing nanopores). In some examples, the methods and systems disclosed herein may not require additional components (e.g., proteins or polynucleotides) to fuse, conjugate, and / or otherwise adhere to the nanopores.

[0096] These goals have been found to be achievable by using a large and dominant cis-to-trans electroosmotic flow (EOF) generated by a large excess of ions flowing through the nanopores, combined with a translocase located on the cis side of the nanopores that can controlly supply and pass a wide variety of analytes through the nanopores from cis to trans, against the direction of electrophoretic force (EPF). In some embodiments, the cis-to-trans osmotic flow can be generated by the flow of ions and solvents from the cis side of the nanopore system to the trans side of the nanopore system.

[0097] This disclosure provides a system that can utilize strong electroosmotic pressure to capture and feed analytes (e.g., peptides, nucleic acid molecules, oligosaccharides, lipids, proteins) from the cis side of nanopores without coupling a motor translocase to the nanopore. In some embodiments, the force within the system is sufficient to hold the translocase motor at the top of the pore. For example, a strong electroosmotic pore combined with a cis-side translocase protein (e.g., a molecular motor protein) may first unwind, then feed analytes of diverse compositions through the nanopore, and then dissociate, allowing the system to process the next molecule. This can be achieved by a strong EOF that pulls at least a portion of the analytes in or near the nanopore, as the translocase may diffuse away from the nanopore after dissociation for a new complex to bind.

[0098] In some embodiments, a strong electroosmotic pressure pulls the analyte as it displaces through the pore, and then transmits this force to the above-mentioned bound translocase motor protein, which acts to hold the translocase at the top of the pore during controlled substrate displacement. The translocase motor then moves through the analyte under translocase activity controlled by nucleotide triphosphates (NTPs), unfolding any three-dimensional structure within the analyte that the translocase encounters, thereby controlling the movement of the analyte within the nanopore at a rate at which changes in current can be measured and characterized.

[0099] Therefore, in one embodiment of the present disclosure, a method for dislocating an analyte through nanopores, wherein the nanopores are contained in a membrane that separates the fluid chamber of the nanopore system into cis and trans sides, (a) To enable protein translocases in solution to selectively capture the transposing analyte in the presence of NTPs and form a complex with it, (b) The translocase-target protein complex is brought into contact with the cis side of the nanopore, enabling the trans side of the analyte, The present invention provides a method in which a nanopore system has a cis-to-trans electroosmotic force (EOF) arising from a net cis-to-trans ionic current flow, thereby trapping the analyte in the nanopores, and a translocase is located at the top of the nanopores, allowing the translocase to control the dislocation.

[0100] For example, a nanopore system has a net ionic current flow from cis to transformer that exceeds a total ionic current flow greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, or greater than 0.35 or less than -0.35 (as specified herein). rel It has an EOF from cis to trans resulting from (also known as).

[0101] In some embodiments, the cis-to-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow, which is at least about -0.99, at least about -0.95, at least about -0.9, at least about -0.8, at least about -0.7, at least about -0.6, at least about -0.5, at least about -0.4, at least about -0.3, at least about -0.2, at least about -0.1, at least about 0.0, at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 0.95, about 0.99, or about 0.99, and is also called the relative net current flow from cis to transformer. In some embodiments, the cis-to-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds a total ionic current flow of less than approximately -0.99, -0.9, -0.8, -0.7, -0.6, -0.5, -0.4, -0.3, -0.2, -0.1, -0.0, -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -0.95, -0.99, or less than approximately -0.99, and is also called the relative net current flow from cis to transformer.

[0102] In some embodiments, the EOF from cis to transformer arises from the net ionic current flow from cis to transformer exceeding the total ionic current flow of about -0.99 to about 0.99, and is also called the relative net current flow from cis to transformer. In some embodiments, the EOF from cis to transformer is 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 -0.99 to about -0.8, about -0.99 to about -0.6, about -0.9 to about -0.4, about -0.9 Approximately -0.2, approximately -0.9 to approximately 0, approximately -0.9 to approximately 0.2, approximately -0.9 to approximately 0.4, approximately -0.9 to approximately 0.6, approximately -0.9 to approximately 0.8, approximately -0.9 to approximately 0.99, approximately -0.8 to approximately -0.6, approximately -0.8 to approximately -0.4, approximately -0.8 to approximately -0.2, approximately -0.8 to approximately 0, approximately -0.8 to approximately 0.2, approximately -0.8 to approximately 0.4, approximately -0.8 to approximately 0.6, approximately -0.8 to approximately 0.8, approximately -0.8 to approximately 0.99, approximately -0.6 to approximately -0.4, approximately -0.6 to approximately -0.2, approximately -0.6 to approximately 0, approximately -0.6 to approximately 0.2, approximately -0.6 to approximately 0.4, approximately -0.6 to approximately 0.6, approximately -0.6 to approximately 0.8, approximately -0.6 to approximately 0.99, approximately -0.4 to approximately -0.2, approximately -0.4 to approximately 0, approximately -0.4 to approximately 0.2, approximately -0.4 to approximately 0.4, approximately -0.4 to approximately 0.6, approximately -0.4 to approximately 0.8, approximately -0.4 to approximately 0.99, approximately -0.2 to approximately 0, approximately -0.2 to approximately 0.2, approximately -0.2 to approximately 0.4, approximately -0.2 to approximately 0.6, approximately -0.2 to approximately 0.8, approximately -0.2 to approximately 0.99, approximately 0 to approximately 0.2, approximately These arise from net ionic current flows from cis to transformer that exceed the total ionic current flow, in the ranges of 0 to approximately 0.4, approximately 0 to approximately 0.6, approximately 0 to approximately 0.8, approximately 0 to approximately 0.99, approximately 0.2 to approximately 0.4, approximately 0.2 to approximately 0.6, approximately 0.2 to approximately 0.8, approximately 0.2 to approximately 0.99, approximately 0.4 to approximately 0.6, approximately 0.4 to approximately 0.8, approximately 0.4 to approximately 0.99, approximately 0.6 to approximately 0.8, approximately 0.6 to approximately 0.99, or approximately 0.8 to approximately 0.99, and are also called relative net current flows from cis to transformer.

[0103] In some embodiments, the cis-transformer EOF is approximately -0.99, approximately -0.95, approximately -0.9, approximately -0.8, approximately -0.7, approximately -0.6, approximately -0.5, approximately -0.4, approximately -0.3, approximately -0.2, approximately -0.1, approximately 0.0, approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 0.95, or approximately 0.99, resulting from the net ionic current flow from cis to transformer exceeding the total ionic current flow, and is also called the relative net current flow from cis to transformer.

[0104] Preferably, the nanopore system may have an ion selectivity P(+) / P(-) greater than 2.0 or less than 0.5, greater than 2.5 or less than 0.4, or greater than 3.0 or less than 0.33.

[0105] In some embodiments, the pores may contain relative ion selectivity P(+) / P(-) of at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or more than about 5, under the difference of the applied voltage across the membrane. In some embodiments, the pores may contain relative ion selectivity P(+) / P(-) of up to about 5, at least about 4, at least about 3, at least about 2, at least about 1, at least about 0.9, at least about 0.8, at least about 0.7, at least about 0.6, at least about 0.5, at least about 0.4, at least about 0.3, at least about 0.2, at least about 0.1, or less than about 0.1, under the difference of the applied voltage across the membrane.

[0106] In some embodiments, the pores may have relative ion selectivity P(+) / P(-) of about 0.1 to about 5 under the applied voltage difference across the membrane. In some embodiments, the pores may have relative ion selectivity P(+) / P(-) of about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 to about 0.5, about 0.1 to about 1, about 0.1 to about 1.5, about 0.1 to about 2, about 0.1 to about 2.5, about 0.1 to about 3, about 0.1 to about 4, about 0.1 to about 5, about 0.2 to about 0.3, and about 0.2 to about 0. 4, approximately 0.2 to approximately 0.5, approximately 0.2 to approximately 1, approximately 0.2 to approximately 1.5, approximately 0.2 to approximately 2, approximately 0.2 to approximately 2.5, approximately 0.2 to approximately 3, approximately 0.2 to approximately 4, approximately 0.2 to approximately 5, approximately 0.3 to approximately 0.4, approximately 0.3 to approximately 0.5, approximately 0.3 to approximately 1, approximately 0.3 to approximately 1.5, approximately 0.3 to approximately 2, approximately 0.3 to approximately 2.5, approximately 0.3 to approximately 3, approximately 0.3 to approximately 4, approximately 0.3 to approximately 5, Approximately 0.4 to 0.5, approximately 0.4 to 1, approximately 0.4 to 1.5, approximately 0.4 to 2, approximately 0.4 to 2.5, approximately 0.4 to 3, approximately 0.4 to 4, approximately 0.4 to 5, approximately 0.5 to 1, approximately 0.5 to 1.5, approximately 0.5 to 2, approximately 0.5 to 2.5, approximately 0.5 to 3, approximately 0.5 to 4, approximately 0.5 to 5, approximately 1 to 1.5, approximately 1 to 2, approximately 1 to 2.5 It may include relative ion selectivity P(+) / P(-) of approximately 1 to 3, approximately 1 to 4, approximately 1 to 5, approximately 1.5 to 2, approximately 1.5 to 2.5, approximately 1.5 to 3, approximately 1.5 to 4, approximately 1.5 to 5, approximately 2 to 2.5, approximately 2 to 3, approximately 2 to 4, approximately 2 to 5, approximately 2.5 to 3, approximately 2.5 to 4, approximately 2.5 to 5, approximately 3 to 4, approximately 3 to 5, or approximately 4 to 5.

[0107] In some embodiments, the pores may have relative ion selectivity P(+) / P(-) of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5 under the applied voltage difference across the membrane.

[0108] Furthermore, it is a nanopore system for dislocating analytes through nanopores, (a) A membrane having nanopores therein, the membrane separating a chamber into a cis side and a trans side, an analyte being added to the cis side and translocating through the nanopores to the trans side, (b) An analyte captured by a protein translocase on the cis side of the chamber, the analyte binding to the analyte and being able to translocate the analyte through the nanopores in a sequential order, (c) including a mechanism for providing a voltage difference between the cis side and the trans side of the membrane, The nanopore system has an electroosmotic force (EOF) from cis to trans resulting from the flow of net ionic current from cis to trans, such that the analyte is captured in the nanopore and the nanopore can be positioned at a translocase that can control the translocation above the nanopore, for example, I 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 from which the nanopore system also results. In some embodiments, I rel can be from about -0.4 to about 0.4. In some cases, I rel is 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 is 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 relThis could be approximately -0.4, approximately -0.35, approximately -0.30, approximately -0.25, approximately -0.20, approximately -0.15, approximately -0.10, approximately -0.05, approximately 0, approximately 0.05, approximately 0.10, approximately 0.15, approximately 0.20, approximately 0.25, approximately 0.30, approximately 0.35, or approximately 0.40.

[0109] In certain embodiments, the nanopore systems of the present disclosure have an ion selectivity P(+) / P(-) greater than 2.0 or less than 0.5, greater than 2.5 or less than 0.4, or greater than 3.0 or less than 0.33.

[0110] This disclosure provides a method, system, or device that may depend on a dominant cis-to-trans EOF in combination with a translocase on the cis side of a nanopore.

[0111] In some cases, EPF can be the dominant process driving trapping and dislocation in nanopore systems. Therefore, all previous demonstrations have shown that, using selected model polypeptides (which have a net charge that aids EPF) or by modifying polypeptides with highly charged tags (e.g., by adding polyanion tags), the resulting EPF force acting on the polypeptide is cis to trans in direction to drive dislocation.

[0112] For example, WO2021 / 111125 relates to a method for characterizing a target polypeptide by forming a conjugate with a highly charged polynucleotide and controlling the movement of the conjugate into a nanopore using a polynucleotide handling protein. Thus, in this approach, the conjugate, which includes a polypeptide section, passes through the nanopore by a polynucleotide handling protein, such as a helicase, which moves along the polynucleotide elongation. This disclosure provides a method that utilizes a protein translocase (in conjunction with electroosmotic flow). In some cases, the peptide does not need to be coupled to the charged portion.

[0113] In some embodiments, EOFs can be used in the nanopore systems of the present disclosure. Where EOFs have been previously used in nanopore systems, it is most frequently either in a trans-to-cis direction acting against cis-to-trans EPFs (slowing EPF-driven dislocations), or in a cis-to-trans direction combined with cis-to-trans EPFs to assist dislocations. Several previous studies have shown the capture of neutral or weakly charged small molecules or small polymers within nanopores via weak electroosmosis (https: / / doi.org / 10.1073 / pnas.2531778100;https: / / pubs.acs.org / doi / full / 10.1021 / ja4026193;https: / / doi.org / 10.1063 / 1.2723088). This disclosure provides a method for capturing and / or dislocating polymer analytes (e.g., long and / or complex polymers) using a cis-to-trans EOF that can overcome a trans-to-cis EPF acting in the opposite direction. The polymer analytes may have contour lengths greater than the length of the nanopores.

[0114] Furthermore, because the chain may not be pulled by force, potentially clogging the nanopores or being expelled back to the cis side of the membrane, it has been considered impossible to control the delivery of analytes into nanopores using motor proteins (e.g., translocases) in solution on the cis side of the nanopores. This approach might be like trying to force cooked spaghetti into a plug hole. See, for example, WO2013 / 123379, which proposes a nanopore system for displacing proteins from cis to trans through nanopores. In this case, the protein translocase is present on at least one side of the nanopore. However, WO2013 / 123379, in particular, provides only experimental data for systems containing the translocase on the trans side, which corresponds to Nivala et al. (Nat. Biotechnol. 2013;31(3):247-250) reporting an alpha-hemolysin (aHL) based nanopore system in which the ClpX translocase is present in trans solution. The theoretical embodiment of WO2013 / 123379 proposed for having a protein translocase (ClpX) located on the cis side requires the fusion of aHL nanopores to a so-called "docking" protein (ClpP) to enable non-covalent docking of the translocase onto the nanopore subunit. For this system to function, the axial pores and nanopores of the translocase can be aligned to the correct orientation. In other words, ClpX can bind to aHL such that the protein substrate is captured from the solution and driven through the central cavity of ClpX, directly entering the upper lumen of aHL hemolysin and ultimately being forced through the entire nanopore.

[0115] Therefore, this disclosure provides for the first time an analyte sensing system in which a translocase functions at the top of a nanopore ("top" refers to the entrance side or surface of the nanopore on the membrane side to which the translocase is added) without modifying the nanopore with docking proteins or other types of accessory elements to displace a diverse repertoire of complex peptides. Rather, the novel system relies on aligning a specific strong electroosmotic mechanism in the direction of dislocation.

[0116] In one embodiment, the present disclosure is a method for transposing a target protein through nanopores, (a) To provide a device that includes nanopores in a membrane that separates the fluid chamber into cis and transform sides, (b) The protein translocase in solution is enabled to capture the target protein to be translocated and to form a complex with it. (c) The translocase-target protein complex is brought into contact with the cis side of the nanopore. (d) The nanopore system has an ion selectivity P+ / P- greater than 3.0 or less than 0.3, and as a result, the target protein is captured within the nanopore using a translocase that controls the rearrangement located at the top of the nanopore.

[0117] In some embodiments, the force-generating electroosmotic flow may pull the analyte through the nanopore against the opposite EPF, while retaining the protein translocase at the top of the nanopore until the analyte is released. Perhaps in the method of the present disclosure, the analyte is pulled through the nanopore against the opposite EPF, while the protein translocase is retained at the top of the nanopore during the dislocation event and then released so that another analyte can bind to it.

[0118] In another aspect of this disclosure, a method comprising providing a nanopore system is provided herein. The nanopore system may include a membrane containing nanopores. In some cases, the membrane may separate a fluid chamber into a cis side and a trans side. Non-nucleic acid polymer analytes may also be provided. The non-nucleic acid polymer analyte may be brought into contact with a trans-locase on the cis side of the fluid chamber. The analyte and the trans-locase may form a complex. The non-nucleic acid polymer analyte may be displaced from the cis side to the trans side using electroosmosis. Electroosmosis may maintain the trans-locase of the complex at the cis-side inlet of the nanopore channel.

[0119] In another aspect of this disclosure, a system comprising providing a nanopore system is provided herein. The nanopore system may include a membrane containing nanopores. In some cases, the membrane may separate a fluid chamber into a cis side and a trans side. Non-nucleic acid polymer analytes may also be provided. The non-nucleic acid polymer analyte may be brought into contact with a translocase on the cis side of the fluid chamber. The analyte and the translocase may form a complex. The non-nucleic acid polymer analyte may be displaced from the cis side to the trans side using electroosmosis. Electroosmosis may couple the translocase of the complex to the cis side inlet 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, additional structures may include proteases. In some cases, the proteases may include serine proteases, thrombin, cysteine ​​proteases, metalloproteinases, chymotrypsin, trypsin, papain, subtilisin, or any combination thereof. In some cases, additional structures may include docking proteins. In some cases, the docking proteins may include ClpP, TatA, TatB, TatC, Tim50, Tim23, Tim17, or any combination thereof.

[0120] In one embodiment, 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 within the cis compartment.

[0121] In one embodiment, net cis-to-trans EOF is achieved by (i) adjusting the pH, (ii) the type and / or concentration of salts across the membrane of the nanopore system, and / or the osmotic pressure, (iii) by modifying (e.g., genetically engineered) or designing the nanopore charge, or by any combination thereof. The dominant EOF is regulated by modifying the nanopores and / or by an asymmetric salt distribution between the cis and trans sides of the chamber.

[0122] In some embodiments, the system has a cation-based relative current EOF of at least 3.0 in the cis-transformer direction. A negatively applied voltage may be present on the transformer side (e.g., the system includes cation-selective (mutant) nanopores).

[0123] In another embodiment, the translocase-analyte complex is formed in solution during a separate operation prior to adding the complex to the cis side of the fluid chamber and bringing it into contact with the nanopores. This approach allows for the use of optimal conditions for the binding (complex formation) of the translocase and analyte. For example, in this pre-loading operation, higher concentrations of both components, different salt conditions, temperature, pH, cofactors, etc., may be selected than those typically used in the cis chamber of the nanopore sensor system. The pre-mixture may be part of a kit that can be coupled to the analyte of interest. The pre-mixture may be added to the cis chamber in a diluted form.

[0124] In one embodiment, the nanopores are solid-state nanopores or biological nanopores having internal pore constriction with a diameter in the range of 0.5 to 2 nm. In some cases, solid-state nanopores may be nanopores fabricated from synthetic materials. In some cases, biological nanopores may be nanopores found in nature.

[0125] In some embodiments, the nanopores may be biological nanopores. The nanopores may be alpha-helical or beta-barrel oligomeric pore-forming toxins or porins. The nanopores are preferably selected from the group consisting of erolysin (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, phage-derived portal proteins (Phi29, G20c, etc.), or their variants. In certain embodiments, the nanopores are selected from the variant CytK nanopores listed in Table 2.

[0126] In some cases, the nanopores may be constructed from elements of existing nanopores (see, e.g., WO2021 / 101378) or newly developed using predictive protein manipulation software (see, e.g., Shimizu et al. 2022, Nature Nanotechnology volume 17, pp. 67-75).

[0127] In some embodiments, the nanopore systems of the Disclosure may utilize any biological nanopores, synthetic nanopores, recombinant nanopores, or any combination thereof. In some cases, biological nanopores, synthetic nanopores, recombinant nanopores, or any combination thereof may function in the nanopore systems of the Disclosure without modification.

[0128] For example, a protein translocase is an AAA+ enzyme that is an unfolderase driven by NTPs. Protein translocases 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 their functional homologs, orthologues, or paralogs. In some cases, the translocase may include ClpX and / or ClpA. In some cases, the translocase may include translocases specific to peptides, proteins, polypeptides, or any combination thereof. In some cases, translocases may include translocases specific to nucleic acid molecules.

[0129] In some embodiments, the non-nucleic acid polymer analytes may be target proteins or peptides. The analytes may be their native, unmodified form. For example, a translocase that is promiscuous in substrate specificity or has been manipulated to remove binding specificity may be mixed with the unmodified analyte to enable binding / loading. Such a loading method may result in a mixture of analyte-translocase complexes, including some translocases loaded at the C-terminus of the analyte and moving in the C-to-N direction, and / or some translocases loaded at the N-terminus of the analyte and moving in the N-to-C direction. The translocases have binding specificity to either the N-terminus or C-terminus of the analyte, and as a result, the complex has translocases moving in a common direction. The translocases acting on the unmodified analyte can increase trapping within nanopores by unfolding the analyte and creating free ends. A complex based on the loading of a translocase onto an unmodified analyte may have a single translocase or multiple translocases positioned at random points along the analyte during capture within the nanopore. When the complex is captured in the nanopore, the bound translocase may act to either feed the analyte into the nanopore or pull it out of the nanopore, depending on which end of the analyte is captured and the orientation of the translocase on the nanopore.

[0130] In another aspect of this disclosure, a method comprising providing a nanopore system is provided herein. The nanopore system may include a membrane containing nanopores. In some cases, the membrane may separate a fluid chamber into a cis side and a trans side. Non-nucleic acid polymer analytes may also be provided. The non-nucleic acid polymer analytes may include a leader construct. The leader construct may include a stall motif, a blocking motif, a coupling motif, a recognition motif, a capture motif, or any combination thereof. In some cases, a translocase may also be provided. The nucleic acid polymer analyte may be rearranged from the cis side to the trans side of the fluid chamber.

[0131] In another aspect of this disclosure, a system comprising providing a nanopore system is provided herein. The nanopore system may include a membrane containing nanopores. In some cases, the membrane may separate a fluid chamber into cis and trans sides. Non-nucleic acid polymer analytes may also be provided. The non-nucleic acid polymer analytes may include a leader construct. The leader construct may include a stall motif, a blocking motif, a coupling motif, a recognition motif, a capture motif, or any combination thereof. In some cases, the leader (or tail) construct may include a membrane-binding motif. In some cases, a translocase may also be provided. The nucleic acid polymer analyte may be rearranged from the cis side to the trans side of the fluid chamber.

[0132] In another aspect of the present disclosure, a system comprising providing a nanopore system is provided herein. The nanopore system may include a membrane containing nanopores. In some cases, the membrane may separate a fluid chamber into a cis side and a trans side. A non-nucleoside polymer analyte may also be provided. In some cases, the cis side may have a first solution. In some cases, the trans side may have a second solution. The first and second solutions may be configured to displace the non-nucleoside polymer analyte. The system may also further include a controller. In some cases, the controller may be operably coupled to the fluid chamber and the nanopores. The controller may be configured to detect one or more signals related to at least one feature of a leader construct. The controller may be configured to detect one or more signals related to at least one feature of a non-nucleoside polymer analyte. The controller may be further configured to detect one of many signals related to at least one feature of a leader construct and one or more signals related to at least one feature of a non-nucleoside polymer analyte. In some cases, one or more signals may be detected during the displacement of the non-nucleoside polymer analyte. In some cases, one or more signals may be detected after the rearrangement of the non-nucleoside polymer analyte. In some cases, one or more signals may be detected during or after the rearrangement of the non-nucleoside polymer analyte. In some cases, the leader construct can be coupled to the non-nucleoside polymer analyte. In some cases, the non-nucleoside polymer analyte can rearrange through nanopores using nanopores. In some cases, the leader construct may include a stalling motif, a blocking motif, a coupling motif, a recognition motif, a capture motif, or any combination thereof.

[0133] In other embodiments of the Disclosure, the analyte includes “leader” and / or “tail” extensions at the ends of the protein. In certain aspects of the Disclosure, the analyte includes a leader construct that can preload and / or optionally stall a protein translocase. For example, preloading occurs outside the nanopore system (the cis chamber of which), after which the analyte-translocase complex is introduced into the cis chamber. In another embodiment, the analyte may be coupled to a leader construct that can load and / or optionally stall a protein translocase when the leader construct and protein translocase are mixed together in the cis chamber of the system. In some embodiments, the analyte may be coupled to a leader construct that can load and / or optionally stall a protein translocase when the leader construct and protein translocase are mixed together in the trans chamber of the system. The leader construct may be an exogenous sequence. The leader construct includes (i) a recognition motif for a protein translocase that supports binding to a specific site and / or enables more efficient binding and loading. It may further include one or more elements of (ii) a capture motif, (iii) a stall motif, (iv) a block motif, and (v) a coupling motif.

[0134] In another embodiment, the translocase is coupled to the nanopore. In the system of the present disclosure, the translocase does not have to be coupled to the top of the nanopore in order to optimally supply the analyte into the nanopore. Instead, the strong cis-to-trans EOF of the present disclosure allows a portion of the analyte extruded from the translocase to be trapped and displaced within the nanopore, and then pulls the translocase to the top of the pore, thereby continuing to control the movement of the extruded analyte. In this embodiment, the analyte does not have a stall motif or trapping motif due to the proximity of the extruded analyte to the nanopore entrance.

[0135] The methods according to this disclosure may further include measuring changes in ionic current caused by dislocations of the analyte passing through nanopores. Changes in current can be measured for (i) open channels, (ii) trapping of the analyte by the nanopores, and / or (iii) the passage of the analyte from (ii) through the nanopores. For example, a method for measuring changes in ionic current includes detecting differences between states (i), (ii), and (iii). In particular embodiments, the measurement includes measuring differences between states (iii) caused by the amino acid composition or structure of the analyte passing through the nanopores. The methods preferably include obtaining one or more measurements characteristic of the analyte. One or more measurements may be one, two, three, four, or five or more characteristics characteristic of the analyte. One or more characteristics may be selected from (i) the length of the analyte, (ii) the identity of the analyte, (iii) the arrangement of the analyte, (iv) the secondary or tertiary structure of the analyte, and (v) whether the analyte has been modified. Any combination of (i) to (v) may be measured according to this disclosure.

[0136] Further embodiments of the present disclosure are nanopore systems for dislocating analytes through nanopores, The present invention relates to a nanopore system comprising: (a) a membrane having nanopores therein, the membrane separating a chamber into a cis side and a trans side, to which an analyte is added on the cis side and displaced through the nanopores to the trans side; (b) an analyte captured on the cis side of the chamber by a protein translocase that can bind to and displace the analyte through the nanopores in a continuous sequence; and (c) an element for providing a voltage difference between the cis side and the trans side of the membrane. In some cases, the element in (c) may include a pair of electrodes.

[0137] In some embodiments, the nanopore system is further characterized by a cis-to-trans electroosmotic force (EOF) arising from a net cis-to-trans ionic current flow, and as a result, the analyte is trapped within the nanopores using a translocase that controls dislocations at the top of the nanopores. The nanopore system has a cis-to-trans EOF arising from a net cis-to-trans ionic current flow exceeding a total ionic current flow of greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, or greater than 0.35 or less than -0.35.

[0138] In certain embodiments, the nanopore system has an ion selectivity 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. (+) / P (-) It holds.

[0139] In some embodiments, the voltage difference can be provided in various ways, for example, by applying a voltage to one circuit and measuring a current, or the system includes a first circuit for applying a voltage and a second circuit for measuring a current. It is also possible to create a voltage difference by an asymmetric salt across the film. For example, the device includes a circuit for providing a voltage between the cis side and the transformer side and measuring the ionic current flowing through the nanopores. See Figure 1. A negative voltage is applied to the transformer side.

[0140] In some embodiments, the system may further include a method for measuring the signal based on the ionic current flowing through the nanopores during the rearrangement. These measurement mechanisms are configured to detect a change in the signal that reflects the characteristics of the analyte as it is rearranged. In some embodiments, the analyte being measured may be a protein. In some cases, the characteristics of the protein being 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, the domain structure of the protein, the length of the protein, the net charge of the protein, or the conformation of the protein. In some embodiments, the analyte being measured may be a nucleic acid molecule. In some cases, the characteristics of the nucleic acid molecule being measured may include the nucleotide sequence of the nucleic acid molecule, nucleotide mutations in the sequence of the nucleic acid molecule, methylation of the nucleic acid molecule, acetylation of the nucleic acid molecule, the length of the nucleic acid molecule, the net charge of the nucleic acid molecule, or the conformation of the nucleic acid molecule. In some embodiments, the analyte being measured may be an oligosaccharide. In some cases, the characteristics of the oligosaccharide being measured may include the sequence of the oligosaccharide, the length of the oligosaccharide, the net charge of the oligosaccharide, the presence or absence of coupled lipids, the presence or absence of coupled peptides, or the structure of the oligosaccharide. In some embodiments, the analyte being measured may be a lipid molecule. In some cases, the characteristics of the lipid being measured may include the length of the lipid, the net charge of the lipid, or the structure of the lipid.

[0141] The system may use alternative mechanisms to measure the voltage-current characteristics of the nanopore system, such as those using ion flux fluorescence probes or field-effect transistor systems, in addition to measuring voltage changes. However, other suitable detection methods also exist, such as tunneling, surface-enhanced Raman, plasmonics, and other spectroscopic methods that do not measure ion current but instead directly measure the properties of the analyte in the nanopores.

[0142] Furthermore, analytical devices are provided that include one or more nanopore systems disclosed herein, for example, in the form of an array.

[0143] Further embodiments relate to the use of methods, nanopore systems, or devices according to the Disclosure for the detection and / or analysis of one or more analytes at the single-molecule level for characterizing at least one feature of an analyte. In one embodiment, the use includes characterizing the amino acid sequence of an undenatured analyte or a mixture of different undenatured analytes.

[0144] In some embodiments, methods for analyzing analytes are provided.

[0145] In some embodiments, methods for analyzing analytes are provided.

[0146] In some embodiments, changes in ion current can be measured while the analyte displaces through the nanopores. In some cases, changes in ion current can be measured by a voltage-based chip. In some cases, the voltage-based chip can measure changes in voltage and / or current across the nanopores. In some cases, the voltage-based chip may be a transformer electrode (e.g., an electrode adjacent to the film / nanopore to measure the voltage across the nanopores).

[0147] The characterization method may involve measuring the flow of ionic current through the pores, typically by measuring the current. Alternatively, the flow of ions through the pores may be measured optically, as disclosed in Heron et al: J.Am.Chem.Soc.9 Vol.131, No.5, 2009. Therefore, the apparatus may also include an electrical circuit capable of applying a potential and measuring the electrical signals across the membrane and pores. The characterization method may be performed using patch clamps or voltage clamps. The characterization method may involve the use of voltage clamps.

[0148] In some embodiments, the analytes include polymer analytes. The analytes may include nucleic acid polymer analytes or non-nucleic acid polymer analytes. The analytes may be synthetic, semi-synthetic, or of biological origin. For example, synthetic analytes may include analytes constructed by non-biological chemical processes, such as polyethylene glycol (PEG) or synthetically constructed DNA molecules. For example, synthetic analytes may include analytes constructed by non-biological chemical processes, such as polyethylene glycol (PEG), synthetically constructed peptides of proteins, or synthetically constructed DNA molecules. Biological analytes may include analytes produced by biological processes, such as proteins produced by cells or by systems using cellular (or cell-derived) components (e.g., enzyme in vitro translation systems). Biological analytes may include analytes produced by biological processes, such as proteins produced by cells. Semi-synthetic analytes may include those of biological and non-biological origin, such as portions made from biologically produced proteins conjugated to PEG molecules. Possible electrical measurements include current measurement, impedance measurement, tunneling, electron tunneling measurement (Ivanov AP et al., Nano Lett. 201 1 Jan 12; 1 l(l): 279-85), FET measurement (International Application WO2005 / 124888), voltage FET measurement, or any combination thereof. In some embodiments, the signal may be an electron tunnel across a solid-state nanopore or a voltage FET measurement across a solid-state nanopore.

[0149] The characterization method may involve measuring the flow of ionic current through the pores by measuring the current. Alternatively, the flow of ions through the pores may be described as follows: Heron et al.: J.Am. Chem.

[0150] As disclosed in Soc.9 Vol.131, No.5, 2009, it can be measured optically. Therefore, the apparatus may also include an electrical circuit capable of applying a potential and measuring electrical signals across the film and pores. The characterization method can be performed using patch clamps or voltage clamps. The characterization method preferably involves the use of voltage clamps.

[0151] The characterization method may be performed on an array of nanopores or wells, each array containing 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more nanopores or wells.

[0152] This characterization method may involve measuring the current flowing through the pores. The method is typically performed using a voltage applied across the membrane and pores. The voltage used is typically +2V to -2V, and typically -400mV to +400mV. The voltage used is preferably within a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, 20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. More preferably, the voltage used is within the range of 20mV to 240mV, and most preferably within the range of 120mV to 220mV. By using an increased applied potential, it is possible to increase the distinction between different analyte features for each pore.

[0153] In some embodiments, the analytes include polymer analytes. The analytes may include nucleic acid-based polymer analytes or non-nucleic acid-based polymer analytes. The analytes may be synthetic, semi-synthetic, or of biological origin. For example, synthetic analytes may include analytes constructed by non-biological chemical processes, such as polyethylene glycol (PEG) or synthetically constructed DNA molecules. Biological analytes may include analytes produced by biological processes, such as proteins produced by cells. Semi-synthetic analytes may include those of biological and non-biological origin, for example, portions created by biologically produced proteins conjugated to PEG molecules.

[0154] In some embodiments, the analyte includes a protein (e.g., polypeptide) or peptide. The protein or peptide may be in a folded state, an unfolded state, or an intermediate state (e.g., a partially unfolded state). The folded state includes a state in which the polymer is in a low-energy state such that the protein or peptide maintains a two-dimensional or three-dimensional structure. This low-energy state may be based on the interaction of amino acids in the peptide or protein. The unfolded state may include a state in which the polymer is in a high-energy state such that the protein or peptide does not maintain a two-dimensional or three-dimensional structure. An intermediate state between the folded and unfolded states may be an energy state in which one or more parts of the peptide or protein can maintain a two-dimensional or three-dimensional structure, while other parts of the peptide or protein cannot. In some embodiments, the protein (e.g., polypeptide) may include a folded protein structure. In some embodiments, the peptide may include a linear structure. In some cases, the peptide may include a portion of a protein.

[0155] In some embodiments, the analyte may include nucleic acid molecules. 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 transfer RNA (tRNA), messenger RNA (mRNA), ribosomal RNA (rRNA), nuclear small RNA (snRNA), nucleolar small RNA (snoRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), microRNA (miRNA), or any combination thereof. In some embodiments, the analyte may include lipids. In some cases, the lipids may be oleic acid, linoleic acid, palmitic acid, docosahexaenoic acid, eicosapentaenoic acid, or any combination thereof. It is possible that the analyte may contain oligosaccharides. In some embodiments, the oligosaccharides may be glycoproteins, inulin, lactose, mannose, sucrose, fructooligosaccharides, monosaccharides, carbohydrates, maltose, prebiotics, galactooligosaccharides, glycans, chitosan, pentasaccharides, or any combination thereof. In some embodiments, the analyte may contain polysaccharides. In some embodiments, the polysaccharides may be starch, glycogen, galactogen, inulin, arabinoxylan, cellulse, chitin, pectin, or any combination thereof.

[0156] In some embodiments, the analyte may include a non-nucleic acid polymer analyte. In some embodiments, a portion of the non-nucleic acid polymer analyte may include nucleic acid molecules. In some cases, a portion of the non-nucleic acid polymer analyte may be 0% to about 100% of the non-nucleic acid polymer analyte. In some cases, a portion of the non-nucleic acid polymer analyte may be at least about 0%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, or up to approximately 0% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be approximately 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% of the non-nucleic acid polymer analyte.

[0157] In some embodiments, a portion of the non-nucleic acid polymer analyte may contain oligosaccharide molecules. In some cases, a portion of the non-nucleic acid polymer analyte may be 0% to about 100% of the non-nucleic acid polymer analyte. In some cases, a portion of the non-nucleic acid polymer analyte may be at least about 0%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, or up to approximately 0% of the non-nucleic acid polymer analyte. In some cases, the portion of the non-nucleic acid polymer analyte may be approximately 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% of the non-nucleic acid polymer analyte.

[0158] 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 its unfolded state. The unfolded analyte may or may not include secondary structural elements. Secondary structural elements may include α-helices, β-helices, coils, or β-sheets. Helices may be counterclockwise or clockwise. The analyte may be unfolded, fully or partially unfolded (e.g., an intermediate unfolded state). The contour length may be the length of the polymer analyte when the two ends of the polymer analyte extend fully from each other. In some embodiments, the analyte may include structured portions, unstructured portions, modified portions, partially modified portions, or a combination thereof. In some embodiments, the analyte may include 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, or more ends. In some embodiments, the analyte may contain 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 fewer. In some embodiments, the analyte may contain 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 may include structured portions, unstructured portions, modified portions, partially modified portions, or a combination thereof.

[0159] The analyte may contain repeating units. These units may include peptide units, sugar units, lipid units, nucleotide units, water-soluble plastic monomers, or combinations thereof. The analyte may contain polypeptides, polysaccharides, nucleic acids, water-soluble plastics, or combinations thereof. In some embodiments, the analyte may contain charges. These charges may be positive or negative. The charges may be distributed uniformly or heterogeneously throughout the analyte. In some embodiments, the charges may be the result of amino acid residues. Amino acid residues may be native or mutant residues. In some cases, mutant amino acid residues may contain one or more additional chemical groups compared to native amino acids. In some embodiments, the analyte may contain peptides. Peptides may include polypeptides or proteins. Proteins may be full-length proteins or cleaved proteins. Cleaved proteins may be shorter than the protein was originally produced. For example, a protein may become shorter due to cleavage (e.g., by peptidases) or degradation (e.g., due to acidic or basic conditions). Proteins may contain sequences that are native protein sequences or modified protein sequences. Sequences can be modified by mutation, deletion, or insertion. Sequences may also be combinations of sequences. For example, a first natural sequence can be added to or inserted into a second natural sequence to form a third sequence which is a combination of the first and second sequences.

[0160] In some embodiments, the analyte includes a protein. In some embodiments, the analyte includes a peptide or polypeptide (e.g., a protein). In some embodiments, a peptide, polypeptide, or protein may be targeted. The targeted analyte may be a target peptide, a target polypeptide, or a target protein. The targeted analyte may be an analyte combined with a translocase. The analyte can be combined with a translocase to form a complex (e.g., a translocase-analyte complex).

[0161] In some embodiments, the analyte and translocase may be located on the cis side of the membrane. In some cases, the analyte and translocase can form a complex on the cis side of the membrane. In some embodiments, the analyte and translocase can form a complex within a nanopore system.

[0162] In some embodiments, the analyte and translocase may be located on the trans side of the membrane. In some cases, the analyte and translocase may form a complex on the trans side of the membrane.

[0163] In some embodiments, the analyte may come into contact with the translocase outside the nanopore system. In some cases, the analyte and translocase may form a complex in a separate container away from the nanopore system. In some cases, the analyte and translocase complex may be added to the nanopore system.

[0164] In some embodiments, the analyte may be the target analyte. In some cases, the target analyte may be a protein. In some cases, the target analyte may be a polypeptide. In some cases, the target analyte may be a peptide. In some cases, the target analyte may be a nucleic acid molecule. In some cases, the nucleic acid molecule may be an RNA molecule. In some cases, the nucleic acid molecule may be a DNA molecule. In some cases, the target analyte may be a polysaccharide. Non-limiting examples of polysaccharides include cellulose, chitin, amylopectin, chitosan, dextran, galactan, lentinan, amylose, amylopectin, starch, hemicellulose, alginic acid, glycosaminoglycans, gelan gum, carrageenan, glycogen, pectin, glucan, inulin, homopolysaccharides, fucoidan, and polydextrose. In some cases, the target analyte may be an oligosaccharide. Non-limiting examples of oligosaccharides include lactose, maltose, sucrose, cellobiose, nigerotriose, maltotriose, melegitose, maltotriulose, raffinose, kestose, nigerotetraose, maltotetraose, lycnose, nystose, sesamose, stachyose, pentasaccharides, fructooligosaccharides, galactooligosaccharides, hexasaccharides, and heptasaccharides. In some embodiments, the target analyte may be glycosylated. In some cases, the glycosylated oligosaccharide may be an N-linked oligosaccharide. In some cases, the glycosylated oligosaccharide may be an O-linked oligosaccharide.

[0165] In some embodiments, electroosmotic flow (also called electroosmotic force, EOF) acts across the membrane in a cis-to-trans direction or trans-to-cis direction. Electroosmotic flow can be a flow resulting from a net flow of ion migration layers along the surface, induced by an applied potential (e.g., an applied voltage potential). For example, a charged surface may form a static layer of ion migrations charged in the opposite direction. Under an applied potential, charged ion migrations can be induced to move in the direction of a higher potential if negative, or in the direction of a lower potential if positive. The flow of charged ions can create a drag force on surrounding solvent (e.g., water) molecules, which can then result in a net flow that exerts a force on surrounding molecules, both charged and neutral. For example, in a negatively charged nanopore lumen, electroosmotic flow may result from a net flow of positive ions in a cis-to-trans direction (due to a lower potential on the trans side, e.g.), causing surrounding water to flow from cis to trans and exert a force on surrounding molecules. The amount of ion flow and the magnitude of the corresponding electroosmotic flow may be influenced by parameters including differences in ion concentration across the membrane, differences in potential, the net charge of the nanopore lumen, the geometry of the nanopore lumen, or a combination thereof.

[0166] In some embodiments, electroosmotic flow may be a flow arising from one or more constrictions present in the nanopore channels. For example, a nanopore constriction may affect the flow of some ions (e.g., larger hydrated ions) more than other ions (e.g., smaller hydrated ions). In some embodiments, electroosmotic flow may be a flow arising from the net flow of moving ions along the surface, induced by an applied potential and one or more constrictions present in the nanopore channels.

[0167] 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 transform side of the membrane. In some cases, the solution on the cis side of the membrane may be a first solution. In some cases, the solution on the transform side of the membrane may be a second solution. The difference may be a difference in the concentration of molecules including ions, electrolytes, or osmolites.

[0168] In some embodiments, the difference between solutions may be due to salt asymmetry or ionic asymmetry, where one side of the membrane (e.g., the cis side) contains different concentrations of ions than the other side (e.g., the trans side). Ionic asymmetry can affect the ionic current across the membrane, as described by the Goldman-Hodgkin-Katz equations.

number

[0169] Here, the ionic current (I(S)) is a function of the applied potential (Vm) and the ionic species S across the membrane. In the equation, P (S) This is the membrane permeability of ion species S, and z s [F is the ion's charge, F is the Faraday constant, R is the gas constant, T is the temperature, [S] シス and [S] トランス These are the cis and transform concentrations of ion species S, respectively. Because the difference in ion concentrations between the cis and transform sides affects the ion flux, ion fluxes combining different species can affect electroosmotic force because ions flow across the membrane in different directions. This can be used to enhance or weaken electroosmotic force by having an ion concentration difference between the cis and transform sides that minimizes or maximizes the contribution of species S's ionic current to the net ion flux.

[0170] A difference in molecular concentration between two sides of a membrane can modify the electroosmotic flux by providing a competing or auxiliary osmotic flux. A concentration difference across the membrane can create an osmotic gradient, and a solvent (e.g., water) can diffuse across the membrane in the direction of higher molecular concentration to minimize the concentration difference between the two sides of the membrane. The osmotic gradient can be oriented to drive the flow of water in the same direction as or different from the electroosmotic force. For example, if the ion concentration is higher on the cis side than on the transform side, the osmotic gradient can drive water from transform to cis, thus creating an osmotic gradient that competes with the electroosmotic force from cis to transform. The ion concentration can also assist the electroosmotic flow from cis to transform, even if it also provides an osmotic gradient.

[0171] In some embodiments, EOF can be generated by an asymmetric salt distribution between the cis and trans sides of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may differ from the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be higher than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the cis side of the membrane may be lower than the concentration of one or more salts on the trans side of the membrane. In some cases, the concentration of one or more salts on the trans side of the membrane may be higher than the concentration of one or more salts on the cis side of the membrane. In some cases, the concentration of one or more salts on the trans side of the membrane may be lower than the concentration of one or more salts on the cis side of the membrane.

[0172] In some cases, the concentration of one or more salts on the cis side of the membrane can be about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more salts on the cis side of the membrane can be about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more salts on the cis side of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, and less At least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed 1,000 nM.In some cases, the concentration of one or more salts on the cis side of the membrane can range from approximately 1,000 nM, 950 nM, 900 nM, 850 nM, 800 nM, 750 nM, 700 nM, 650 nM, 600 nM, 550 nM, 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, and more. The maximum value is approximately 150 nM, the maximum is approximately 100 nM, the maximum is approximately 95 nM, the maximum is approximately 90 nM, the maximum is approximately 85 nM, the maximum is approximately 80 nM, the maximum is approximately 75 nM, the maximum is approximately 70 nM, the maximum is approximately 65 nM, the maximum is approximately 60 nM, the maximum is approximately 55 nM, the maximum is approximately 45 nM, the maximum is approximately 40 nM, the maximum is approximately 35 nM, the maximum is approximately 30 nM, the maximum is approximately 25 nM, the maximum is approximately 20 nM, the maximum is approximately 15 nM, the maximum is approximately 10 nM, the maximum is approximately 5 nM, the maximum is approximately 1 nM, or it may be less than 1 nM. In some cases, the concentration of one or more salts on the cis side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM. M may be approximately 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, or 1,000 nM.

[0173] In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte may be at least about 0.01 M, at least about 0.05 M, at least about 0.10 M, at least about 0.20 M, at least about 0.30 M, at least about 0.40 M, at least about 0.50 M, at least about 0.60 M, at least about 0.70 M, at least about 0.80 M, at least about 0.90 M, at least about 1.00 M, at least about 1.10 M, at least about 1.25 M, at least about 1.50 M, at least about 1.75 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 3.5 M, at least about 4 M, at least about 4.5 M, at least about 5 M, or greater than about 5 M. In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte may be up to about 5 M, up to about 4.5 M, up to about 4 M, up to about 3.5 M, up to about 3 M, up to about 2.5 M, up to about 2 M, up to about 1.75 M, up to about 1.50 M, up to about 1.25 M, up to about 1 M, up to about 0.90 M, up to about 0.80 M, up to about 0.70 M, up to about 0.60 M, up to about 0.50 M, up to about 0.40 M, up to about 0.30 M, up to about 0.20 M, up to about 0.10 M, up to about 0.05 M, up to about 0.01 M, or less than about 0.01 M.

[0174] In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte can be about 0.01 M to about 5 M. In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte can be about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to about 0.5 M, Approximately 0.1M to approximately 1M, approximately 0.1M to approximately 1.5M, approximately 0.1M to approximately 2M, approximately 0.1M to approximately 2.5M, approximately 0.1M to approximately 3M, approximately 0.1M to approximately 3.5M, approximately 0.1M to approximately 4M, approximately 0.1M to approximately 4.5M, approximately 0.1 M ~ about 5M, about 0.5M - about 1M, about 0.5M - about 1.5M, about 0.5M - about 2M, about 0.5M - about 2.5M, about 0.5M - about 3M, about 0.5M - about 3.5M, about 0.5M - about 4M, about 0.5M - about 4.5M , about 0.5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2M , about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3.5 M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, It can be about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M, or about 4.5M to about 5M.

[0175] In some embodiments, the concentration of the cis-side salt, ion, osmolite, or electrolyte may be about 0.01 M, about 0.05 M, about 0.10 M, about 0.20 M, about 0.30 M, about 0.40 M, about 0.50 M, about 0.60 M, about 0.70 M, about 0.80 M, about 0.90 M, about 1.00 M, about 1.10 M, about 1.25 M, about 1.50 M, about 1.75 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M.

[0176] In some cases, the concentration of one or more salts on the transform side of the membrane can be about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more salts on the transform side of the membrane can be about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more salts on the transform side of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, At least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed about 1,000 nM.In some cases, the concentration of one or more salts on the trans side of the membrane is up to approximately 1,000 nM, up to approximately 950 nM, up to approximately 900 nM, up to approximately 850 nM, up to approximately 800 nM, up to approximately 750 nM, up to approximately 700 nM, up to approximately 650 nM, up to approximately 600 nM, up to approximately 550 nM, up to approximately 500 nM, up to approximately 450 nM, up to approximately 400 nM, up to approximately 350 nM, up to approximately 300 nM, up to approximately 250 nM, up to approximately 200 nM. The maximum nM is approximately 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less than 1 nM. In some cases, the concentration of one or more salts on the trans side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM. It may be nM, approximately 100nM, approximately 150nM, approximately 200nM, approximately 250nM, approximately 300nM, approximately 350nM, approximately 400nM, approximately 450nM, approximately 500nM, approximately 550nM, approximately 600nM, approximately 650nM, approximately 700nM, approximately 750nM, approximately 800nM, approximately 850nM, approximately 900nM, approximately 950nM, or approximately 1,000nM.

[0177] In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be at least about 0.01 M, at least about 0.05 M, at least about 0.10 M, at least about 0.20 M, at least about 0.30 M, at least about 0.40 M, at least about 0.50 M, at least about 0.60 M, at least about 0.70 M, at least about 0.80 M, at least about 0.90 M, at least about 1.00 M, at least about 1.10 M, at least about 1.25 M, at least about 1.50 M, at least about 1.75 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 3.5 M, at least about 4 M, at least about 4.5 M, at least about 5 M, or greater than about 5 M. In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be up to about 5 M, up to about 4.5 M, up to about 4 M, up to about 3.5 M, up to about 3 M, up to about 2.5 M, up to about 2 M, up to about 1.75 M, up to about 1.50 M, up to about 1.25 M, up to about 1 M, up to about 0.90 M, up to about 0.80 M, up to about 0.70 M, up to about 0.60 M, up to about 0.50 M, up to about 0.40 M, up to about 0.30 M, up to about 0.20 M, up to about 0.10 M, up to about 0.05 M, up to about 0.01 M, or less than about 0.01 M.

[0178] In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be about 0.01 M to about 5 M. In some embodiments, the concentration of the cis-side salt, ions, osmolite, or electrolyte may be about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to about 0.5 M. Approximately 0.1M to approximately 1M, approximately 0.1M to approximately 1.5M, approximately 0.1M to approximately 2M, approximately 0.1M to approximately 2.5M, approximately 0.1M to approximately 3M, approximately 0.1M to approximately 3.5M, approximately 0.1M to approximately 4M, approximately 0.1M to approximately 4.5M, approximately 0.1 M ~ about 5M, about 0.5M - about 1M, about 0.5M - about 1.5M, about 0.5M - about 2M, about 0.5M - about 2.5M, about 0.5M - about 3M, about 0.5M - about 3.5M, about 0.5M - about 4M, about 0.5M - about 4.5M , about 0.5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2M , about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3.5 M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, It can be about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M, or about 4.5M to about 5M.

[0179] In some embodiments, the concentration of the transformer-side salt, ions, osmolite, or electrolyte may be about 0.01 M, about 0.05 M, about 0.10 M, about 0.20 M, about 0.30 M, about 0.40 M, about 0.50 M, about 0.60 M, about 0.70 M, about 0.80 M, about 0.90 M, about 1.00 M, about 1.10 M, about 1.25 M, about 1.50 M, about 1.75 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M.

[0180] In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis side and the trans side may be at least about 0.01 M, at least about 0.05, at least about 0.10, at least about 0.20, at least about 0.30, at least about 0.40, at least about 0.50, at least about 0.60, at least about 0.70, at least about 0.80, at least about 0.90, at least about 1.00, at least about 1.10, at least about 1.25, at least about 1.50, at least about 1.75, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5 M, or greater than about 5 M. In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis and trans sides may be up to about 5 M, up to about 4.5 M, up to about 4 M, up to about 3.5 M, up to about 3 M, up to about 2.5 M, up to about 2 M, up to about 1.75 M, up to about 1.50 M, up to about 1.25 M, up to about 1 M, up to about 0.90 M, up to about 0.80 M, up to about 0.70 M, up to about 0.60 M, up to about 0.50 M, up to about 0.40 M, up to about 0.30 M, up to about 0.20 M, up to about 0.10 M, up to about 0.05 M, up to about 0.01 M, or less than about 0.01 M.

[0181] In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis and trans sides can be about 0.01 M to about 5 M. In some embodiments, the difference in concentration of salt, ions, or electrolytes between the cis and trans sides can be about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, and about 0.1 M to about 0. 5M, about 0.1M to about 1M, about 0.1M to about 1.5M, about 0.1M to about 2M, about 0.1M to about 2.5M, about 0.1M to about 3M, about 0.1M to about 3.5M, about 0.1M to about 4M, about 0.1M to about 4.5M, about 0 .1M to about 5M, about 0.5M to about 1M, about 0.5M to about 1.5M, about 0.5M to about 2M, about 0.5M to about 2.5M, about 0.5M to about 3M, about 0.5M to about 3.5M, about 0.5M to about 4M, about 0.5M to about 4. 5M, about 0.5M to about 5M, about 1M to about 1.5M, about 1M to about 2M, about 1M to about 2.5M, about 1M to about 3M, about 1M to about 3.5M, about 1M to about 4M, about 1M to about 4.5M, about 1M to about 5M, about 1.5M to about 2 M, about 1.5M to about 2.5M, about 1.5M to about 3M, about 1.5M to about 3.5M, about 1.5M to about 4M, about 1.5M to about 4.5M, about 1.5M to about 5M, about 2M to about 2.5M, about 2M to about 3M, about 2M to about 3. 5M, about 2M to about 4M, about 2M to about 4.5M, about 2M to about 5M, about 2.5M to about 3M, about 2.5M to about 3.5M, about 2.5M to about 4M, about 2.5M to about 4.5M, about 2.5M to about 5M, about 3M to about 3.5M, It can be about 3M to about 4M, about 3M to about 4.5M, about 3M to about 5M, about 3.5M to about 4M, about 3.5M to about 4.5M, about 3.5M to about 5M, about 4M to about 4.5M, about 4M to about 5M, or about 4.5M to about 5M.

[0182] In some embodiments, the difference in concentration of salt, ion, or electrolyte between the cis side and the trans side may be about 0.01 M, about 0.05 M, about 0.10 M, about 0.20 M, about 0.30 M, about 0.40 M, about 0.50 M, about 0.60 M, about 0.70 M, about 0.80 M, about 0.90 M, about 1.00 M, about 1.10 M, about 1.25 M, about 1.50 M, about 1.75 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M.

[0183] In some embodiments, one or more salts may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide chloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc hydroxide chloride monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, potassium glutamate, sodium ferricyanide, sodium ferrocyanide, potassium ferricyanide, potassium ferrocyanide, or any combination thereof.

[0184] In some embodiments, one or more salts on the cis side of the membrane may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide chloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof. In some embodiments, one or more salts on the trans side of the membrane may include sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc hydroxide chloride, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr's salt, sodium potassium sulfate, potassium permanganate, copper tetraaminosulfate, zinc hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, or any combination thereof.

[0185] In some embodiments, one or more salts on the cis side of the membrane may be the same as one or more salts on the transform side of the membrane. In some cases, one or more salts on the cis side of the membrane may be of the same type as the salts on the transform side of the membrane. In some embodiments, one or more salts on the cis side of the membrane may be different from one or more salts on the transform side of the membrane. In some cases, one or more types of salts on the cis side of the membrane may be different from one or more types of salts on the transform side of the membrane.

[0186] In some embodiments, one or more salts may comprise about one to about ten types of salts. In some cases, one or more salts may comprise at least about one type of salt, at least about two types of salts, at least about three types of salts, at least about four types of salts, at least about five types of salts, at least about six types of salts, at least about seven types of salts, at least about eight types of salts, at least about nine types of salts, at least about ten types of salts, or more than ten types of salts. In some cases, one or more salts may comprise up to about ten types of salts, up to about nine types of salts, up to about eight types of salts, up to about seven types of salts, up to about six types of salts, up to about five types of salts, up to about four types of salts, up to about three types of salts, up to about two types of salts, up to about one type of salt, or less than one type of salt. In some cases, one or more salts may include one type of salt, about two types of salt, about three types of salt, about four types of salt, about five types of salt, about six types of salt, about seven types of salt, about eight types of salt, about nine types of salt, or about ten types of salt.

[0187] In some embodiments, one or more salts on the cis side of the membrane may be the same type of salt as one or more salts on the trans side of the membrane. In some cases, the same type of salt present on the cis and trans sides of the membrane may be present at the same concentration. In some cases, the same type of salt present on the cis and trans sides of the membrane may be present at different concentrations.

[0188] In some embodiments, one or more salts on the cis side of the membrane may be of a different type than one or more salts on the trans side of the membrane. In some embodiments, the different types of salts present on the cis and trans sides of the membrane may be present at the same concentration. In some cases, the different types of salts present on the cis and trans sides of the membrane may be present at different concentrations.

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

[0190] In some cases, the concentration of one or more salts on the cis side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even higher than 500%.

[0191] In some cases, the concentration of one or more salts on the cis side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the trans side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be less than 0.1% higher.

[0192] In some cases, the concentration of one or more salts on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% of the concentration of one or more salts on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

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

[0194] In some cases, the concentration of one or more salts on the cis side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even lower than 500%.

[0195] In some cases, the concentration of one or more salts on the cis side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the trans side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be lower than 0.1%.

[0196] In some cases, the concentration of one or more salts on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% of the concentration of one or more salts on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0197] In some embodiments, the concentration of one or more salts on the trans side of the membrane may be about 0.1% to about 500% higher than the concentration of one or more salts on the cis side of the membrane. In some cases, the concentration of one or more salts on the trans side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% Approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% ~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~ It may be approximately 360%, approximately 360% to approximately 370%, approximately 370% to approximately 380%, approximately 380% to approximately 390%, approximately 390% to approximately 400%, approximately 400% to approximately 410%, approximately 410% to approximately 420%, approximately 420% to approximately 430%, approximately 430% to approximately 440%, approximately 440% to approximately 450%, approximately 450% to approximately 460%, approximately 460% to approximately 470%, approximately 470% to approximately 480%, approximately 480% to approximately 490%, or approximately 490% to approximately 500% higher.

[0198] In some cases, the concentration of one or more salts on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even higher than 500%.

[0199] In some cases, the concentration of one or more salts on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the cis side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be less than 0.1% higher.

[0200] In some cases, the concentration of one or more salts on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more salts on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

[0201] In some embodiments, the concentration of one or more salts on the transform side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more salts on the cis side of the membrane. In some cases, the concentration of one or more salts on the transform side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% Approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% ~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~ It may be approximately 360%, approximately 360% to approximately 370%, approximately 370% to approximately 380%, approximately 380% to approximately 390%, approximately 390% to approximately 400%, approximately 400% to approximately 410%, approximately 410% to approximately 420%, approximately 420% to approximately 430%, approximately 430% to approximately 440%, approximately 440% to approximately 450%, approximately 450% to approximately 460%, approximately 460% to approximately 470%, approximately 470% to approximately 480%, approximately 480% to approximately 490%, or even 490% to approximately 500% lower.

[0202] In some cases, the concentration of one or more salts on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, less At least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or even lower than 500%.

[0203] In some cases, the concentration of one or more salts on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 3% higher than the concentration of one or more salts on the cis side of the membrane. 90%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75% , up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be lower than 0.1%.

[0204] In some cases, the concentration of one or more salts on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more salts on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0205] In some embodiments, EOF can be generated by an asymmetric salt distribution. An asymmetric salt distribution may occur when the concentration of one or more salts on the cis side of the membrane is greater than or less than the concentration of one or more salts on the trans side of the membrane.

[0206] Alternatively, EOF can be generated by a symmetric salt distribution between the cis and trans sides of the membrane. A symmetric salt distribution may occur when the concentration of one or more salts on the cis side of the membrane is the same as the concentration of one or more salts on the trans side of the membrane. In some embodiments, the concentration of one or more salts on the cis side of the membrane may be the same as the concentration of one or more salts on the trans side of the membrane.

[0207] In some embodiments, EOF may be generated by an asymmetric ion distribution between the cis and trans sides of the membrane. An asymmetric ion distribution may occur when the concentration of one or more ions on the cis side of the membrane is greater than or less than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be greater than or less than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be higher than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be lower than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the trans side of the membrane may be higher than the concentration of one or more ions on the cis side of the membrane. In some cases, the concentration of one or more ions on the trans side of the membrane may be lower than the concentration of one or more ions on the cis side of the membrane.

[0208] In some cases, the concentration of one or more ions on the cis side of the membrane can be about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more ions on the cis side of the membrane can be about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more ions on the cis side of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, At least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed 1,000 nM.In some cases, the concentration of one or more ions on the cis side of the membrane is up to approximately 1,000 nM, up to approximately 950 nM, up to approximately 900 nM, up to approximately 850 nM, up to approximately 800 nM, up to approximately 750 nM, up to approximately 700 nM, up to approximately 650 nM, up to approximately 600 nM, up to approximately 550 nM, up to approximately 500 nM, up to approximately 450 nM, up to approximately 400 nM, up to approximately 350 nM, up to approximately 300 nM, up to approximately 250 nM, and up to approximately 200 nM. The maximum nM is approximately 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less than 1 nM. In some cases, the salt concentration on the cis side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, and approximately It may be 100 nM, approximately 150 nM, approximately 200 nM, approximately 250 nM, approximately 300 nM, approximately 350 nM, approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, approximately 800 nM, approximately 850 nM, approximately 900 nM, approximately 950 nM, or approximately 1,000 nM.

[0209] In some cases, the concentration of one or more ions on the transform side of the membrane can range from about 1 nanomolar (nM) to about 1,000 nM. In some cases, the concentration of one or more ions on the transform side of the membrane can range from about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 100 nM to about 1,000 nM. In some cases, the concentration of one or more ions on the transform side of the membrane can range from at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, and at least about 95 nM. , at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or may exceed 1,000 nM.In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 1,000 nM, 950 nM, 900 nM, 850 nM, 800 nM, 750 nM, 700 nM, 650 nM, 600 nM, 550 nM, 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, and 200 nM. The maximum values ​​may be approximately 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or less than 1 nM. In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, and 90 nM. It may be 5 nM, approximately 100 nM, approximately 150 nM, approximately 200 nM, approximately 250 nM, approximately 300 nM, approximately 350 nM, approximately 400 nM, approximately 450 nM, approximately 500 nM, approximately 550 nM, approximately 600 nM, approximately 650 nM, approximately 700 nM, approximately 750 nM, approximately 800 nM, approximately 850 nM, approximately 900 nM, approximately 950 nM, or approximately 1,000 nM.

[0210] In some embodiments, one or more ions may include chlorides, carbonic acid, chlorous acid, chloric acid, phosphoric acid, bicarbonate, bromide, ammonium sulfate, ammonium, sulfuric acid, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, oxide, or any combination thereof.

[0211] In some embodiments, one or more ions on the cis side of the membrane may include chloride, carbonic acid, chlorous acid, chloric acid, phosphoric acid, bicarbonate, bromide, ammonium sulfate, ammonium, sulfuric acid, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, oxide, or any combination thereof.

[0212] In some embodiments, one or more ions on the transformer side of the membrane may include chloride, carbonic acid, chlorous acid, chloric acid, phosphoric acid, bicarbonate, bromide, ammonium sulfate, ammonium, sulfuric acid, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, oxide, or any combination thereof.

[0213] In some embodiments, one or more ions on the cis side of the membrane may be of the same type as one or more ions on the trans side of the membrane. In some embodiments, one or more ions on the cis side of the membrane may be of a different type than one or more ions on the trans side of the membrane.

[0214] In some embodiments, one or more ions may contain about 1 to about 10 ions. In some cases, one or more ions may contain at least about 1 ion, at least about 2 ions, at least about 3 ions, at least about 4 ions, at least about 5 ions, at least about 6 ions, at least about 7 ions, at least about 8 ions, at least about 9 ions, at least about 10 ions, or more than 10 ions. In some cases, one or more ions may contain up to about 10 ions, up to about 9 ions, up to about 8 ions, up to about 7 ions, up to about 6 ions, up to about 5 ions, up to about 4 ions, up to about 3 ions, up to about 2 ions, up to about 1 ion, or less than 1 ion. In some cases, one or more ions may contain about 1 ion, about 2 ions, about 3 ions, about 4 ions, about 5 ions, about 6 ions, about 7 ions, about 8 ions, about 9 ions, or about 10 ions.

[0215] 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.

[0216] In some embodiments, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 500% higher than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 7 0%~approx. 75%, approx. 75%~approx. 80%, approx. 80%~approx. 85%, approx. 85%~approx. 90%, approx. 90%~approx. 95%, approx. 95%~approx. 100%, approx. 100%~approx. 110%, approx. 110%~approx. 120%, approx. 120%~approx. 130%, approx. 130%~approx. 140%, approx. 140%~approx. 150%, approx. 150%~approx. 160%, approx. 160%~approx. 170%, approx. 170%~approx. 180%, approx. 180%~approx. 190%, approx. 190%~approx. 200%, approx. 20 0% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% It may be approximately 360%, 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or even 490% to 500% higher.

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

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

[0219] In some cases, the concentration of one or more ions on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

[0220] In some embodiments, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 7 0%~approx. 75%, approx. 75%~approx. 80%, approx. 80%~approx. 85%, approx. 85%~approx. 90%, approx. 90%~approx. 95%, approx. 95%~approx. 100%, approx. 100%~approx. 110%, approx. 110%~approx. 120%, approx. 120%~approx. 130%, approx. 130%~approx. 140%, approx. 140%~approx. 150%, approx. 150%~approx. 160%, approx. 160%~approx. 170%, approx. 170%~approx. 180%, approx. 180%~approx. 190%, approx. 190%~approx. 200%, approx. 20 0% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% It may be approximately 360%, 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or even 490% to 500% lower.

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

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

[0223] In some cases, the concentration of one or more ions on the cis side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the trans side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0224] In some embodiments, the concentration of one or more ions on the transform side of the membrane may be about 0.1% to about 500% higher than the concentration of salts on the cis side of the membrane. In some cases, the concentration of salts on the transform side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 70% to about 70% 5%, approximately 75% to 80%, approximately 80% to 85%, approximately 85% to 90%, approximately 90% to 95%, approximately 95% to 100%, approximately 100% to 110%, approximately 110% to 120%, approximately 120% to 130%, approximately 130% to 140%, approximately 140% to 150%, approximately 150% to 160%, approximately 160% to 170%, approximately 170% to 180%, approximately 180% to 190%, approximately 190% to 200%, approximately 200% to 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 350%~approximately It may be 360%, approximately 360% to 370%, approximately 370% to 380%, approximately 380% to 390%, approximately 390% to 400%, approximately 400% to 410%, approximately 410% to 420%, approximately 420% to 430%, approximately 430% to 440%, approximately 440% to 450%, approximately 450% to 460%, approximately 460% to 470%, approximately 470% to 480%, approximately 480% to 490%, or even 490% to 500% higher.

[0225] In some cases, the concentration of one or more ions on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, and at least about 180%. , at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or may be higher than 500%.

[0226] In some cases, the concentration of one or more ions on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 420%, up to approximately 410%, and up to approximately 400%. At approximately 390%, at a maximum of approximately 380%, at a maximum of approximately 370%, at a maximum of approximately 360%, at a maximum of approximately 350%, at a maximum of approximately 340%, at a maximum of approximately 330%, at a maximum of approximately 320%, at a maximum of approximately 310%, at a maximum of approximately 300%, at a maximum of approximately 290%, at a maximum of approximately 280%, at a maximum of approximately 270%, at a maximum of approximately 260%, at a maximum of approximately 250%, at a maximum of approximately 240%, at a maximum of approximately 230%, and at a maximum At approximately 220%, with a maximum of approximately 210%, with a maximum of approximately 200%, with a maximum of approximately 190%, with a maximum of approximately 180%, with a maximum of approximately 170%, with a maximum of approximately 160%, with a maximum of approximately 150%, with a maximum of approximately 140%, with a maximum of approximately 130%, with a maximum of approximately 120%, with a maximum of approximately 110%, with a maximum of approximately 100%, with a maximum of approximately 95%, with a maximum of approximately 90%, with a maximum of approximately 85%, with a maximum of approximately 80%, with a maximum of approximately 75% %, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be less than 0.1% higher.

[0227] In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% higher.

[0228] In some embodiments, the concentration of one or more ions on the transform side of the membrane may be about 0.1% to about 500% lower than the concentration of one or more ions on the cis side of the membrane. In some cases, the concentration of one or more ions on the transform side of the membrane may be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, and about 7 0%~approx. 75%, approx. 75%~approx. 80%, approx. 80%~approx. 85%, approx. 85%~approx. 90%, approx. 90%~approx. 95%, approx. 95%~approx. 100%, approx. 100%~approx. 110%, approx. 110%~approx. 120%, approx. 120%~approx. 130%, approx. 130%~approx. 140%, approx. 140%~approx. 150%, approx. 150%~approx. 160%, approx. 160%~approx. 170%, approx. 170%~approx. 180%, approx. 180%~approx. 190%, approx. 190%~approx. 200%, approx. 20 0% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% It may be approximately 360%, 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or even 490% to 500% lower.

[0229] In some cases, the concentration of one or more ions on the trans side of the membrane is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, and at least about 180%. , at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or lower than 500%.

[0230] In some cases, the concentration of one or more ions on the trans side of the membrane is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, and up to approximately 420%, up to approximately 410%, and up to approximately 400%. At approximately 390%, at a maximum of approximately 380%, at a maximum of approximately 370%, at a maximum of approximately 360%, at a maximum of approximately 350%, at a maximum of approximately 340%, at a maximum of approximately 330%, at a maximum of approximately 320%, at a maximum of approximately 310%, at a maximum of approximately 300%, at a maximum of approximately 290%, at a maximum of approximately 280%, at a maximum of approximately 270%, at a maximum of approximately 260%, at a maximum of approximately 250%, at a maximum of approximately 240%, at a maximum of approximately 230%, and at a maximum At approximately 220%, with a maximum of approximately 210%, with a maximum of approximately 200%, with a maximum of approximately 190%, with a maximum of approximately 180%, with a maximum of approximately 170%, with a maximum of approximately 160%, with a maximum of approximately 150%, with a maximum of approximately 140%, with a maximum of approximately 130%, with a maximum of approximately 120%, with a maximum of approximately 110%, with a maximum of approximately 100%, with a maximum of approximately 95%, with a maximum of approximately 90%, with a maximum of approximately 85%, with a maximum of approximately 80%, with a maximum of approximately 75% %, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or may be lower than 0.1%.

[0231] In some cases, the concentration of one or more ions on the trans side of the membrane is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, and 170% higher than the concentration of one or more ions on the cis side of the membrane. It may be approximately 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or even 500% lower.

[0232] Alternatively, EOF can be generated by a symmetric ion distribution between the cis and trans sides of the membrane. A symmetric ion distribution can occur when the concentration of one or more ions on the cis side of the membrane is the same as the concentration of one or more ions on the trans side of the membrane. In some cases, the concentration of one or more ions on the cis side of the membrane is the same as the concentration of one or more ions on the trans side of the membrane.

[0233] In some embodiments, the EOF may be generated by asymmetric concentrations of one or more salts and one or more ions between the cis side and the trans side of the membrane.

[0234] In some embodiments, the electroosmotic force may act in the same direction as the electrophoretic force, or in the opposite direction to the electrophoretic force. In some embodiments, the electroosmotic force may be greater than the electrophoretic force. In some embodiments, the electroosmotic force may be less than the electrophoretic force.

[0235] In some embodiments, the cis-to-trans EOF may include a net ionic current flow from the cis side of the membrane to the transform side of the membrane. In some embodiments, the transform-to-cis EOF may include a net ionic current flow from the transform side of the membrane to the cis side of the membrane. In some cases, the nanopore system may include a total ionic current flow. In some cases, the net ionic current flow may include a flow less than the total of all ions in the nanopore system. In some cases, the net ionic current flow may include a flow less than the total of all ions in the nanopore system in a particular direction. In some cases, the particular direction may be from the cis side of the membrane to the transform side of the membrane. In some cases, the particular direction may be from the transform side of the membrane to the cis side of the membrane. In some cases, the total ionic current flow may include the total flow of all ions in the nanopore system. In some cases, the total flow of all ions in the nanopore system may be from the cis side of the membrane to the transform side of the membrane. In some cases, the total flow of all ions within the nanopore system may be in the direction from the transformer side of the membrane to the cis side of the membrane. In some cases, the total ion current flow may include the total flow of all ions within the nanopore system in a specific direction. In some cases, the specific direction may be from the cis side of the membrane to the transformer side of the membrane. In some cases, the specific direction may be from the transformer side of the membrane to the cis side of the membrane.

[0236] In some embodiments, the net ionic current flow may comprise about 0.001% to about 100% of the total ionic current flow. In some cases, the net ionic current flow may comprise about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 0.1% to about 1%, about 1% to about 10%, or about 10% to about 100% of the total ionic current flow. In some cases, the net ionic current flow may include at least about 0.001%, at least about 0.005%, at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 100% of the total ionic current flow. In some cases, the net ionic current flow is approximately 100% of the total ionic current flow, at most about 99.5%, at most about 99%, at most about 98%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most This may include approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, up to approximately 0.05%, up to approximately 0.01%, up to approximately 0.005%, up to approximately 0.001%, or less than 0.001%.In some cases, the net ionic current flow may consist of approximately 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 100% of the total ionic current flow.

[0237] In some embodiments, the cis-to-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow of about 0.0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 0.95, or about 0.99, and is also called the relative net current flow from cis to transformer. In some embodiments, the cis-transformer EOF arises from a net ionic current flow from cis to transformer that exceeds the total ionic current flow, which is less than about 0.0, less than about -0.1, less than about -0.2, less than about -0.3, less than about -0.4, less than about -0.5, less than about -0.6, less than about -0.7, less than about -0.8, less than about -0.9, less than about -0.95, or less than about -0.99, and is also called the relative net current flow from cis to transformer.

[0238] In some embodiments, the net electroosmotic current (I) is absolute relative to the applied voltage. relV ) is greater than approximately 0.01, greater than approximately 0.02, greater than approximately 0.03, greater than approximately 0.04, greater than approximately 0.05, greater than approximately 0.06, greater than approximately 0.07, greater than approximately 0.08, greater than approximately 0.09, greater than approximately 0.10, greater than approximately 0.15, greater than approximately 0.2, greater than approximately 0.3, greater than approximately 0.4, greater than approximately 0.5, greater than approximately 0.6, greater than approximately 0.7, greater than approximately 0.8, greater than approximately 0.9, or greater than approximately 1 picoampere / millivolt (pA / mV). In some embodiments, the net electroosmotic current (I) is the absolute relative value of the applied voltage. relV) is less than approximately 0.01, less than approximately 0.02, less than approximately 0.03, less than approximately 0.04, less than approximately 0.05, less than approximately 0.06, less than approximately 0.07, less than approximately 0.08, less than approximately 0.09, less than approximately 0.10, less than approximately 0.15, less than approximately 0.2, less than approximately 0.3, less than approximately 0.4, less than approximately 0.5, less than approximately 0.6, less than approximately 0.7, less than approximately 0.8, less than approximately 0.9, or less than approximately 1 pA / mV.

[0239] 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 can move the analyte through the translocase. In some cases, the translocase can move the analyte through the translocase into the nanopores. In some cases, the translocase can move the analyte (ananlyte) through the translocase into the nanopores and through the nanopore channels. The molecular motor may be driven by NTP, or by ATP, 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). Translocases may include ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, their functional homologs, orthologues, or paralogs, or any combination thereof. Translocases can bind to or form complexes with analytes (e.g., translocase-analyte complexes). Translocases can extrude analytes through them. Translocases can rearrange analytes through nanopores. Extrusion of analytes through translocases can disrupt (e.g., unfold) the quaternary, tertiary, or secondary structure of the analyte (e.g., protein). Disruption of the quaternary, tertiary, or secondary structure can aid in the rearrangement of analytes through nanopores. The translocase may form a complex with the analyte (e.g., peptide, protein) at its N-terminus or C-terminus. The translocase may form a translocase-analyte complex with the analyte on the cis side of the fluid chamber. The translocase-analyte complex may form outside the fluid chamber or inside the fluid chamber (e.g., on the cis side of the fluid chamber).The translocase-analyte complex can be formed before being added to the cis side of the fluid chamber (e.g., by preloading with a preloading solution). The translocase can control the rate of translocation. The rate of translocation of the translocase can be adjusted. The adjustment can be performed by changing the concentration of the energy source of the translocase (e.g., NTP, ATP).

[0240] In some embodiments, the translocase can move an analyte. In some embodiments, the translocase may not be able to separate the strands of double-stranded nucleic acid. In some cases, the translocase may not be a helicase. In some embodiments, the translocase may not be able to replicate nucleic acid. In some cases, the translocase may not be a nucleic acid polymerase. In some cases, the translocase may not be a DNA polymerase or an RNA polymerase. In some embodiments, the translocase may not be involved in nucleic acid replication. In some embodiments, the translocase may not be able to cleave an analyte. In some embodiments, the translocase may not be a topoisomerase.

[0241] In some embodiments, the translocase can be coupled to a nanopore. The translocase can be coupled covalently (e.g., by gene fusion) or non-covalently (e.g., by recognition elements).

[0242] In some embodiments, the translocase may not be coupled to a 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 adjacent to the nanopore.

[0243] In some embodiments, the translocase may not be bound to the nanopores. In some cases, the translocase may not be bound to the openings of the nanopores. In some cases, the translocase may not be bound to the membrane adjacent to the nanopores.

[0244] In some embodiments, electroosmosis can capture the translocase-analyte complex (e.g., the translocase-analyte complex). Capture may result in pulling a portion of the analyte not within the translocase of the translocase-analyte complex into the nanopores. Capture of a portion of the analyte by the nanopore channels can cause a portion of the analyte in the translocase-analyte complex to displace through the nanopores, as the analyte may be further pulled into the nanopore channels by electroosmosis. Displacement may occur against or in combination with electrophoresis. Displacement of the analyte portion occurs when the portion of the analyte being displaced approaches the portion of the analyte within the translocase of the translocase-analyte complex. Translocase can be positioned adjacent to nanopore channels. If the electroosmotic force acts from cis to trans, the translocase can be positioned adjacent to nanopores on the cis side of the nanopore, or if the electroosmotic force acts from trans to cis, it can be positioned adjacent to nanopores on the trans side of the nanopore. Electroosmotic force can retain the translocase in the translocase-analyte complex adjacent to the nanopores of the nanopore channel by, for example, continuously drawing the analyte through the nanopore and transferring the electroosmotic force to the attached translocase. Adjacent to a nanopore channel means being near the opening of the nanopore channel, also called the "upper part" of the nanopore, or near portions of the nanopore that are not within the membrane.

[0245] In some embodiments, the EOF may retain the translocase at the top of the analyte-free nanopores. In some cases, a portion of the translocase may be trapped in the nanopores. In some cases, a portion of the translocase may be trapped in the nanopores due to electroosmosis. In some cases, a portion of the translocase may be trapped in the nanopores due to electrophoretic force. In some cases, a portion of the translocase may be trapped in the nanopores due to both electroosmosis and electrophoretic force. In some cases, the portion of the translocase trapped in the nanopores may be a charged linker or peptide extension of the translocase.

[0246] When held adjacent to a nanopore channel, the translocase of the translocase-analyte complex may be held oriented so that the translocase channel is adjacent to the nanopore channel. This orientation may be provided by the electroosmotic attraction of the analyte into the nanopore, thereby allowing the connection point of the analyte to the translocase (e.g., the translocase channel) to be adjacent to the nanopore channel, and the translocase channel to align. In some embodiments, the translocase can control the rate of analyte rearrangement. The rearrangement rate may be a result of the translocase acting on the analyte as a molecular motor. In some embodiments, the rearrangement rate may be about 0.1 amino acids / second (aa / s) to about 1,000 aa / s. In some cases, the dislocation rate may be at least about 0.1 aa / s, at least about 0.5 aa / s, at least about 1 aa / s, at least about 5 aa / s, at least about 10 aa / s, at least about 50 aa / s, at least about 100 aa / s, at least about 500 aa / s, at least about 1,000 aa / s, or more than 1,000 aa / s. In some cases, the dislocation rate may be at least up to about 1,000 aa / s, up to about 500 aa / s, up to about 100 aa / s, up to about 50 aa / s, up to about 10 aa / s, up to about 5 aa / s, up to about 1 aa / s, up to about 0.5 aa / s, up to about 0.1 aa / s, or less than 0.1 aa / s. In some cases, the dislocation rate may be approximately 0.1 aa / s, 0.5 aa / s, 1 aa / s, 5 aa / s, 10 aa / s, 50 aa / s, 100 aa / s, 500 aa / s, or 1,000 aa / s.

[0247] The orientation of the dislocation may be such that the supply direction of the translocase aligns with the channels of the nanopores. The supply direction may be oriented from cis to trans, or from 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 may supply the analyte so that the translocase can supply the analyte through the nanopores in the direction of electroosmosis, or it may retain the analyte so that it pulls it through the nanopores against electroosmosis.

[0248] Translocase can deliver analytes through nanopores in the direction of electroosmosis so that they disassemble at a faster or slower rate than analytes that disassemble solely by electroosmosis.

[0249] In some embodiments, the rate of analyte dislocation through nanopores using translocase can be made faster than the rate of analyte dislocation through nanopores without translocase. In some cases, the rate of analyte dislocation through nanopores using translocase can be made about 0.1% to about 500% faster than the rate of analyte dislocation through nanopores without translocase. In some cases, the rate of analyte dislocation through nanopores using 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%, and about 60% to about 6 5%, approximately 65% ​​to 70%, approximately 70% to 75%, approximately 75% to 80%, approximately 80% to 85%, approximately 85% to 90%, approximately 90% to 95%, approximately 95% to 100%, approximately 100% to 110%, approximately 110% to 120%, approximately 120% to 130%, approximately 130% to 140%, approximately 140% to 150%, approximately 150% to 160%, approximately 160% to 170%, approximately 170% to 180%, approximately 180% to 190%, approximately 190% to 200%, approximately 200%~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 35 It can be made faster by 0% to approximately 360%, approximately 360% to approximately 370%, approximately 370% to approximately 380%, approximately 380% to approximately 390%, approximately 390% to approximately 400%, approximately 400% to approximately 410%, approximately 410% to approximately 420%, approximately 420% to approximately 430%, approximately 430% to approximately 440%, approximately 440% to approximately 450%, approximately 450% to approximately 460%, approximately 460% to approximately 470%, approximately 470% to approximately 480%, approximately 480% to approximately 490%, or approximately 490% to approximately 500%.

[0250] In some cases, the rate of analyte dislocation through nanopores using translocase is at least approximately 0.1%, at least approximately 0.5%, at least approximately 1%, at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, at least approximately 110%, at least approximately 120%, at least approximately 130%, at least approximately 140%, at least approximately 150%, at least approximately 160%, and at least approximately 170% less than the rate of analyte dislocation through nanopores without translocase. It can be made faster by at least approximately 180%, at least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or more than 500%.In some cases, the rate of dislocation of analytes through nanopores using translocase is up to approximately 500%, 490%, 480%, 470%, 460%, 450%, 440%, 430%, 420%, and 41% higher than the rate of dislocation of analytes through nanopores without translocase. 0%, up to approximately 400%, up to approximately 390%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to Approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately It can be made 75% faster, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or less than 0.1% faster.In some cases, the rate of analyte dislocation through nanopores using translocase is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, and 100%. It can be made 60%, approximately 170%, approximately 180%, approximately 190%, approximately 200%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, or approximately 500% faster.

[0251] In some embodiments, the rate of analyte dislocation through nanopores using translocase can be slower than the rate of analyte dislocation through nanopores without translocase. In some cases, the rate of analyte dislocation through nanopores using translocase can be slower by about 0.1% to about 500% than the rate of analyte dislocation through nanopores without translocase. In some cases, the rate of analyte dislocation through nanopores using translocase can be slower by about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, and about 60% to about 6 5%, approximately 65% ​​to 70%, approximately 70% to 75%, approximately 75% to 80%, approximately 80% to 85%, approximately 85% to 90%, approximately 90% to 95%, approximately 95% to 100%, approximately 100% to 110%, approximately 110% to 120%, approximately 120% to 130%, approximately 130% to 140%, approximately 140% to 150%, approximately 150% to 160%, approximately 160% to 170%, approximately 170% to 180%, approximately 180% to 190%, approximately 190% to 200%, approximately 200%~approximately 210%, approximately 210%~approximately 220%, approximately 220%~approximately 230%, approximately 230%~approximately 240%, approximately 240%~approximately 250%, approximately 250%~approximately 260%, approximately 260%~approximately 270%, approximately 270%~approximately 280%, approximately 280%~approximately 290%, approximately 290%~approximately 300%, approximately 300%~approximately 310%, approximately 310%~approximately 320%, approximately 320%~approximately 330%, approximately 330%~approximately 340%, approximately 340%~approximately 350%, approximately 35 It can be slowed down by approximately 0% to 360%, approximately 360% to 370%, approximately 370% to 380%, approximately 380% to 390%, approximately 390% to 400%, approximately 400% to 410%, approximately 410% to 420%, approximately 420% to 430%, approximately 430% to 440%, approximately 440% to 450%, approximately 450% to 460%, approximately 460% to 470%, approximately 470% to 480%, approximately 480% to 490%, or approximately 490% to 500%.

[0252] In some cases, the rate of analyte dislocation through nanopores using translocase is at least approximately 0.1%, at least approximately 0.5%, at least approximately 1%, at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, at least approximately 110%, at least approximately 120%, at least approximately 130%, at least approximately 140%, at least approximately 150%, at least approximately 160%, and at least approximately 170% less than the rate of analyte dislocation through nanopores without translocase. It can be slowed down by at least approximately 180%, at least approximately 190%, at least approximately 200%, at least approximately 210%, at least approximately 220%, at least approximately 230%, at least approximately 240%, at least approximately 250%, at least approximately 260%, at least approximately 270%, at least approximately 280%, at least approximately 290%, at least approximately 300%, at least approximately 310%, at least approximately 320%, at least approximately 330%, at least approximately 340%, at least approximately 350%, at least approximately 360%, at least approximately 370%, at least approximately 380%, at least approximately 390%, at least approximately 400%, at least approximately 410%, at least approximately 420%, at least approximately 430%, at least approximately 440%, at least approximately 450%, at least approximately 460%, at least approximately 470%, at least approximately 480%, at least approximately 490%, at least approximately 500%, or more than 500%.In some cases, the rate of dislocation of analytes through nanopores using translocase is up to approximately 500%, 490%, 480%, 470%, 460%, 450%, 440%, 430%, 420%, and 41% higher than the rate of dislocation of analytes through nanopores without translocase. 0%, up to approximately 400%, up to approximately 390%, up to approximately 380%, up to approximately 370%, up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to Approximately 230%, up to approximately 220%, up to approximately 210%, up to approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately It can be slowed down by 75%, up to approximately 70%, up to approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1%, or less than 0.1%.In some cases, the rate of analyte dislocation through nanopores using translocase is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, and 100%. It can be slowed down by 60%, approximately 170%, approximately 180%, approximately 190%, approximately 200%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, or approximately 500%.

[0253] In some embodiments, the primary force in analyte dislocation through nanopores may be EOF. In some embodiments, analyte dislocation through nanopores may occur using EOF. In some cases, analyte dislocation through nanopores may occur in the absence of translocase. In some cases, analyte dislocation through nanopores may occur using EOF and in the absence of translocase.

[0254] In some embodiments, analyte dislocations through the nanopores do not occur in the absence of EOF. In some cases, analyte dislocations through the nanopores do not occur in the presence of translocase. In some cases, analyte dislocations through the nanopores do not occur in the absence of EOF and in the presence of translocase.

[0255] The translocase can be retained on the cis or trans side of the nanopore without needing to be coupled to the nanopore. Electroosmotic force can retain the translocase adjacent to the nanopore without additional coupling to the nanopore channel. Electroosmotic force can retain the translocase adjacent to the nanopore so that the nanopore can couple with the translocase. The translocase can be retained adjacent to the nanopore channel while the analyte displaces through the nanopore. After the analyte has completely displaced through the nanopore, the translocase may continue to be retained adjacent to the nanopore, or it may be released from its position adjacent to the nanopore. The released translocase can then form a translocase-analyte complex with another analyte. In some embodiments, the translocase does not need to be coupled to the nanopore. In some cases, the translocase does not need to be coupled adjacent to the nanopore. In some cases, the translocase does not need to be coupled to the membrane adjacent to the nanopore.

[0256] Alternatively, in some embodiments, the translocase may be coupled to the cis or trans side of the nanopore. In some cases, the translocase may be coupled to the nanopore via covalent bonds. In some cases, the covalent bonds are polar covalent bonds. In some cases, the covalent bonds are nonpolar covalent bonds. In some cases, the translocase may be coupled to the nanopore via non-covalent bonds. In some cases, the non-covalent bonds may include electrostatic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, or any combination thereof. In some cases, the translocase may be coupled to the nanopore via a linker. In some cases, the linker is (GGGGS)3, (GGGGS) n , (SG) n , (Gly)8, (Gly)6, (EAAAK)3, (EAAAK) n、VSQTSKLTRAETVFPDV、PLGLWA、RVLAEA、EDVVCCSNSY、GGIEGRGS、TRHRQPRGWE、AGNRVR RSVG、RRRRRRRRR、GFLG、A(EAAAK)4ALEA(EAAAK)4A、PAPAP、AEAAAAKEAAAKA、(Ala-Pro) nThe linker may include disulfide bonds, cysteine ​​bonds, or any combination thereof. In some embodiments, the linker may include any combination of amino acids. In some cases, the amino acids may be standard amino acids. In some cases, the standard amino acids may include alanine, arginine, asparagine, 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 non-standard amino acids. In some cases, non-natural amino acids include hydroproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, pyroglutamic acid, naphthylalanine, Abu, DAB, methionine sulfoxide, methionine sulfone, alpha-amino-n-butyric acid, norvaline, alloisoleucine, t-leucine, alpha-amino-n-heptanoic acid, pipecolic acid, allosreonine, homocysteine, homoserine, and alpha,beta-diaminopropionic acid. The linker may include α,γ-diaminobutyric acid, beta-alanine, beta-amino-n-butyric acid, beta-aminoisobutyric acid, beta-aminoisobutyric acid, γ-aminobutyric acid, α-aminoisobutyric acid, isovaline, sarcosine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, N-methyl-beta-alanine, N-ethyl-beta-alanine, isoserine, α-hydroxy-γ-aminobutyric acid, or any combination thereof. In some cases, the linker may include any combination of standard amino acids and non-natural amino acids. In some cases, the linker may be ethylene glycol. In some cases, the linker may be polyethylene glycol. In some cases, the linker may be biotin. In some cases, the linker may be streptavidin. In some cases, the linker may be a cysteine ​​bond. In some cases, the linker may be formed using Spytag / Spycatcher, Halo-tag, Snap-tag, or other bioconjugation methods.In some cases, linkers can be formed from click chemistry. In other cases, linkers attach to unnatural amino acids.

[0257] 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 transform side. The cis side may contain a first fluid solution. The transform side may contain a second fluid solution. In some embodiments, one or more solutions on either the cis or transform side of the fluid chamber may be configured to have a set pH. One or more solutions may have a pH 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, greater than about 12, greater than about 13, or greater than about 14, and these can be utilized. One or more solutions may have a pH of less than approximately 1, less than approximately 2, less than approximately 3, less than approximately 3.8, less than approximately 4, less than approximately 4.5, less than approximately 6, less than approximately 7, less than approximately 8, less than approximately 9, less than approximately 10, less than approximately 10.5, less than approximately 11, less than approximately 12, less than approximately 13, or less than approximately 14, and these can be used.

[0258] In some embodiments, the first solution and the second solution may be different solutions. In some embodiments, the first solution and the second solution may be different solutions and may have different concentrations of one or more ions. In some embodiments, the first solution and the second solution may be different solutions and may have different concentrations of one or more salts. In some embodiments, the first solution and the second solution may be different solutions and may have different concentrations of one or more salts and different concentrations of one or more ions.

[0259] Alternatively, in some embodiments, the first solution and the second solution may be different solutions having the same concentration of one or more salts. In some embodiments, the first solution and the second solution may be different solutions having the same concentration of one or more ions. In some embodiments, the first solution and the second solution may be different solutions having the same concentration of one or more ions and the same concentration of one or more salts.

[0260] In some embodiments, the first solution and the second solution may be the same solution. In some embodiments, the first solution and the second solution may be the same solution and have different concentrations of one or more ions. In some embodiments, the first solution and the second solution may be the same solution and have different concentrations of one or more salts. In some embodiments, the first solution and the second solution may be the same solution and have different concentrations of one or more ions and different concentrations of one or more salts.

[0261] Alternatively, in some embodiments, the first solution and the second solution may be the same solution and may have the same concentration of one or more salts. In some embodiments, the first solution and the second solution may be the same solution and may have the same concentration of one or more ions. In some embodiments, the first solution and the second solution may be the same solution and may have the same concentration of one or more ions and the same concentration of one or more salts.

[0262] A fluid solution may be configured to provide electroosmotic flow (also called electroosmotic force). In some embodiments, the electroosmotic flow may be generated by having an asymmetric distribution of one or more salts between the cis side and the transform side of the membrane. In some embodiments, the electroosmotic flow may be generated by having an asymmetric distribution of one or more ions between the cis side and the transform side of the membrane. In some embodiments, the electroosmotic flow may be generated by having an asymmetric distribution of one or more ions and one or more salts between the cis side and the transform side of the membrane. The electroosmotic force may act across the membrane. In some embodiments, the membrane includes nanopores. In some embodiments, a pair of electrodes is provided. The pair of electrodes may be arranged by one electrode on the cis side of the fluid chamber and the other electrode on the transform side of the fluid chamber. In some embodiments, the pair of electrodes may be configured to provide an applied voltage. The applied voltage may be across the membrane. The applied voltage can result in electrophoretic force. In some embodiments, the pair of electrodes may be configured to provide electrophoretic force across the membrane. The pair of electrodes may be configured to measure a signal.

[0263] In some embodiments, the applied voltage across the membrane can be on the order of 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 1000 millivolts (mV). The applied voltage can be at most on the order of about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, or about 1000 mV. In some embodiments, the voltage is negative from cis to trans. In some embodiments, the voltage is positive from cis to trans.

[0264] In some embodiments, translocation of an analyte through the nanopore occurs in the direction from cis to trans. In some embodiments, translocation of an analyte through the nanopore occurs in the direction from trans to cis. In some embodiments, translocation of an analyte through the nanopore occurs in the direction of the electroosmotic force (EOF). In some embodiments, translocation of an analyte through the nanopore occurs in the direction opposite to the electrophoretic force (EPF). In some embodiments, translocation of an analyte through the nanopore occurs in the direction of the EOF and in the direction opposite to the direction of the EPF.

[0265] In some embodiments, EOF may be greater than EPF. In some cases, EOF is about 0.1% to about 500% greater than EPF. In some cases, EOF is about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, and about 80%. ~approximately 85%, approximately 85%~approximately 90%, approximately 90%~approximately 95%, approximately 95%~approximately 100%, approximately 100%~approximately 110%, approximately 110%~approximately 120%, approximately 120%~approximately 130%, approximately 130%~approximately 140%, approximately 140%~approximately 150%, approximately 150%~approximately 160%, approximately 160%~approximately 170%, approximately 170%~approximately 180%, approximately 180%~approximately 190%, approximately 190%~approximately 200%, approximately 200%~approximately 210%, approximately 210%~ Approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% to approximately 360%, Approximately 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or 490% to 500% longer and larger.

[0266] In some cases, EOF is at least approximately 0.1%, at least approximately 0.5%, at least approximately 1%, at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, at least approximately 110%, at least approximately 120%, at least approximately 130%, at least approximately 140%, at least approximately 150%, at least approximately 160%, at least approximately 170%, at least approximately 180%, at least approximately 190%, and at least approximately 20%. It may be 0%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or at least about 500% greater.

[0267] In some cases, EOF is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, up to approximately 390%, up to approximately 380%, and up to approximately 370% compared to EPF. Up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to Approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to It may be approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1% larger, or up to less than 0.1% larger.

[0268] In some cases, EOF is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and 2% higher than EPF. 00%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, or may be approximately 500% larger.

[0269] In some embodiments, analyte dislocations through the nanopores occur in the direction of the EOF. In some embodiments, analyte dislocations through the nanopores occur in the direction of the EPF. In some embodiments, analyte dislocations through the nanopores occur in the direction of both the EOF and the EPF.

[0270] Alternatively, in some embodiments, analyte dislocations through the nanopores occur in the direction of the EPF. In some embodiments, analyte dislocations through the nanopores occur in the direction opposite to the EOF. In some embodiments, analyte dislocations through the nanopores occur in the direction of the EPF and opposite to the EOF.

[0271] Alternatively, in some embodiments, EPF may be greater than EOF. In some embodiments, EPF may be greater than EOF. In some cases, EPF is about 0.1% to about 500% greater than EOF. In some cases, EPF is about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, and about 80%. ~approximately 85%, approximately 85%~approximately 90%, approximately 90%~approximately 95%, approximately 95%~approximately 100%, approximately 100%~approximately 110%, approximately 110%~approximately 120%, approximately 120%~approximately 130%, approximately 130%~approximately 140%, approximately 140%~approximately 150%, approximately 150%~approximately 160%, approximately 160%~approximately 170%, approximately 170%~approximately 180%, approximately 180%~approximately 190%, approximately 190%~approximately 200%, approximately 200%~approximately 210%, approximately 210%~ Approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% to approximately 360%, Approximately 360% to 370%, 370% to 380%, 380% to 390%, 390% to 400%, 400% to 410%, 410% to 420%, 420% to 430%, 430% to 440%, 440% to 450%, 450% to 460%, 460% to 470%, 470% to 480%, 480% to 490%, or 490% to 500% longer and larger.

[0272] In some cases, EPF is at least approximately 0.1%, at least approximately 0.5%, at least approximately 1%, at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 100%, at least approximately 110%, at least approximately 120%, at least approximately 130%, at least approximately 140%, at least approximately 150%, at least approximately 160%, at least approximately 170%, at least approximately 180%, at least approximately 190%, and at least approximately 2%. 00%, may be greater than or greater than 500%, or at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%.

[0273] In some cases, EPF is up to approximately 500%, up to approximately 490%, up to approximately 480%, up to approximately 470%, up to approximately 460%, up to approximately 450%, up to approximately 440%, up to approximately 430%, up to approximately 420%, up to approximately 410%, up to approximately 400%, up to approximately 390%, up to approximately 380%, and up to approximately 370% compared to EOF. Up to approximately 360%, up to approximately 350%, up to approximately 340%, up to approximately 330%, up to approximately 320%, up to approximately 310%, up to approximately 300%, up to approximately 290%, up to approximately 280%, up to approximately 270%, up to approximately 260%, up to approximately 250%, up to approximately 240%, up to approximately 230%, up to approximately 220%, up to approximately 210%, up to Approximately 200%, up to approximately 190%, up to approximately 180%, up to approximately 170%, up to approximately 160%, up to approximately 150%, up to approximately 140%, up to approximately 130%, up to approximately 120%, up to approximately 110%, up to approximately 100%, up to approximately 95%, up to approximately 90%, up to approximately 85%, up to approximately 80%, up to approximately 75%, up to approximately 70%, up to It may be approximately 65%, up to approximately 60%, up to approximately 55%, up to approximately 50%, up to approximately 45%, up to approximately 40%, up to approximately 35%, up to approximately 30%, up to approximately 25%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 5%, up to approximately 1%, up to approximately 0.5%, up to approximately 0.1% larger, or up to less than 0.1% larger.

[0274] In some cases, EPF is approximately 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, and 2% higher than EOF. 00%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, or may be approximately 500% larger.

[0275] In some embodiments, a signal is measured. The signal may include an electrical signal. The signal may be related to or caused by dislocations of the analyte. The signal may include an ionic current or a change in ionic current. The signal may include a measurement of the change in current between nanopore states. The nanopore states may include an open channel, trapping of the analyte by the nanopore, or passage of the polymer from the trapped state through the nanopore. In some embodiments, measuring the signal may include comparing the signals between different states of the nanopore.

[0276] In some embodiments, electrophoretic force is provided. In some embodiments, the method involves dissociating the analyte through nanopores. Dissociation may be assisted by electroosmosis, electrophoretic force, or a combination thereof. Dissociation may be opposed to electroosmosis, electrophoretic force, or a combination thereof. In some embodiments, the analyte is in a pre-modified state before dissociation. In some embodiments, the method involves measuring a signal. The signal may be caused by or affected by the dissociation of the analyte. In some embodiments, one or more analytes dissociate. The signal of one or more dissociated analytes may be measured. In some embodiments, multiple analytes may be measured. One or more signals from multiple analytes may be used to characterize them. For the purposes of the frequency method, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 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 1000, at least about 1500, at least about 2000, at least about 2500, at least about 3500, at least about 4000, at least about 4500, at least about 5000, at least about 5500, at least about 6000, at least about 6500, at least about 7000, at least about 7500, at least about 8000, at least about 8500, at least about 9000, at least about 9500, or at least about 10,000 analytes can be characterized.In some embodiments, the maximum is approximately 2, the maximum is approximately 3, the maximum is approximately 4, the maximum is approximately 5, the maximum is approximately 6, the maximum is approximately 7, the maximum is approximately 8, the maximum is approximately 9, the maximum is approximately 10, the maximum is approximately 20, the maximum is approximately 30, the maximum is approximately 50, the maximum is approximately 100, the maximum is approximately 200, the maximum is approximately 300, the maximum is approximately 400, the maximum is approximately 500, the maximum is approximately 600, the maximum is approximately 700, the maximum is approximately 800, the maximum is approximately 900, the maximum is approximately 1000, Up to approximately 1500, 2000, 2500, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or up to approximately 10000 analytes can be characterized.

[0277] In some embodiments, the cis and transformer solutions in a fluid chamber are configured to generate electroosmosis. Electroosmosis can be generated due to the difference in solute concentration between the cis and transformer solutions. The solute may be ions or osmolites. These ions or osmolites can flow across the membrane through nanopores. These ions may be high-mobility or low-mobility ions.

[0278] In some embodiments, the electrophoretic force may act in the cis-to-trans direction or from trans-to-cis direction. The electrophoretic force may act in the same direction as the electroosmotic force or in the opposite direction to the electroosmotic force. The electrophoretic force may exert a greater or lesser force on the analyte than the electroosmotic force. The electrophoretic force may assist or oppose the dislocation of the analyte.

[0279] In some embodiments, high-mobility ions may be present in amounts of less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 15%, less than 20%, less than 25%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, or less than 95% of the salt content on the membrane side through which they flow. In some embodiments, high-mobility ions may be present in amounts exceeding approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the salt content on the side of the membrane through which they flow.

[0280] In some embodiments, low-mobility ions may be present in amounts of less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 15%, less than 20%, less than 25%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, or less than 95% of the salt content on the membrane side through which they flow. In some embodiments, low-mobility ions may be present in amounts exceeding approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the salt content on the side of the membrane through which they flow.

[0281] In some embodiments, the concentrations of the cis-side salt, ion, osmolite, or electrolyte are approximately 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 greater than approximately 5M. In some embodiments, the difference in concentration between the cis side and the trans side of the cis side of the salt, ions, osmolite, or electrolyte is approximately 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 greater than approximately 5 M.

[0282] In some embodiments, the solute concentration is greater on the cis side than on the trans side.

[0283] In some embodiments, the analyte is an unmodified analyte. In some embodiments, the analyte is an analyte without a label or tag. In some embodiments, the analyte includes a reader construct. The reader construct may include molecules that conjugate or couple to the analyte. Conjugation or coupling of molecules to the analyte can improve the performance features of the method provided for characterizing the analyte. Performance features may include read length, throughput, processing speed, sequence accuracy, sequence coverage, or a combination thereof. The reader construct may include a label, barcode, or a combination thereof. In some embodiments, the reader construct may be configured to modify the features of the analyte. The reader construct may be configured to modify the binding of the analyte to a translocase, the binding of the analyte to an unfoldase, the binding of the analyte to a membrane, the capture of the analyte by nanopores, or a combination thereof. In some embodiments, the reader construct may be configured to couple the analyte with a translocase. In some cases, the reader construct may be configured to bind the analyte with a translocase. In some cases, the reader construct may be bound to the analyte via a covalent bond. In some cases, the leader construct may be configured to bind to the analyte via non-covalent bonds. In some cases, the leader construct may be configured to bind to the analyte via a linker. In some embodiments, the leader construct may be configured to couple the analyte with an unfoldase. In some embodiments, the leader construct may not be configured to couple to nanopores. In some cases, the leader construct may not couple to nanopores. In some cases, the leader construct may not bind to nanopores. In some cases, the leader construct may not be configured to assist in the capture of the analyte by nanopores.

[0284] In some embodiments, the leader construct may include nucleic acids. In some cases, the nucleic acids may include DNA, RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), cross-linked nucleic acid (BNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), or any combination thereof. In some embodiments, the leader construct may include one or more peptides or proteins. In some embodiments, the leader construct may include nucleic acids, proteins, peptides, or any combination thereof.

[0285] In some embodiments, the leader construct may be oriented from 5' to 3'. In some embodiments, the leader construct may be oriented from N-terminus to C-terminus.

[0286] In some embodiments, the leader construct may include a coupling motif, a stall motif, a block motif, a recognition motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a stall motif, a block motif, a recognition motif, and a capture motif in an N-terminal to C-terminal orientation.

[0287] In some embodiments, the leader construct may include a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0288] In some embodiments, the leader construct may include a coupling motif, a stall motif, a recognition motif, a block motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0289] In some embodiments, the leader construct may include a coupling motif, a stall motif, a recognition motif, a capture motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0290] In some embodiments, the leader construct may include a coupling motif, a stall motif, a capture motif, a block motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a stall motif, a capture motif, a block motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0291] In some embodiments, the leader construct may include a coupling motif, a stall motif, a capture motif, a recognition motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a stall motif, a capture motif, a recognition motif, and a block motif in an N-terminus to C-terminus orientation.

[0292] In some embodiments, the leader construct may include a coupling motif, a blocking motif, a stall motif, a recognition motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a blocking motif, a stall motif, a recognition motif, and a capture motif in an N-terminal to C-terminal orientation.

[0293] In some embodiments, the leader construct may include a coupling motif, a blocking motif, a stall motif, a capture motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a blocking motif, a stall motif, a capture motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0294] In some embodiments, the leader construct may include a coupling motif, a block motif, a recognition motif, a stall motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a block motif, a recognition motif, a stall motif, and a capture motif in an N-terminal to C-terminal orientation.

[0295] In some embodiments, the leader construct may include a coupling motif, a block motif, a recognition motif, a capture motif, and a stall motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a block motif, a recognition motif, a capture motif, and a stall motif in an N-terminal to C-terminal orientation.

[0296] In some embodiments, the leader construct may include a coupling motif, a blocking motif, a capture motif, a recognition motif, and a stall motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a blocking motif, a capture motif, a recognition motif, and a stall motif in an N-terminal to C-terminal orientation.

[0297] In some embodiments, the leader construct may include a coupling motif, a blocking motif, a capture motif, a stall motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a blocking motif, a capture motif, a stall motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0298] In some embodiments, the leader construct may include a coupling motif, a recognition motif, a capture motif, a stall motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a recognition motif, a capture motif, a stall motif, and a block motif in an N-terminal to C-terminal orientation.

[0299] In some embodiments, the leader construct may include a coupling motif, a recognition motif, a capture motif, a blocking motif, and a stall motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a recognition motif, a capture motif, a blocking motif, and a stall motif in an N-terminus to C-terminus orientation.

[0300] In some embodiments, the leader construct may include a coupling motif, a recognition motif, a stall motif, a block motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a recognition motif, a stall motif, a block motif, and a capture motif in an N-terminal to C-terminal orientation.

[0301] In some embodiments, the leader construct may include a coupling motif, a recognition motif, a stall motif, a capture motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a recognition motif, a stall motif, a capture motif, and a block motif in an N-terminal to C-terminal orientation.

[0302] In some embodiments, the leader construct may include a coupling motif, a recognition motif, a blocking motif, a stall motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a recognition motif, a blocking motif, a stall motif, and a capture motif in an N-terminal to C-terminal orientation.

[0303] In some embodiments, the leader construct may include a coupling motif, a recognition motif, a blocking motif, a capture motif, and a stall motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a recognition motif, a blocking motif, a capture motif, and a stall motif in an N-terminal to C-terminal orientation.

[0304] In some embodiments, the leader construct may include a coupling motif, a capture motif, a block motif, a stall motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a capture motif, a block motif, a stall motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0305] In some embodiments, the leader construct may include a coupling motif, a capture motif, a block motif, a recognition motif, and a stall motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a capture motif, a block motif, a recognition motif, and a stall motif in an N-terminal to C-terminal orientation.

[0306] In some embodiments, the leader construct may include a coupling motif, a capture motif, a recognition motif, a blocking motif, and a stall motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a capture motif, a recognition motif, a blocking motif, and a stall motif in an N-terminal to C-terminal orientation.

[0307] In some embodiments, the leader construct may include a coupling motif, a capture motif, a recognition motif, a stall motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a capture motif, a recognition motif, a stall motif, and a block motif in an N-terminal to C-terminal orientation.

[0308] In some embodiments, the leader construct may include a coupling motif, a capture motif, a stall motif, a block motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a capture motif, a stall motif, a block motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0309] In some embodiments, the leader construct may include a coupling motif, a capture motif, a stall motif, a recognition motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a coupling motif, a capture motif, a stall motif, a recognition motif, and a block motif in an N-terminus to C-terminus orientation.

[0310] In some embodiments, the leader construct may include a stall motif, a block motif, a recognition motif, a capture motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a block motif, a recognition motif, a capture motif, and a coupling motif in an N-terminal to C-terminal orientation.

[0311] In some embodiments, the leader construct may include a stall motif, a block motif, a recognition motif, a coupling motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a block motif, a recognition motif, a coupling motif, and a capture motif in an N-terminal to C-terminal orientation.

[0312] In some embodiments, the leader construct may include a stall motif, a block motif, a capture motif, a recognition motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a block motif, a capture motif, a recognition motif, and a coupling motif in an N-terminus to C-terminus orientation.

[0313] In some embodiments, the leader construct may include a stall motif, a block motif, a capture motif, a coupling motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a block motif, a capture motif, a coupling motif, and a recognition motif in an N-terminus to C-terminus orientation.

[0314] In some embodiments, the leader construct may include a stall motif, a block motif, a coupling motif, a recognition motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a block motif, a coupling motif, a recognition motif, and a capture motif in an N-terminal to C-terminal orientation.

[0315] In some embodiments, the leader construct may include a stall motif, a block motif, a coupling motif, a capture motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a block motif, a coupling motif, a capture motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0316] In some embodiments, the leader construct may include a stall motif, a recognition motif, a block motif, a capture motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a recognition motif, a block motif, a capture motif, and a coupling motif in an N-terminus to C-terminus orientation.

[0317] In some embodiments, the leader construct may include a stall motif, a recognition motif, a block motif, a coupling motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a recognition motif, a block motif, a coupling motif, and a capture motif in an N-terminus to C-terminus orientation.

[0318] In some embodiments, the leader construct may include a stall motif, a recognition motif, a capture motif, a block motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a recognition motif, a capture motif, a block motif, and a coupling motif in an N-terminus to C-terminus orientation.

[0319] In some embodiments, the leader construct may include a stall motif, a recognition motif, a capture motif, a coupling motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a recognition motif, a capture motif, a coupling motif, and a block motif in an N-terminus to C-terminus orientation.

[0320] In some embodiments, the leader construct may include a stall motif, a recognition motif, a coupling motif, a block motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a recognition motif, a coupling motif, a block motif, and a capture motif in an N-terminal to C-terminal orientation.

[0321] In some embodiments, the leader construct may include a stall motif, a recognition motif, a coupling motif, a capture motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a recognition motif, a coupling motif, a capture motif, and a block motif in an N-terminus to C-terminus orientation.

[0322] In some embodiments, the leader construct may include a stall motif, a capture motif, a block motif, a recognition motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a capture motif, a block motif, a recognition motif, and a coupling motif in an N-terminus to C-terminus orientation.

[0323] In some embodiments, the leader construct may include a stall motif, a capture motif, a block motif, a coupling motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a capture motif, a block motif, a coupling motif, and a recognition motif in an N-terminus to C-terminus orientation.

[0324] In some embodiments, the leader construct may include a stall motif, a capture motif, a recognition motif, a block motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a capture motif, a recognition motif, a block motif, and a coupling motif in an N-terminus to C-terminus orientation.

[0325] In some embodiments, the leader construct may include a stall motif, a capture motif, a recognition motif, a coupling motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a capture motif, a recognition motif, a coupling motif, and a block motif in an N-terminus to C-terminus orientation.

[0326] In some embodiments, the leader construct may include a stall motif, a capture motif, a coupling motif, a block motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a capture motif, a coupling motif, a block motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0327] In some embodiments, the leader construct may include a stall motif, a capture motif, a coupling motif, a recognition motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a capture motif, a coupling motif, a recognition motif, and a block motif in an N-terminus to C-terminus orientation.

[0328] In some embodiments, the leader construct may include a stall motif, a coupling motif, a block motif, a recognition motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a coupling motif, a block motif, a recognition motif, and a capture motif in an N-terminus to C-terminus orientation.

[0329] In some embodiments, the leader construct may include a stall motif, a coupling motif, a block motif, a capture motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a coupling motif, a block motif, a capture motif, and a recognition motif in an N-terminus to C-terminus orientation.

[0330] In some embodiments, the leader construct may include a stall motif, a coupling motif, a recognition motif, a capture motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a coupling motif, a recognition motif, a capture motif, and a block motif in an N-terminus to C-terminus orientation.

[0331] In some embodiments, the leader construct may include a stall motif, a coupling motif, a recognition motif, a block motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a coupling motif, a recognition motif, a block motif, and a capture motif in an N-terminus to C-terminus orientation.

[0332] In some embodiments, the leader construct may include a stall motif, a coupling motif, a capture motif, a recognition motif, and a block motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a coupling motif, a capture motif, a recognition motif, and a block motif in an N-terminus to C-terminus orientation.

[0333] In some embodiments, the leader construct may include a stall motif, a coupling motif, a capture motif, a block motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a stall motif, a coupling motif, a capture motif, a block motif, and a recognition motif in an N-terminus to C-terminus orientation.

[0334] In some embodiments, the leader construct may include a block motif, a stall motif, a recognition motif, a capture motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a stall motif, a recognition motif, a capture motif, and a coupling motif in an N-terminal to C-terminal orientation.

[0335] In some embodiments, the leader construct may include a block motif, a stall motif, a recognition motif, a coupling motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a stall motif, a recognition motif, a coupling motif, and a capture motif in an N-terminal to C-terminal orientation.

[0336] In some embodiments, the leader construct may include a block motif, a stall motif, a capture motif, a recognition motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a stall motif, a capture motif, a recognition motif, and a coupling motif in an N-terminal to C-terminal orientation.

[0337] In some embodiments, the leader construct may include a block motif, a stall motif, a capture motif, a coupling motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a stall motif, a capture motif, a coupling motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0338] In some embodiments, the leader construct may include a block motif, a stall motif, a coupling motif, a recognition motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a stall motif, a coupling motif, a recognition motif, and a capture motif in an N-terminal to C-terminal orientation.

[0339] In some embodiments, the leader construct may include a block motif, a stall motif, a coupling motif, a capture motif, and a recognition motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a stall motif, a coupling motif, a capture motif, and a recognition motif in an N-terminal to C-terminal orientation.

[0340] In some embodiments, the leader construct may include a block motif, a recognition motif, a stall motif, a capture motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a recognition motif, a stall motif, a capture motif, and a coupling motif in an N-terminal to C-terminal orientation.

[0341] In some embodiments, the leader construct may include a block motif, a recognition motif, a stall motif, a coupling motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a recognition motif, a stall motif, a coupling motif, and a capture motif in an N-terminal to C-terminal orientation.

[0342] In some embodiments, the leader construct may include a block motif, a recognition motif, a capture motif, a stall motif, and a coupling motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a recognition motif, a capture motif, a stall motif, and a coupling motif in an N-terminal to C-terminal orientation.

[0343] In some embodiments, the leader construct may include a block motif, a recognition motif, a capture motif, a coupling motif, and a stall motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a recognition motif, a capture motif, a coupling motif, and a stall motif in an N-terminal to C-terminal orientation.

[0344] In some embodiments, the leader construct may include a block motif, a recognition motif, a coupling motif, a stall motif, and a capture motif in a 5' to 3' orientation. In some embodiments, the leader construct may include a block motif, a recognition motif, a coupling motif, a stall motif, and a capture motif in an N-terminal to C-terminal orientation.

[0345] In some embodiments, the leader construct may include a block motif, a recognition motif, a coupling motif, a capture motif, and a stall motif in a 5' to 3' orientation. ...

Claims

1. It is a method, (a) (i) To provide a nanopore system comprising (1) a fluid chamber and (2) a membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side and a transform side. (b) Contacting the cis side of the nanopore with a complex containing a non-nucleic acid polymer analyte and a translocase, (c) A method comprising using electroosmotic force from the cis side to the trans side to dislocate the non-nucleic acid polymer analyte to the trans side of a fluid channel, wherein the electroosmotic force from the cis side to the trans side dislocates the translocase of the complex to the cis side inlet of the nanopore channel.

2. The method according to claim 1, further comprising contacting the non-nucleic acid polymer analyte with the translocase to produce the complex before (c).

3. The method according to claim 2, wherein the composite is generated 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 transformer side includes a net flow of ionic current from the cis side to the transformer side.

5. The method according to any one of claims 1 to 4, wherein the electroosmotic force from the cis side to the transformer side is adjusted by pH, type of salt, concentration of salt, osmotic pressure across the membrane, modification of the nanopores, or any combination thereof.

6. The method according to claim 5, wherein the modification of the nanopores includes modification of the charge of the nanopores.

7. The method according to claim 5, wherein the electroosmotic force from the cis side to the transformer side is regulated by the asymmetric salt distribution between the cis side and the transformer side of the fluid chamber.

8. The method according to any one of claims 1 to 7, wherein the composite is formed in the solution on the cis side of the fluid chamber.

9. The method according to any one of claims 1 to 8, wherein the composite is formed before the composite is brought into contact with the cis side of the nanopore.

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

11. The method according to any one of claims 1 to 10, wherein the translocase comprises an unfoldase driven by nucleotide triphosphates (NTPs).

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

13. The aforementioned AAA+ enzymes include casein-degrading mitochondrial matrix peptidase chaperone subunit X (ClpX), casein-degrading mitochondrial matrix peptidase chaperone subunit A (ClpA), proteasome-activating nucleotidase (PAN), LON, and thermoplasma. The method according to claim 12, comprising a protein selected from the group consisting of acidophilus balocin-containing protein-like ATPase (VAT), AMA, 854, membrane-bound AAA (MBA), small-molecular-weight archaeal ubiquitin-like modified protein (SAMP), casein-degrading mitochondrial matrix peptidase chaperone subunit C (ClpC), casein-degrading mitochondrial matrix peptidase chaperone subunit E (ClpE), HsIU, casein-degrading mitochondrial matrix peptidase chaperone subunit Y (ClpY), LonA, LonB, FtsH, Mpa, actinomycete Cdc48-like protein (Cpa), Msp1, SecA, and functional homologs, orthologs, or paralogs thereof.

14. The method according to any one of claims 1 to 13, wherein the system further includes a pair of electrodes.

15. The method according to claim 14, wherein the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force.

16. The method according to claim 15, wherein the applied voltage is a negative voltage relative to the transformer side.

17. The method according to claim 15, wherein the applied voltage is a positive voltage relative to the transformer side.

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

19. The method according to any one of claims 15 to 18, wherein the magnitude of the applied voltage exceeds 20 mV.

20. The method according to any one of claims 15 to 19, wherein the absolute relative net electroosmotic current with respect to the applied voltage is greater than about 0.10 picoamperes / millivolts (pA / mV).

21. The method according to any one of claims 1 to 20, wherein the non-nucleic acid polymer analyte comprises a leader construct at its N-terminus or C-terminus.

22. The method according to claim 21, wherein the leader construct is configured to couple one or more translocases to the non-nucleic acid polymer analyte.

23. The method according to claim 21 or 22, wherein the leader structure is configured to stall one or more translocases.

24. The method according to any one of claims 21 to 23, wherein the reader construct includes a recognition motif.

25. The method according to any one of claims 21 to 24, wherein the leader structure further comprises a capture motif, a stall motif, a block motif, or a combination thereof.

26. It is a system, Fluid chamber and The fluid chamber includes a membrane with nanopores that separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, A system comprising the first solution and the second solution configured to generate electroosmotic force, wherein the electroosmotic force is configured to couple the translocase of the complex to the cis-side inlet of the nanopore channel, and the complex comprises a non-nucleic acid polymer analyte and the translocase.

27. The system according to claim 26, further comprising a translocase.

28. The system according to claim 27, wherein the translocase includes an unfoldase driven by ATP.

29. The system according to claim 27, wherein the translocase includes an unfoldase driven by NTP.

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

31. The system according to 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, Msp1, SecA, and their functional homologs, orthologs, or paralogs.

32. The system according to any one of claims 26 to 31, wherein the translocase is configured to displace the non-nucleic acid polymer analyte through the nanopores in a continuous sequence.

33. The system according to any one of claims 26 to 32, wherein the first solution contains a solute at a first concentration, and the second solution contains a solute at a second concentration.

34. The system according to claim 33, wherein the solute comprises ions or osmolite.

35. The system according to claim 33, wherein the difference between the first concentration of the solute and the second concentration of the solute is configured to generate the electroosmotic force.

36. The system according to any one of claims 26 to 35, wherein the electroosmotic force includes a net ion current flow from the cis side to the transformer side.

37. The system according to any one of claims 26 to 36, wherein the electroosmotic force is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the system, modification of the nanopores, or any combination thereof.

38. The system according to any one of claims 26 to 37, wherein the electroosmotic force is regulated by modifying the charge of the nanopores.

39. The system according to any one of claims 26 to 38, wherein the electroosmotic force is regulated by the asymmetric salt distribution between the cis side and the transform side of the membrane.

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

41. The system according to claim 40, wherein the first electrode of the pair of electrodes is positioned on the cis side, and the second electrode of the pair of electrodes is positioned on the transformer side of the film.

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

43. The system according to claim 42, wherein the signal is related to the characteristics of the non-nucleic acid polymer analyte.

44. The system according to any one of claims 40 to 43, wherein the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force.

45. The system according to claim 44, wherein the applied voltage is a negative voltage relative to the transformer side.

46. The system according to claim 44, wherein the applied voltage is a positive voltage relative to the transformer side.

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

48. The system according to any one of claims 44 to 47, wherein the magnitude of the applied voltage exceeds 20 mV.

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

50. The system according to any one of claims 44 to 49, wherein the signal includes an ion current or a change thereof.

51. It is a method, (a) (i) A nanopore system comprising (1) a fluid chamber and (2) a membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side and a transform side. (ii) Non-nucleic acid polymer analytes, wherein the non-nucleic acid polymer analytes are coupled to a leader construct comprising a stall motif, a block motif, a coupling motif, or a combination thereof, and (iii) To provide translocase, (b) A method comprising dislocating the non-nucleic acid polymer analyte from the cis side to the trans side of the fluid chamber.

52. The method according to claim 51, wherein the leader construct comprises nucleic acid.

53. The method according to claim 51 or 52, wherein the leader construct comprises a peptide.

54. The method according to any one of claims 51 to 53, wherein the leader construct comprises nucleic acids and peptides.

55. The method according to any one of claims 51 to 54, wherein the stall motif is configured to disrupt the interaction between the translocase and the non-nucleic acid polymer analyte.

56. The method according to any one of claims 51 to 55, wherein the stall motif comprises an amino acid sequence.

57. The method according to claim 56, wherein the amino acid sequence includes n repeats of (glycine)n, (serine-glycine)n, (glycine-serine)n, (alanine)n, (valine)n, (alanine-serine)n, (serine-alanine)n, (valine-serine)n, or (serine-valine)n.

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

59. The method according to any one of claims 51 to 58, wherein the stall motif includes a non-amino acid chemistry region.

60. The method according to claim 59, wherein the non-amino acid chemical region includes polyethylene glycol.

61. The method according to any one of claims 51 to 60, wherein the block motif is configured to prevent the translocase from rearranging the non-nucleic acid polymer analyte beyond the block motif.

62. The method according to claim 61, wherein the block motif is configured to prevent the translocase from rearranging the non-nucleic acid polymer analyte beyond the leader construct.

63. The method according to claim 61, wherein the block motif is configured to prevent the translocase from displacing the non-nucleic acid polymer analyte through the nanopores.

64. The method according to any one of claims 51 to 63, wherein the block motif includes a three-dimensional obstruction.

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

66. The method according to claim 65, wherein the one or more bulky amino acids include histidine, phenylalanine, tyrosine, or tryptophan.

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

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

69. The method according to claim 64, wherein the steric hindrance comprises at least a portion of the unfolding resistance protein.

70. The method according to claim 69, wherein the expansion resistance protein comprises a maltose-binding protein, titin, dihydrofolate reductase, burnase, or a combination thereof.

71. The method according to claim 69 or 70, wherein the expansion-resistant protein comprises a disulfide bond.

72. The method according to claim 64, wherein the steric hindrance includes a large binding molecule.

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

74. The method according to any one of claims 51 to 73, wherein the coupling motif is configured to couple the leader construct to the non-nucleic acid polymer analyte.

75. The method according to claim 74, wherein the non-nucleic acid polymer analyte includes a peptide.

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

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

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

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

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

81. The method according to any one of claims 51 to 80, wherein the coupling motif includes an enzyme coupling region.

82. The method according to claim 81, wherein the enzyme coupling region attaches the coupling motif to the enzyme.

83. The method according to claim 82, wherein the enzyme comprises peptiligase, omniligase, or saltase.

84. The method according to any one of claims 51 to 83, wherein the coupling motif of the leader construct is coupled to the non-nucleic acid polymer analyte via bonding.

85. The method according to any one of claims 51 to 84, wherein the coupling motif of the leader construct is coupled to the non-nucleic acid polymer analyte via a linker.

86. The aforementioned leader structure, (i) Recognition motif, (ii) The method according to any one of claims 51 to 85, further comprising at least one of the capture motifs.

87. The method according to claim 86, wherein the capture motif includes a polycation tag.

88. The method according to claim 87, wherein the polycation tag comprises n repetitions of (serine-glycine-arginine)n, (serine-arginine)n, (arginine)n.

89. The method according to claim 86, wherein the capture motif includes a polyanion tag.

90. The method according to claim 89, wherein the polyanion tag comprises n repetitions of (serine-glycine-aspartic acid)n, (serine-aspartic acid)n, (aspartic acid)n.

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

92. The method according to claim 86, wherein the sequence of recognition motifs includes one or more of sequence numbers 201 to 206.

93. The method according to any one of claims 51 to 92, wherein the leader construct is attached to the C-terminus or N-terminus of the non-nucleic acid polymer analyte.

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

95. The method according to claim 94, wherein the leader construct is coupled to the N-terminus of the polypeptide.

96. The method according to claim 94, wherein the leader construct is coupled to the C-terminus of the polypeptide.

97. The method according to any one of claims 51 to 96, wherein the non-nucleic acid polymer analyte comprises another leader construct.

98. The method according to claim 97, wherein the leader construct and the other leader construct are configured to displace the non-nucleic acid polymer analyte through the nanopores in both the C-terminus-to-N-terminus direction and the N-terminus-to-C-terminus direction.

99. The method according to any one of claims 51 to 98, wherein the non-nucleic acid polymer analyte is rearranged using electroosmosis.

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

101. The method according to claim 100, wherein the electroosmotic force extrudes the non-nucleic acid polymer analyte through the nanopores against the electrophoretic force.

102. The method according to claim 99, wherein the electroosmotic force includes a net ion current flow from the cis side to the transformer side.

103. The method according to any one of claims 99 to 102, wherein the electroosmotic force is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the nanopore system, modification of the nanopores, or any combination thereof.

104. The method according to any one of claims 99 to 103, wherein the electroosmotic force is adjusted by modifying the charge of the nanopores.

105. The method according to any one of claims 99 to 104, wherein the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the transform side of the membrane.

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

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

108. The method according to claim 107, wherein the applied voltage is a negative voltage relative to the transformer side.

109. The method according to claim 107, wherein the applied voltage is a positive voltage to the transformer side.

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

111. The method according to any one of claims 107 to 110, wherein the magnitude of the applied voltage exceeds 20 mV.

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

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

114. The method according to claim 113, wherein the translocase comprises an unfoldase driven by ATP.

115. The method according to claim 113, wherein the translocase comprises an unfoldase driven by NTP.

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

117. The method according to 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 their functional homologs, orthologs, or paralogs.

118. It is a system, Fluid chamber and A membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, and the first solution and the second solution are configured to rearrange a non-nucleic acid polymer analyte. Translocase and, A system comprising a leader construct comprising at least one of a stall motif, a block motif, or a coupling motif, or a combination thereof, wherein the leader construct is configured to couple to the non-nucleic acid polymer analyte.

119. It is a system, Fluid chamber and A membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, and the first solution and the second solution are configured to rearrange a non-nucleic acid polymer analyte. A system comprising a fluid chamber and a controller operably coupled to the nanopores, the controller configured to detect one or more signals related to at least one feature of the leader construct and one or more signals related to at least one feature of the non-nucleoside polymer analyte during or after the rearrangement of the non-nucleoside polymer analyte coupled to the leader construct through the nanopores using a translocase, wherein the leader construct comprises at least one of a stall motif, a block motif, or a coupling motif, or a combination thereof.

120. The system according to claim 119, wherein the controller is further configured to use a pair of electrodes to detect one or more signals relating to at least one feature of the reader construct and one or more signals relating to at least one feature of the non-nucleoside polymer analyte.

121. The system according to claim 119 or 120, wherein the controller is further configured to separate the one or more signals relating to the at least one feature of the reader construct from the one or more signals relating to the at least one feature of the non-nucleic acid polymer analyte.

122. The system according to any one of claims 118 to 121, wherein the leader construct comprises one or more nucleic acid molecules.

123. The system according to any one of claims 118 to 122, wherein the leader construct comprises one or more peptides.

124. The system according to any one of claims 118 to 123, wherein the leader construct comprises one or more nucleic acid molecules and one or more peptides.

125. The system according to any one of claims 118 to 124, wherein the stall motif is configured to disrupt the interaction between the translocase and the non-nucleic acid polymer analyte.

126. The system according to any one of claims 118 to 125, wherein the stall motif comprises an amino acid sequence.

127. The system according to claim 126, wherein the amino acid sequence includes n repeats of (glycine)n, (serine-glycine)n, (glycine-serine)n, (alanine)n, (valine)n, (alanine-serine)n, (serine-alanine)n, (valine-serine)n, or (serine-valine)n.

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

129. The system according to any one of claims 118 to 128, wherein the stall motif includes a non-amino acid chemistry region.

130. The system according to claim 129, wherein the non-amino acid chemical region includes polyethylene glycol.

131. The system according to any one of claims 118 to 130, wherein the block motif is configured to prevent the translocase from rearranging the non-nucleic acid polymer analyte beyond the block motif.

132. The system according to claim 131, wherein the block motif is configured to prevent the translocase from displacing the non-nucleic acid polymer analyte beyond the leader construct.

133. The system according to claim 131, wherein the block motif is configured to prevent the translocase from displacing the non-nucleic acid polymer analyte through the nanopores.

134. The system according to any one of claims 118 to 133, wherein the block motif includes a three-dimensional obstruction.

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

136. The system according to claim 135, wherein the one or more bulky amino acids include histidine, phenylalanine, tyrosine, or tryptophan.

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

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

139. The system according to any one of claims 134 to 138, wherein the steric hindrance comprises at least a portion of the expansion resistance protein.

140. The system according to claim 139, wherein the expansion resistance protein comprises a maltose-binding protein, titin, dihydrofolate reductase, burnase, or a combination thereof.

141. The system according to claim 139, wherein the expansion resistance protein comprises a disulfide bond.

142. The system according to any one of claims 134 to 141, wherein the steric obstruction includes a large binding molecule.

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

144. The system according to any one of claims 118 to 143, wherein the coupling motif is configured to couple the leader construct to the non-nucleic acid polymer analyte.

145. The system according to claim 144, wherein the non-nucleic acid polymer analyte includes a peptide.

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

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

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

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

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

151. The system according to any one of claims 118 to 150, wherein the coupling motif includes an enzyme coupling region.

152. The system according to claim 151, wherein the enzyme coupling region attaches the coupling motif to the enzyme.

153. The system according to claim 152, wherein the enzyme comprises peptiligase, omniligase, or saltase.

154. The system according to any one of claims 118 to 153, wherein the coupling motif of the leader construct is coupled to the non-nucleic acid polymer analyte via bonding.

155. The system according to any one of claims 118 to 154, wherein the coupling motif of the leader construct is coupled to the non-nucleic acid polymer analyte via a linker.

156. The aforementioned leader structure, (i) Recognition motif, (ii) The system according to any one of claims 118 to 155, further comprising at least one of the capture motifs.

157. The system according to claim 156, wherein the capture motif includes a polycation tag.

158. The system according to claim 157, wherein the polycation tag comprises n repeats of (serine-glycine-arginine)n, (serine-arginine)n, (arginine)n, and the capture motif comprises a polyanion tag.

159. The system according to claim 156, wherein the capture motif includes a polyanion tag.

160. The system according to claim 159, wherein the polyanion tag comprises n repetitions of (serine-glycine-aspartic acid)n, (serine-aspartic acid)n, (aspartic acid)n.

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

162. The system according to any one of claims 156 to 161, wherein the sequence of recognition motifs includes one or more of sequence numbers 201 to 206.

163. The system according to any one of claims 118 to 161, wherein the leader construct is attached to the C-terminus or N-terminus of the non-nucleic acid polymer analyte.

164. The system according to claim 163, wherein the non-nucleic acid polymer analyte comprises a polypeptide, and the leader construct is attached to the N-terminus of the polypeptide.

165. The system according to claim 163, wherein the non-nucleic acid polymer analyte comprises a polypeptide, and the leader construct is attached to the C-terminus of the polypeptide.

166. The system according to any one of claims 118 to 165, wherein the non-nucleic acid polymer analyte comprises a second leader construct.

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

168. The system according to claim 167, wherein the first solution contains a solute at a first concentration, and the second solution contains a solute at a second concentration.

169. The system according to claim 168, wherein the solute comprises ions or osmolite.

170. The system according to claim 169, wherein the difference between the first concentration of the solute and the second concentration of the solute is configured to generate the electroosmotic force.

171. The system according to any one of claims 167 to 170, wherein the electroosmotic force includes the flow of net ion current from the cis side of the membrane to the transformer side.

172. The system according to any one of claims 167 to 171, wherein the electroosmotic force is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the system, modification of the nanopores, or any combination thereof.

173. The system according to any one of claims 167 to 172, wherein the electroosmotic force is regulated by modifying the charge of the nanopores.

174. The system according to any one of claims 167 to 173, wherein the electroosmotic force is regulated by the asymmetric salt distribution between the cis side and the transform side of the membrane.

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

176. The system according to claim 175, wherein the first electrode is located on the cis side of the fluid chamber and the second electrode is located on the transformer side of the fluid chamber.

177. The system according to claim 175 or 176, wherein the pair of electrodes are configured to provide a voltage applied to generate an electrophoretic force across the membrane in the opposite direction to the electroosmotic force.

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

179. The system according to claim 177 or 178, wherein the applied voltage is a negative voltage relative to the transformer side.

180. The applied voltage is a positive voltage to the transformer side and the transformer side. (ii) Non-nucleic acid polymer analytes, and (b) The non-nucleic acid polymer analyte is displaced from the cis side to the trans side of the fluid chamber, The system according to claim 177 or 178, wherein the nanopores include an adapter, and at least a portion of the adapter is located within the channels of the nanopores.

181. The method according to claim 180, wherein the adapter is configured to modify the geometric shape of the channel of the nanopore.

182. The method according to claim 181, wherein the adapter is configured to narrow the channel of the nanopore.

183. The method according to any one of claims 180 to 182, wherein the adapter is configured to modify the charge of the channel in the nanopore.

184. The method according to claim 183, wherein the adapter is configured to modify the channels of the nanopores to have a positive net charge.

185. The method according to claim 183, wherein the adapter is configured to modify the channels of the nanopores to have a negative net charge.

186. The method according to any one of claims 180 to 185, wherein the adapter includes a protein-based adapter or a chemical adapter.

187. The method according to claim 186, wherein the protein adapter comprises a CsgF subunit, a CsgF subunit cleavage, or a CsgF subunit homolog.

188. The method according to claim 186, wherein the chemical adapter comprises cyclodextrin, cucurbituryl, crown ether, calixsalen, porphyrin, cyclosporine, cyclom, or cyclom.

189. The method according to any one of claims 180 to 188, wherein the adapter is coupled to the channel of the nanopore.

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

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

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

193. The method according to any one of claims 180 to 192, wherein the nanopore system includes electroosmotic force from the cis side to the transformer side arising from a net ionic current flow from the cis side to the transformer side.

194. The method according to claim 193, further comprising providing an electrophoretic force acting in the opposite direction to the electroosmotic force from the cis side to the transformer side.

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

196. The method according to any one of claims 180 to 195, wherein the non-nucleic acid polymer analyte is displaced through the nanopores using electroosmosis.

197. The method according to claim 196, further comprising providing an electrophoretic force acting in the opposite direction to the electroosmotic force.

198. The method according to claim 197, wherein the electroosmotic force extrudes the non-nucleic acid polymer analyte through the nanopores against the electrophoretic force.

199. The method according to any one of claims 196 to 198, wherein the electroosmotic force includes a net ion current flow from the cis side to the transformer side.

200. The method according to any one of claims 196 to 199, wherein the electroosmotic force is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the nanopore system, modification of the nanopores, or any combination thereof.

201. The method according to any one of claims 196 to 200, wherein the electroosmotic force is adjusted by modifying the charge of the nanopores.

202. The method according to any one of claims 196 to 201, wherein the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the transform side of the membrane.

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

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

205. The method according to claim 204, wherein the applied voltage is a negative voltage relative to the transformer side.

206. The method according to claim 204, wherein the applied voltage is a positive voltage relative to the transformer side.

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

208. The method according to any one of claims 204 to 207, wherein the magnitude of the applied voltage exceeds 20 mV.

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

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

211. The method according to claim 210, wherein the translocase comprises an unfoldase driven by ATP.

212. The method according to claim 210, wherein the translocase includes an unfoldase driven by NTP.

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

214. The method according to 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 their functional homologs, orthologs, or paralogs.

215. It is a system, (a) Fluid chamber and (b) A membrane comprising nanopores, wherein the membrane separates the fluid chamber into a cis side containing a first solution and a trans side containing a second solution, and the first solution and the second solution are configured to rearrange a non-nucleic acid polymer analyte, A system in which the nanopores include an adapter within the channel of the nanopores.

216. The system according to claim 215, wherein the adapter includes a protein-based adapter or a chemical adapter.

217. The system according to claim 216, wherein the protein adapter comprises a CsgF subunit, a CsgF subunit cleavage, or a CsgF subunit homolog.

218. The system according to claim 216, wherein the chemical adapter comprises cyclodextrin, cucurbituryl, crown ether, calixsalen, porphyrin, cyclosporine, cyclom, or cyclom.

219. The system according to any one of claims 215 to 218, wherein the adapter is coupled to the channel of the nanopore.

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

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

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

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

224. The system according to claim 223, wherein the solute comprises ions or osmolite.

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

226. The system according to any one of claims 215 to 225, wherein the first solution and the second solution are configured to generate electroosmotic force across the membrane.

227. The system according to claim 226, wherein the electroosmotic force arises from the flow of net ionic current from the cis side of the membrane to the transformer side.

228. The system according to claim 226 or 227, further comprising an electrophoretic force acting in the opposite direction to the electroosmotic force, wherein the electroosmotic force is strong enough to push the non-nucleic acid polymer analyte through the nanopores against the electrophoretic force.

229. The system according to any one of claims 226 to 228, wherein the electroosmotic force is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the system, modification of the nanopores, or any combination thereof.

230. The system according to any one of claims 226 to 229, wherein the electroosmotic force is regulated by modifying the charge of the nanopores.

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

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

233. The system according to claim 232, wherein the first electrode of the pair of electrodes is positioned on the cis side, and the second electrode of the pair of electrodes is positioned on the transformer side of the film.

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

235. The system according to claim 234, wherein the signal is related to the characteristics of the non-nucleic acid polymer analyte.

236. The system according to any one of claims 232 to 235, wherein the pair of electrodes are configured to provide a voltage applied to generate electrophoretic force.

237. The system according to claim 236, wherein the applied voltage is a negative voltage relative to the transformer side.

238. The system according to claim 236, wherein the applied voltage is a positive voltage relative to the transformer side.

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

240. The system according to any one of claims 236 to 239, wherein the magnitude of the applied voltage exceeds 20 mV.

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

242. It is a method, (a) (i) To provide a nanopore system comprising (1) a fluid chamber and (2) a membrane containing nanopores, wherein the membrane separates the fluid chamber into a cis side and a transform side. (b) Adding the combined solution to the cis side of the fluid chamber, wherein the combined solution includes a non-nucleic acid polymer analyte and a pre-loading solution. (c) A method comprising dislocating the non-nucleic acid polymer analyte from the cis side to the trans side of the fluid chamber.

243. The method of claim 242, further comprising combining a sample containing a non-nucleic acid polymer analyte with a pre-loading solution before (b).

244. The method according to claim 242 or 243, wherein the preloading solution comprises translocase.

245. The method according to claim 244, wherein the non-nucleic acid polymer analyte is rearranged using the translocase.

246. The method according to claim 245, wherein the translocase comprises an unfoldase driven by ATP.

247. The method according to claim 245, wherein the translocase includes an unfoldase driven by NTP.

248. The method according to claim 247, wherein the translocase comprises an AAA+ enzyme.

249. The method according to 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 their functional homologs, orthologs, or paralogs.

250. The method according to any one of claims 245 to 249, wherein a non-nucleic acid polymer analyte-translocase complex is formed by combining the sample and the preloading solution.

251. The method according to any one of claims 245 to 250, wherein a non-nucleic acid polymer analyte-leader construct composite is formed by combining the sample and the preloading solution.

252. The method according to any one of claims 242 to 251, wherein the preloading solution comprises a leader construct.

253. The method according to any one of claims 242 to 252, wherein the preloading solution contains a chemical substance that enhances the binding of the non-nucleic acid polymer analyte to the components of the preloading solution.

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

255. The method according to any one of claims 242 to 253, wherein the preloading solution comprises one or more cofactors.

256. The one or more cofactors mentioned above are NTP, M ２＋ The method according to claim 255, comprising NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, Ficol, iron manganese, cobalt, copper, penicillamine, trientine, calcium sodium edetate, or ethylenediaminetetraacetic acid.

257. The method according to any one of claims 242 to 256, wherein the non-nucleic acid polymer analyte is rearranged using electroosmosis.

258. The method according to claim 257, wherein the dislocation provides an electrophoretic force acting in the opposite direction to the electroosmotic force.

259. The method according to claim 258, wherein the electroosmotic force extrudes the non-nucleic acid polymer analyte through the nanopores against the electrophoretic force.

260. The method according to any one of claims 257 to 259, wherein the electroosmotic force includes a net ion current flow from the cis side to the transformer side.

261. The method according to any one of claims 257 to 260, wherein the electroosmotic force is controlled by pH, type of salt, concentration of salt, osmotic pressure across the membrane of the nanopore system, modification of the nanopores, or any combination thereof.

262. The method according to any one of claims 257 to 261, wherein the electroosmotic force is adjusted by modifying the charge of the nanopores.

263. The method according to any one of claims 257 to 262, wherein the electroosmotic force is regulated by an asymmetric salt distribution between the cis side and the transform side of the membrane.

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

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

266. The method according to claim 265, wherein the applied voltage is a negative voltage relative to the transformer side.

267. The method according to claim 265, wherein the applied voltage is a positive voltage relative to the transformer side.

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

269. The method according to any one of claims 265 to 268, wherein the magnitude of the applied voltage exceeds 20 mV.

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

271. It is a system, Fluid chamber and A membrane containing nanopores, wherein the membrane separates the fluid chamber into (i) a cis side containing a first solution and (ii) a trans side containing a second solution, and the first and second solutions are configured to displace non-nucleic acid polymer analytes across the nanopores, A system comprising a preloading solution configured to interact with the aforementioned non-nucleic acid polymer analyte.

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

273. The system according to claim 272, wherein the solute comprises ions or osmolite.

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

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

276. The system according to claim 275, wherein the electroosmotic force arises from the flow of net ionic current from the cis side of the membrane to the transformer side.

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

278. The system according to any one of claims 275 to 277, wherein the electroosmotic force is regulated by modifying the charge of the nanopores.

279. The system according to any one of claims 275 to 278, wherein the electroosmotic force is regulated by the asymmetric salt distribution between the cis side and the transform side of the membrane.

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

281. The system according to claim 280, wherein the applied voltage is a negative voltage relative to the transformer side.

282. The system according to claim 280, wherein the applied voltage is a positive voltage relative to the transformer.

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

284. The system according to any one of claims 280 to 283, wherein the magnitude of the applied voltage exceeds 20,300 mV.

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

286. The system according to any one of claims 271 to 285, wherein the preloading solution comprises one or more cofactors.

287. The one or more cofactors mentioned above are NTP, M ２＋ The system according to claim 286, comprising NblA / B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, Ficol, iron manganese, cobalt, copper, penicillamine, trientine, calcium sodium edetate, or ethylenediaminetetraacetic acid.

288. The system according to any one of claims 271 to 287, wherein the preloading solution comprises a translocase.

289. The system according to claim 288, wherein the translocase includes an unfoldase driven by ATP.

290. The system according to claim 288, wherein the translocase includes an unfoldase driven by NTP.

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

292. The system according to 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, Msp1, and SecA.

293. The system according to any one of claims 271 to 292, wherein the preloading solution comprises a leader construct.

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

295. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, or 242 to 270, wherein the nanopores have an ion selectivity P(+) / P(-) greater than 2.

0.

296. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295, wherein the nanopores have an ion selectivity P(+) / P(-) of less than 0.

50.

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

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

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

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

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

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

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

304. The method according to claim 303, wherein the at least 30 peptide units include positively charged residues.

305. The method according to claim 303, wherein the at least 30 peptide units include negatively charged residues.

306. The method according to claim 303, wherein the at least 30 peptide units include positively charged residues and negatively charged residues.

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

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

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

310. The method according to claim 309, wherein the measurement includes measuring a signal of (i) the open channel of the nanopore, (ii) the capture of the non-nucleoside polymer analyte by the nanopore, or (iii) the passage of the non-nucleoside polymer analyte through the nanopore.

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

312. The method according to claim 309, wherein the signal includes an ion current, a change in ion current, or a derivative thereof.

313. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 312, wherein the nanopores include biological nanopores.

314. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 313, wherein the nanopores include internal pore constriction of about 0.5 nm to about 2 nanometers (nm).

315. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 314, wherein the nanopores include an alpha-helical oligomer pore structure.

316. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 315, wherein the nanopores include a beta-barrel oligomer pore structure.

317. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 316, wherein the nanopores include recombinant nanopores.

318. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 317, wherein the nanopores contain proteins of erolysine (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, modified variants thereof, or ion-selective variants thereof.

319. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 318, wherein the nanopores include biological nanopores.

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

321. The method according to claim 320, wherein the biological nanopore restricts the passage of one or more ions through the channels of the nanopore by altering the charge of the channels of the nanopore.

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

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

324. The method according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 323, wherein the nanopores are mutant CytK nanopores.

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

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

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

328. The mutant CytK nanopores (i) K128D and K155D, (ii) K128D, K155D, and T116D, (iii) T147D or S151D, (iv) K128D, K155D, and S120D, (v) Q122D, T147D, or S155D, and (vi) The method according to claim 324, comprising one of the amino acid substitution combinations of K128D, K155D, Q145D, and S151D.

329. The mutant CytK nanopores (i) S120D, G122D, or K155D, (ii) S120D combined with K128F / K128D, (iii) Q122D or S151D, (iv) K128D or K128F, (v) S120D, K115D, and Q122D, (vi) K128F, S120D, and G122D, and (vii) The method according to claim 324, comprising one or more combinations of amino acid substitutions of K128F, S120D, G122D, and K155D.

330. The system according to any one of claims 26 to 50, 118 to 180, 219 to 244, or 273 to 296, wherein the nanopores have an ion selectivity P(+) / P(-) greater than 2.

0.

331. The system according to any one of claims 26 to 50, 118 to 180, 215 to 241, 271 to 294, or 330, wherein the nanopores have an ion selectivity P(+) / P(-) of less than 0.

50.

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

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

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

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

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

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

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

339. The system according to claim 337, wherein the at least 30 peptide units include positively charged residues.

340. The system according to claim 337, wherein the at least 30 peptide units include negatively charged residues.

341. The system according to claim 337, wherein the at least 30 peptide units include positively charged residues and negatively charged residues.

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

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

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

345. The system according to claim 344, wherein the measurement includes measuring a signal about (i) the open channels of the nanopores, (ii) the capture of the non-nucleoside polymer analyte by the nanopores, or (iii) the passage of the non-nucleoside polymer analyte through the nanopores.

346. The system according to claim 345, wherein the measurement includes detecting the difference between states (i), (ii), and (iii).

347. The system according to claim 344, wherein the signal includes an ion current, a change in ion current, or a derivative thereof.

348. The system according to any one of claims 26-50, 118-180, 215-241, 271-294, or 330-347, wherein the nanopores include biological nanopores.

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

350. The system according to any one of claims 26-50, 118-180, 215-241, 271-294, or 330-349, wherein the nanopores include an alpha-helical oligomer pore structure.

351. The system according to any one of claims 26-50, 118-180, 215-241, 271-294, or 330-350, wherein the nanopores include a beta-barrel oligomer pore structure.

352. The system according to any one of claims 26 to 50, 118 to 180, 215 to 241, 271 to 294, or 330 to 351, wherein the nanopores include recombinant nanopores.

353. The system according to any one of claims 26-50, 118-180, 245-241, 271-294, or 330-352, wherein the nanopores contain proteins of erolysine (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, OmpF, OmpG, FhuA, phage-derived portal proteins, modified variants thereof, or ion-selective variants thereof.

354. The system according to any one of claims 26-50, 118-180, 215-241, 271-294, or 330-353, wherein the nanopores include biological nanopores.

355. The system according to claim 354, wherein the biological nanopores are modified to restrict the passage of one or more ions through the channels of the nanopores.

356. The system according to claim 355, wherein the biological nanopore restricts the passage of one or more ions through the channels of the nanopore by altering the charge of the channels of the nanopore.

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

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

359. The system according to any one of claims 26-50, 118-180, 215-241, 271-294, or 330-358, wherein the nanopores are mutant CytK nanopores.

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

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

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

363. The mutant CytK nanopores (v) K128D and K155D, (vi) K128D, K155D, and T116D, (vii) T147D or S151D, (viiii) K128D, K155D, and S120D, (v) Q122D, T147D, or S155D, and (vi) The system according to claim 359, comprising one of the amino acid substitution combinations of K128D, K155D, Q145D, and S151D.

364. The mutant CytK nanopores (i) S120D, G122D, or K155D, (ii) S120D combined with K128F / K128D, (iii) Q122D or S151D, (vi) K128D or K128F, (vii) S120D, K115D, and Q122D, (vi) K128F, S120D, and G122D, and (vii) The system according to claim 359, comprising one or more combinations of amino acid substitutions of K128F, S120D, G122D, and K155D.

365. A device including 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 according to any one of claims 1 to 25, 51 to 117, 180 to 214, 242 to 270, or 295 to 329 for characterizing at least one structural feature of the non-nucleic acid polymer analyte.

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

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

369. Use of the system according to 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 non-nucleic acid polymer analytes at the single-molecule level.

370. A method for dislocating a target protein through nanopores, wherein the nanopores are contained within a membrane that separates the fluid chamber of the nanopore system into cis and trans sides. (a) Enables protein translocases in solution to selectively capture the target proteins that are to be rearranged in the presence of NTPs and to form complexes with them. (b) The translocase-target protein complex is brought into contact with the cis side of the nanopore, enabling the transposition of the target protein to the trans side, A method wherein the nanopore system has a cis-to-trans electroosmotic force (EOF) resulting from a net cis-to-trans flow of ionic current, and as a result, the target protein is trapped in the nanopore using the protein translocase that controls the rearrangement located at the top of the nanopore.

371. The nanopore system has a net ion current flow from cis to transformer (I) that exceeds the total ion current flow of greater than 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, most preferably greater than 0.35 or less than -0.

35. ｒｅｌ The method according to claim 370, comprising having an EOF from cis to transform resulting from ).

372. The method according to claim 370 or 371, wherein the cis-to-trans EOF is regulated by modifying the charge of the nanopores (e.g., genetically engineered) or any combination thereof, by adjusting the pH, type and / or concentration of salts and / or osmotic pressure across the membrane of the nanopore system, preferably by modifying the nanopores and / or by an asymmetric salt distribution between the cis and trans sides of the chamber.

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

374. The method according to any one of claims 370 to 373, wherein the translocase-target protein complex is formed in solution during a separate step prior to adding the complex to the cis side of the fluid chamber and bringing it into contact with the nanopores.

375. The method according to any one of claims 370 to 374, wherein the target protein comprises a leader construct at its N-terminus and / or C-terminus, enabling preloading and optionally stalling one or more protein translocases.

376. The method according to claim 375, wherein the leader construct comprises (i) a recognition motif for the protein translocase, and more preferably further comprises one or more elements of (ii) a capture motif, (iii) a stall motif, and (iv) a block motif.

377. A nanopore system for dislocating target proteins through nanopores, (a) A membrane having nanopores therein, wherein the membrane separates the chamber into a cis side and a trans side, the target protein is added to the cis side and translocated to the trans side through the nanopores, (b) A target protein that is captured by a protein translocase on the cis side of the chamber, which binds to the target protein and can displace the target protein through the nanopores in a continuous sequence, (c) A device for providing a voltage difference between the cis side and the transformer side of the film, The nanopore system has an electroosmotic flow (EOF) from cis to trans resulting from a net ionic current flow from cis to trans, and as a result, the target protein is trapped in the nanopores by the translocase controlling the rearrangement located at the top of the nanopores, preferably the nanopore system has an EOF from cis to trans resulting from a net ionic current flow from cis to trans exceeding a total ionic current flow of 0.2 or less than -0.2, preferably greater than 0.3 or less than -0.3, most preferably greater than 0.35 or less than -0.

35.

378. The nanopore system according to claim 377, further comprising a method for measuring a signal based on an ionic current flowing through the nanopores during a dislocation period, wherein the measuring method detects a change in the signal that reflects the characteristics of the protein as it is dislocated.

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

33. （＋） / P （－） A method or nanopore system according to any one of claims 370 to 378, comprising:

380. The method or nanopore system according to any one of claims 370 to 379, wherein the nanopores are biological nanopores, preferably biological nanopores having internal pore constriction in the range of 0.5 to 2 nm, and more preferably the nanopores are alpha-helical or beta-barrel oligomer pore-forming toxins or porins.

381. The method or nanopore system according to claim 380, wherein the nanopores are selected from the group consisting of erolysine (Aer), cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, fragaseatoxin C (FraC), lysenin, phage-derived portal proteins, and modified variants thereof, and preferably the nanopores are modified to have a net charge in the region facing the lumen greater than 21, preferably greater than 28, more preferably greater than 35, and most preferably the net charge is negative.

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

383. The method or nanopore system according to any one of claims 370 to 382, ​​wherein the protein translocase is an unfoldase driven by NTP, preferably an AAA+ enzyme.

384. The method or nanopore system according to 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 their functional homologs, orthologues, or paralogs.

385. The nanopore system has an ion selectivity P greater than 2.0, preferably greater than 2.5, and more preferably greater than 3.

0. （＋） / P （－） The method or nanopore system according to any one of claims 370 to 384, wherein a negative voltage is applied to the transformer side.

386. The method or nanopore system according to claim 385, wherein the system comprises cation-selective (mutant) nanopores.

387. An analytical device comprising an array of nanopore systems according to any one of claims 377 to 386.

388. Use of a nanopore system or device according to any one of claims 377 to 387 for characterizing at least 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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