Nanobody functionalized biological nanopores and related tools and methods

Abstract

The present invention relates to tools and methods for analyzing analytes using nanopore-based sensors, for example, to methods, nanopore systems, and devices for the random detection of (unlabeled) analytes in complex samples (e.g., specific detection of protein biomarkers in body samples). There is provided a method of detecting the presence of at least one analyte in a sample using a nanopore system comprising a cis-chamber comprising a first conductive liquid medium in liquid communication with a trans-chamber comprising a second conductive liquid medium via a modified nanopore, the method comprises: (a) adding a sample of the presence of an analyte to be analyzed to a cis chamber; (b) optionally applying an electrical potential across the modified nanopore; (c) measuring the ionic current through the modified nanopore, where the modified nanopore is a biological nanopore functionalized with a recognition element (e.g., a protein recognition element) R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa capable of specifically binding to the analyte.

Description

[0001] Incorporated by Reference

[0002] This application claims the benefit of European application number EP22211193.2, filed on December 2, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Determining an analyte (eg, an analyte of interest) in a sample is an important aspect of scientific research. The presence or absence of an analyte in a sample may be clinically important. Summary of the Invention

[0004] In one aspect, the present disclosure provides a method for detecting the presence of at least one target analyte in a sample using a nanopore system, the nanopore system comprising a cis chamber in fluid communication with a trans chamber through a modified nanopore, the cis chamber comprising a first conductive liquid medium, the trans chamber comprising a second conductive liquid medium, the method comprising (a) adding a sample to be analyzed for the presence of the target analyte to the cis chamber, (b) optionally applying a potential across the modified nanopore, and (c) measuring an ionic current through the modified nanopore, wherein the modified nanopore is a biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa (e.g., 10 kDa to 40 kDa) that is capable of specifically binding to the target analyte, and wherein R dynamically moves in and out of the nanopore to cause transient current blockage events, and wherein binding of R to the target analyte modulates its dynamic movement, thereby causing a change in the frequency and / or amplitude of the current blockage events, and wherein the change in the frequency and / or amplitude of the current blockage events indicates the presence of the target analyte in the sample.

[0005] In some embodiments, the modified nanopore is an oligomeric assembly comprising or consisting of monomers of the general formula NLR, wherein N is a monomer of a pore-forming toxin having a maximum inner diameter (e.g., lumen diameter) of 5 nm to 20 nm, and L is a flexible linker attached to the cis-entrance of the pore.

[0006] In some embodiments, binding of R to the target analyte increases the time that R resides outside the pore, thereby reducing the frequency and / or amplitude of current blockade events.

[0007] In some embodiments of any of the foregoing embodiments, the biological nanopore is functionalized with at least two different protein recognition elements R' and R", eg, where R' and R" bind to different sites on the target analyte.

[0008] In some embodiments of any of the preceding embodiments, the target analyte is a protein, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof, wherein when the protein is selected from the group consisting of a folded / native protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, the target analyte is a protein.

[0009] In some embodiments, the target analyte is a protein, preferably selected from a folded / native protein, a clinically relevant protein, a biomarker, a pathogenic protein, or a cell surface protein.

[0010] In some embodiments of any of the foregoing embodiments, the sample is a complex sample comprising a mixture of proteins, wherein the sample comprises a clinical sample, such as a body fluid, such as whole blood, plasma, urine, feces, saliva, cerebrospinal fluid, breast milk, and sputum.

[0011] In one aspect, the present disclosure provides a modified protein nanopore having a minimum pore diameter of 5 nm, functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, via a flexible linker, that specifically reacts with a target analyte, preferably a target protein. In a preferred embodiment, R can move in and out of the pore to induce a blockade current.

[0012] In one aspect, the present disclosure provides a sensor system for protein analysis, comprising a fluid-filled compartment separated by a membrane into a first chamber and a second chamber, an electrode capable of applying a potential across the membrane, and at least one biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, which is capable of specifically binding to a target analyte, and wherein R is positioned at the top of the nanopore by a flexible linker to allow movement in and out of the nanopore, thereby causing a transient current blockade event.

[0013] In one aspect, the present disclosure provides a nanopore sensor system comprising a cis chamber in liquid communication with a trans chamber via a modified nanopore, the cis chamber comprising a first conductive liquid medium, the trans chamber comprising a second conductive liquid medium, wherein the modified nanopore is a biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, capable of specifically binding to a target analyte, wherein R is tethered to the top of the nanopore and capable of internalizing in the pore and dynamically moving in and out of the lumen of the nanopore to cause a transient current blockade event.

[0014] In some embodiments of any of the foregoing embodiments, R is an IgG-based portion or a non-IgG-based portion, a nanobody, a scFv fragment, a Fab fragment, an affimer, a monobody, an affibody, an Adnectin, a DARPin, or an anticalin, more preferably R is a nanobody.

[0015] In some embodiments of any of the aforementioned embodiments, the biological nanopore is a pore-forming toxin, preferably having a maximum inner diameter (e.g., lumen diameter) of 5 nm to 20 nm, more preferably selected from cytolysin A (ClyA), pleurotolysin (PlyAB), YaxAB, perforin-2 (PFN2, PDB_ID 6SB3), tripartite alpha-pore forming toxin (Ah1B, PDB_ID 6GRJ), C9 (PDB_ID 6DLW), GspD secretin (PDB_ID 5WQ7), Helicobacter pylori OMC (PDB_ID 6X6S), SpoIIIAG (PDB_ID 5WC3), Gasdermin-A3 (PDB_ID 6CB8), or mutants thereof enabling site-specific functionalization with a protein recognition element. In some embodiments, the biological nanopore is ClyA, preferably a mutant ClyA, more preferably ClyA comprising the mutation S110C.

[0016] In some embodiments of any of the foregoing embodiments, the flexible linker is an oligonucleotide, preferably duplex DNA, or a chemically modified RNA.

[0017] In some embodiments of any of the preceding embodiments, the nanopore is (reversibly) functionalized with R via a flexible linker L, preferably by nucleic acid hybridization between a first oligonucleotide, which is conjugated to the nanopore, and a second oligonucleotide, which is conjugated to R, which is complementary to the first oligonucleotide.

[0018] In one aspect, the present disclosure provides an array comprising a plurality of sensor systems according to any of the preceding embodiments, wherein the array comprises a plurality of discrete reservoirs, each of the plurality of discrete reservoirs comprising a nanopore modified with a different R element to allow detection of a different analyte.

[0019] In one aspect, the present disclosure provides a kit for preparing an array according to the aforementioned embodiments, comprising a nanopore pre-modified with a linker moiety, preferably as part of a double-stranded DNA complex consisting of an original strand and a complementary protection strand.

[0020] In some embodiments of any of the foregoing embodiments, the method, nanopore or sensor system array, or kit can be used for single protein detection, preferably in combination with high throughput analysis. In some embodiments, the sensor system is integrated into a portable device comprising multiple sensor systems.

[0021] In one aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a first side and a second side, wherein the nanopore is coupled to a recognition element, and (b) contacting the recognition element with an analyte.

[0022] In some embodiments, the recognition element is configured to move between the inner region of the nanopore and the outer region of the nanopore. In some embodiments, the recognition element is coupled to the nanopore via a connexon. In some embodiments, the length of the connexon is about 4 nanometers to about 8 nanometers. In some embodiments, the connexon comprises an oligonucleotide, a duplex DNA molecule, a chemically modified RNA molecule, or any combination thereof. In some embodiments, the nanopore is coupled to at least a portion of the connexon. In some embodiments, the nanopore is coupled to a first oligonucleotide, and the connexon is coupled to a second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide are coupled together via nucleic acid hybridization.

[0023] In some embodiments of any of the foregoing embodiments, the nanopore system further comprises an electrode pair. In some embodiments, the electrode pair is configured to generate an electric potential across the nanopore. In some embodiments, the movement of the recognition element between the inner region of the nanopore and the outer region of the nanopore realizes a change in the current of the nanopore system. In some embodiments, the method further comprises (c) measuring an ion current passing through the inner region of the nanopore. In some embodiments, the method further comprises (d) detecting the presence or absence of an analyte via a change in the ion current.

[0024] In some embodiments of any one of the foregoing embodiments, recognition element is about 5 kilodaltons to about 50 kilodaltons. In some embodiments of any one of the foregoing embodiments, recognition element is coupled to analyte. In some embodiments, recognition element is coupled to analyte to realize the movement of recognition element. In some embodiments, the movement of recognition element is realized to produce the following (i) or (ii) change: (i) the mobile frequency of recognition element or (ii) the noise or amplitude of the electric current of nanopore system. In some embodiments, when coupled to analyte, recognition element can not move between the inner area of nanopore and the outer area of nanopore. In some embodiments, when coupled to analyte, recognition element moves between the inner area of nanopore and the outer area of nanopore. In some embodiments, when coupled to analyte, recognition element moves between the inner area of nanopore and the outer area of nanopore. In some embodiments, when recognition element is coupled to analyte, the change of following (i) or (ii) is reduced: (i) the mobile frequency of recognition element or (ii) the noise or amplitude of electric current blockage.

[0025] In some embodiments of any of the foregoing embodiments, the nanopore is coupled to another recognition element. In some embodiments, the recognition element and the another recognition element bind to different regions of the analyte. In some embodiments, the recognition element and the another recognition element bind to different analytes.

[0026] In some embodiments of any of the preceding embodiments, the analyte is a protein, a peptide, a small molecule, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof.

[0027] In some embodiments of any of the foregoing embodiments, the analyte is a protein. In some embodiments, the protein is a folded protein, a native protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, or any combination thereof.

[0028] In some embodiments of any of the foregoing embodiments, the analyte is from a sample. In some embodiments, the sample is a complex sample. In some embodiments, the complex sample comprises a mixture of proteins. In some embodiments, the sample is a clinical sample. In some embodiments, the clinical sample comprises a body fluid. In some embodiments, the body fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

[0029] In some embodiments of any of the foregoing embodiments, the recognition element is a protein recognition element. In some embodiments, the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monoclonal antibody, an affimer, an affimer, an Adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

[0030] In some embodiments of any of the foregoing embodiments, the nanopore comprises an oligomeric assembly. In some embodiments, at least one subunit of the oligomeric assembly comprises a subunit of a nanopore coupled to a recognition element. In some embodiments, the recognition element is coupled to at least one subunit of the nanopore via a connector. In some embodiments, at least one subunit of the nanopore comprises a monomer of a pore-forming toxin. In some embodiments, the pore-forming toxin comprises cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2, a triple α-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof. In some embodiments, the pore-forming toxin comprises one or more mutations. In some embodiments, the pore-forming toxin is ClyA. In some embodiments, ClyA comprises an S110C mutation.

[0031] In some embodiments of any of the foregoing embodiments, the interior region of the nanopore comprises an inner diameter of about 5 nanometers to about 20 nanometers.

[0032] In one aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a first side and a second side, wherein the nanopore is coupled to a protein recognition element, wherein the protein recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore, and (b) contacting the protein recognition element with an analyte.

[0033] In one aspect, the present disclosure provides a system comprising: (a) a fluid chamber, and (b) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into (1) a first side and (2) a second side, wherein the nanopore is coupled to a recognition element.

[0034] In some embodiments, the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

[0035] In some embodiments, the recognition element is coupled to the nanopore via a connexon. In some embodiments, the connexon is about 4 nanometers to about 8 nanometers in length. In some embodiments, the connexon comprises an oligonucleotide, a duplex DNA molecule, a chemically modified RNA molecule, or any combination thereof. In some embodiments, the nanopore is coupled to at least a portion of the connexon. In some embodiments, the nanopore is coupled to a first oligonucleotide, and the connexon is coupled to a second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide are coupled together via nucleic acid hybridization.

[0036] In some embodiments of any of the foregoing embodiments, the system further comprises an electrode pair. In some embodiments, the electrode pair is configured to generate an electric potential across the nanopore. In some embodiments, the movement of the recognition element between the interior region of the nanopore and the exterior region of the nanopore achieves a current change in the system.

[0037] In some embodiments of any of the foregoing embodiments, the recognition element is about 5 kilodaltons to about 50 kilodaltons.

[0038] In some embodiments of any one of the foregoing embodiments, recognition element is configured to be coupled with analyte.In some embodiments, the recognition element coupled with analyte is configured to realize the movement of recognition element.In some embodiments, the movement of recognition element produces the change of following (i) or (ii): the noise or the amplitude of the electric current of (i) recognition element or (ii) system.In some embodiments, recognition element is not configured to move between the inner area of nanopore and the outer area of nanopore when coupled with analyte.In some embodiments, recognition element is configured to move between the inner area of nanopore and the outer area of nanopore when coupled with analyte.In some embodiments, when recognition element is coupled with analyte, the change of following (i) or (ii) reduces: the noise or the amplitude of the mobile frequency of (i) recognition element or (ii) electric current blockage. In some embodiments, the analyte is a protein, a peptide, a small molecule, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof. In some embodiments, the analyte is a protein. In some embodiments, the protein is a folded protein, a native protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, or any combination thereof. In some embodiments, the analyte is from a sample. In some embodiments, the sample is a complex sample. In some embodiments, the complex sample comprises a mixture of proteins. In some embodiments, the sample is a clinical sample. In some embodiments, the clinical sample comprises a body fluid. In some embodiments, the body fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

[0039] In some embodiments of any of the foregoing embodiments, the nanopore is configured to couple with another recognition element. In some embodiments, the recognition element and the another recognition element are configured to bind to different regions of the analyte. In some embodiments, the recognition element and the another recognition element are configured to bind to different analytes.

[0040] In some embodiments of any of the foregoing embodiments, the recognition element is a protein recognition element. In some embodiments, the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monoclonal antibody, an affimer, an affimer, an Adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

[0041] In some embodiments of any of the foregoing embodiments, the nanopore includes an oligomeric assembly. In some embodiments, at least one subunit of the oligomeric assembly includes a subunit of a nanopore coupled to a recognition element. In some embodiments, the recognition element is configured to couple to at least one subunit of the nanopore via a connector. In some embodiments, at least one subunit of the nanopore includes a monomer of a pore-forming toxin. In some embodiments, the pore-forming toxin includes cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2, a triple α-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof. In some embodiments, the pore-forming toxin includes one or more mutations. In some embodiments, the pore-forming toxin is ClyA. In some embodiments, ClyA includes an S110C mutation.

[0042] In some embodiments of any of the foregoing embodiments, the interior region of the nanopore comprises an inner diameter of about 5 nanometers to about 20 nanometers.

[0043] In one aspect, the present disclosure provides a system comprising: (a) a fluid chamber; and (b) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into (1) a first side and (2) a second side, wherein the nanopore is coupled to a protein recognition element, wherein the protein recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

[0044] In one aspect, the present disclosure provides a nanopore comprising a region configured to couple to a recognition element, wherein the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

[0045] In some embodiments, the recognition element is coupled to the nanopore via a connexon. In some embodiments, the connexon is about 4 nanometers to about 8 nanometers in length. In some embodiments, the connexon comprises an oligonucleotide, a duplex DNA complex, a chemically modified RNA complex, or any combination thereof. In some embodiments, the nanopore is coupled to at least a portion of the connexon. In some embodiments, the nanopore is coupled to a first oligonucleotide, and the connexon is coupled to a second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide are coupled together via nucleic acid hybridization.

[0046] In some embodiments of any of the foregoing embodiments, the recognition element is about 5 kilodaltons to about 50 kilodaltons.

[0047] In some embodiments of any one of the foregoing embodiments, the recognition element is configured to be coupled with the analyte. In some embodiments, the recognition element is coupled with the analyte to achieve movement of the recognition element. In some embodiments, the recognition element is configured to not move between the inner region of the nanopore and the outer region of the nanopore when coupled with the analyte. In some embodiments, the recognition element is configured to move between the inner region of the nanopore and the outer region of the nanopore when coupled with the analyte. In some embodiments, the analyte is a protein, a peptide, a small molecule, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof. In some embodiments, the analyte is a protein. In some embodiments, the protein is a folded protein, a native protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, or any combination thereof. In some embodiments, the analyte is from a sample. In some embodiments, the sample is a complex sample. In some embodiments, the complex sample includes a mixture of proteins. In some embodiments, the sample is a clinical sample. In some embodiments, the clinical sample comprises a body fluid. In some embodiments, the body fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

[0048] In some embodiments of any of the foregoing embodiments, the nanopore is configured to couple with another recognition element. In some embodiments, the recognition element and the another recognition element are configured to bind to different regions of the analyte. In some embodiments, the recognition element and the another recognition element are configured to bind to different analytes.

[0049] In some embodiments of any of the foregoing embodiments, the recognition element is a protein recognition element. In some embodiments, the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monoclonal antibody, an affimer, an affimer, an Adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

[0050] In some embodiments of any of the foregoing embodiments, the nanopore includes an oligomeric assembly. In some embodiments, at least one subunit of the oligomeric assembly includes a subunit of a nanopore coupled to a recognition element. In some embodiments, the recognition element is configured to couple to at least one subunit of the nanopore via a connector. In some embodiments, at least one subunit of the nanopore includes a monomer of a pore-forming toxin. In some embodiments, the pore-forming toxin includes cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2, a triple α-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof. In some embodiments, the pore-forming toxin includes one or more mutations. In some embodiments, wherein the pore-forming toxin is ClyA. In some embodiments, ClyA includes an S110C mutation.

[0051] In one aspect, the present disclosure provides an array comprising a plurality of nanopore systems according to any one of the preceding embodiments, wherein the array comprises a plurality of discrete reservoirs, wherein one or more of the nanopore systems comprises nanopores modified with different recognition elements to allow detection of different analytes.

[0052] In one aspect, the present disclosure provides a kit for preparing the system of any of the preceding embodiments, the kit comprising a nanopore pre-modified with a linker, wherein the linker is part of a double-stranded DNA complex consisting of an original strand and a complementary protection strand.

[0053] In one aspect, the present disclosure provides use of a method, nanopore system, nanopore, array or kit according to any one of the preceding embodiments for single protein detection, wherein the single protein detection is combined with high throughput analysis. In some embodiments, the sensor system is integrated into a portable device comprising multiple sensor systems.

[0054] Another aspect of the present disclosure provides a non-transitory computer-readable medium containing machine-executable code that, when executed by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0055] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled to the computer processors, wherein the computer memory comprises machine executable code that, when executed by the one or more computer processors, implements any of the methods described above or elsewhere herein.

[0056] For those skilled in the art, additional aspects and advantages of the present disclosure will become apparent from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure admits of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0057] Incorporated by Reference

[0058] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. To the extent that an incorporated-by-reference publication, patent, or patent application conflicts with the disclosure contained in this specification, the specification is intended to supersede and / or take precedence over any such conflicting material. DETAILED DESCRIPTION

[0059] The present invention relates to tools and methods for analyzing analytes (e.g., target analytes) using nanopore-based sensors. For example, the present invention relates to methods, nanopore systems, and devices for stochastically detecting analytes in complex samples (e.g., detecting (label-free) protein biomarkers in bodily samples).

[0060] Nanopores can sense single molecules stochastically and in real time and have been used to detect various analytes such as metal ions. 1 2 , biomolecules 3 4 , nucleic acids 5 , peptides 8 9 For example, protein sensing via this technique 10 11 12 It has additional advantages over other existing techniques such as enzyme-linked immunosorbent assay (ELISA) and mass spectrometry because it can be used for protein characterization 13 and quantitative 14 , and it can also provide insights into protein unfolding kinetics 15 , conformational changes 16 17 16 and ligand binding affinity 18 19 In addition, nanopores can be easily integrated into small portable devices 20 , which makes it very suitable for point-of-care diagnosis.

[0061] To date, various nanopore-based strategies have been explored for protein sensing. Directly, protein detection can be achieved by monitoring the current modulation caused by their direct binding / translocation inside / through the lumen of the pore. The key to this strategy is to select a pore with an appropriate geometry that can accommodate the analyte. In the past decade, nanopores with large lumen areas, such as FraC 21 , cytolysin (ClyA) 22 and PlyAB 23 24 They have been explored for studying folded proteins. For example, ClyA, which has a relatively large (approximately 6 x 6 x 10 nm) cylindrical interior, has been shown to capture and characterize different folded proteins. 25 and distinguish the interaction between peptide or DNA ligands and the protein 22 Although these biological nanopores have been proven to be effective, the fixed size and limited types of protein pores available in nature limit their general application in sensing folded proteins of various variable sizes. In contrast, binder-assisted indirect detection of proteins outside nanopores has formed a more general strategy for folded protein sensing. 14 25 27 28 29 30 Compared to bare nanopores, these approaches in various ways enable nanopores to detect large proteins that do not fit inside the nanopore and enhance the specificity of protein sensing by exploiting specific binding interactions with proteins. These strategies in various ways involve trapping proteins close to the nanopore entrance so that the presence of the analyte causes a change in the current or involve transferring binding interactions that occur outside the nanopore to the interior of the pore, which results in a change in the ion flow through the pore. To date, various binders such as biotin have been used to 10 , aptamers 29 , peptides 26 , protein domain 30 Nanopores have been functionalized chemically or genetically and have been widely used for protein detection or protein-ligand binding studies.

[0062] In one example, Thakur and Movileanu established a platform for studying protein-protein interactions. 30 In this study, a protein domain containing a flexible 12-amino acid peptide adapter at its N-terminus (the RNase barnase, Bn) was fused to the monomeric pore t-FhuA. When the cognate ligand protein (Barstar, Bs) binds to the protein binder, the adapter is pulled away from the pore opening, which induces a distinguishable unblocking current event. 30This nanopore sensor has demonstrated the ability to detect and quantify protein analytes in the presence of small amounts of serum; however, it has several drawbacks that limit its application in protein sensing. First, constructing nanopores with different protein ligands genetically linked (i.e., fused) to the nanopore is laborious, making this approach suboptimal for the detection of a wide variety of different proteins. Furthermore, the preparation of the nanopore requires refolding the protein in urea and detergent, which carries the risk of rendering many protein ligands ineffective.

[0063] In another example, Bayley et al. (which is incorporated herein by reference in its entirety) demonstrated that aptamer-modified α-hemolysin (α-HL) nanopores, in which a 15mer DNA aptamer (TBA) is hybridized to an oligonucleotide covalently attached to a cysteine near the pore mouth, allow for the detection of thrombin 29 Notably, the anchoring of the DNA adaptor to the pore confers modularity, so that by varying the aptamer, the same nanopore construct can be used to detect a variety of analytes. However, it is important to note that the diversity in structure and length (ranging from 15 to 80 bases) of aptamers for different analytes means that different aptamers cannot be expected to behave in the same way every time without extensive experimentation (e.g., the linker on each aptamer must be carefully tuned to enable detection), and therefore, it is not possible to create a universal system that employs different aptamer binders for different desired analytes.

[0064] Soskine et al. 18 (which is incorporated herein by reference in its entirety) A ligand was conjugated to the top of the ClyA nanopore to detect folded proteins by selective external association and pore entry. In this method, proteins bound to the aptamer above the nanopore are allowed to enter the nanopore, while non-proteins that do not bind to the aptamer are blocked from entering the nanopore.

[0065] Crucially, this can be problematic when applying the platform to biological samples such as blood, as aptamers can be rapidly degraded by nucleases.

[0066] In some embodiments, the present disclosure provides novel modular nanopore sensors that allow for stochastic sensing (label-free) of protein targets that is not subject to the drawbacks of known nanopore-based sensors. In some embodiments, the present disclosure provides universal and multipurpose systems that allow for specific and sensitive detection of proteins and protein-containing pathogens. The present disclosure provides novel modular nanopore sensors that allow for stochastic sensing of analytes (e.g., proteins), which in some embodiments are universal and multipurpose systems that allow for specific and sensitive detection of proteins and protein-containing analytes (e.g., viruses, bacteria) in complex samples (e.g., blood or serum), including analytes that are larger than the pore diameter.

[0067] In some embodiments, the nanopore can be functionalized with a small (up to about 50 kDa) recognition element (e.g., protein recognition element) (e.g., nanobody) at the top (or mouth) of a large vestibule nanopore that can be moved in and out of the hole to cause a blockade current. An analyte (e.g., a target analyte) is added to a solution outside the hole (at the first end or cis end) and forms a target-recognition element complex, resulting in a change in the capture of the recognition element inside the nanopore, which in turn results in a change in the ionic current through the open hole. Nanobodies (or other small binding molecules) block the hole in the resting state, limiting the entry of other proteins. Importantly, in the resting state, the recognition element is primarily located inside the nanopore, and proteins and other unwanted nonspecific background molecules from the solution cannot enter the nanopore. Therefore, this method is not affected by background noise from non-related proteins in the solution, which might otherwise interrupt the signal or block the nanopore.

[0068] In some embodiments, the recognition element can be a small recognition element (e.g., a protein recognition element), which is highly specific for the detection of a wide range of entities (e.g., proteins). Secondly, the recognition element is appropriately tethered to the nanopore as a replaceable module, for example, by hybridization of complementary chains. Therefore, compared to existing methods, nanopores functionalized with different recognition elements (e.g., nanobodies) can be easily obtained, and the preparation method is less laborious. Third, this nuclease-resistant nanopore design makes the sensing of proteins in biological fluids independent of their size, where large proteins are detected outside the nanopore, while small proteins are detected within the cavity of the hole.

[0069] In some embodiments, nanobodies are used as replaceable modules fixed to ClyA dodecamers via the formation of DNA duplexes to illustrate pore design. By simply changing the modules, four different nanobody-functionalized nanopores were constructed, and all nanopore constructs showed the ability to detect analytes. For example, thanks to the multivalent interaction between the SARS-CoV-2 spike protein and the multimeric Ty1 nanobody, this method enables us to detect proteins in the picomolar to low nanomolar range, even in the presence of blood. Here, the present invention provides a novel and versatile strategy that allows highly specific and sensitive detection of various proteins in biological fluids, regardless of their size, shape, and charge.

[0070] Therefore, in one embodiment, the present invention provides a nanopore sensor system comprising a cis chamber in liquid communication with a trans chamber via a modified nanopore, the cis chamber comprising a first conductive liquid medium, the trans chamber comprising a second conductive liquid medium, wherein the modified nanopore is a biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, which is capable of specifically binding to a target analyte. In one embodiment, the present invention provides a nanopore sensor system comprising a first side of a fluid chamber (e.g., a cis chamber) in liquid communication with a second side of a fluid chamber (e.g., a trans chamber) via a modified nanopore, the first side of the fluid chamber comprising a first conductive liquid medium and the second side of the fluid chamber comprising a second conductive liquid medium, wherein the modified nanopore is a nanopore (e.g., a biological nanopore) functionalized with a recognition element (e.g., a protein recognition element) R of 5 kDa to 50 kDa (e.g., 10 kDa to 40 kDa) that is capable of specifically binding to an analyte (e.g., a target analyte). The recognition element R is suitably tethered to the top of the nanopore and, based on its small size relative to the larger luminal size of the pore, is capable of internalization within the pore. It dynamically moves in and out of the nanopore cavity (vestibule) to cause transient current blockade events. Binding of R to the analyte modulates its dynamic movement, thereby causing a change in the frequency and / or amplitude of the current blockade events. Changes in the frequency and / or amplitude of the current blockade events indicate the presence of the analyte in the sample.

[0071] In some embodiments, the nanopore of the nanopore sensor system of the present disclosure may be a biological nanopore. Alternatively, in some embodiments, the nanopore of the nanopore sensor system of the present disclosure may be a solid-state nanopore.

[0072] In some embodiments, the identification element can be less than the internal diameter (for example, lumen diameter) of nanopore.In some cases, the identification element can be less than about 0.1% to about 500% of internal diameter (for example, lumen diameter).In some cases, the identification element can be less than about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210 %, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% To about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490%, or about 490% to about 500%.In some cases, the recognition element can be at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 40%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or more than 500%.In some cases, the recognition element can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 220%, at most about 245%, at most about 260%, at most about 270%, at most about 280%, at most about 29 ... %, up to about 210%, up to about 200%, up to about 190%, up to about 180%, up to about 170%, up to about 160%, up to about 150%, up to about 140%, up to about 130%, up to about 120%, up to about 110%, up to about 100%, up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, up to about 1%, up to about 0.5%, up to about 0.1%, or less than 0.1%. In some cases, the recognition element can be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180% smaller than the inner diameter (e.g., lumen diameter) of the nanopore. , about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500%.

[0073] In some embodiments, the recognition element can move through the outer region of the nanopore and the inner region of the nanopore. In some cases, the inner region of the nanopore can be the channel of the nanopore. In some cases, the inner region of the nanopore can be the lumen of the nanopore on the first side of the fluid chamber. In some cases, the outer region can be any region outside the channel of the nanopore or the lumen of the nanopore. In some cases, the recognition element can move freely between the inner region and the outer region of the nanopore. In some cases, free movement can include movement of the recognition element without being hindered by a connector.

[0074] In some embodiments, movement of the recognition element between the interior region of the nanopore and the exterior region of the nanopore can effect a change in the ionic current of the nanopore. In some embodiments, movement of the recognition element into the interior region of the nanopore can reduce the ionic current (e.g., the amplitude or noise of the ionic current) moving through the channel of the nanopore. In some cases, the reduction in the ionic current moving through the channel of the nanopore can be measured.

[0075] In some embodiments, movement of the recognition element into the interior region of the nanopore can block at least a portion of the channel of the nanopore. In some cases, movement of the recognition element into the interior region of the nanopore can increase the ionic current moving through the channel of the nanopore.

[0076] In some embodiments, movement of the recognition element into the outer region of the nanopore can cause the channel of the nanopore to open. In some cases, movement of the recognition element into the outer region of the nanopore can increase the ionic current moving through the channel of the nanopore. In some cases, the increase in the ionic current moving through the channel of the nanopore can be measured.

[0077] In some embodiments, the nanopore system can exist in two states: (i) the recognition element is in the interior region of the nanopore, and (ii) the recognition element is in the exterior region of the nanopore. In some cases, the change in the nanopore system from (i) to (ii) can be measured. In some cases, the frequency of the nanopore system can be a measurement of the movement between (i) and (ii). In some cases, the nanopore system can measure the change in the frequency of the movement of the recognition element. In some cases, the change in frequency can indicate the presence of an analyte. In some cases, the change in frequency can indicate the absence of an analyte. In some cases, the change in frequency can be used to determine the concentration of the analyte in a solution. In some cases, the frequency of the movement of the recognition element in the nanopore system can be 0.1 kilohertz (kHz) to about 1 megahertz (MHz). In some cases, the frequency of the nanopore system can be 0.1 kHz to about 1 kHz, about 1 kHz to about 100 kHz, or about 100 kHz to about 1 MHz. In some cases, the frequency of movement of the recognition element in the nanopore system can be at least about 0.1 kHz, at least about 1 kHz, at least about 5 kHz, at least about 10 kHz, at least about 20 kHz, at least about 30 kHz, at least about 40 kHz, at least about 50 kHz, at least about 60 kHz, at least about 70 kHz, at least about 80 kHz, at least about 90 kHz, at least about 100 kHz, at least about 200 kHz, at least about 300 kHz, at least about 400 kHz, at least about 500 kHz, at least about 600 kHz, at least about 700 kHz, at least about 800 kHz, at least about 900 kHz, at least about 1,000 kHz, at least about 1 MHz, or greater than 1 MHz. In some cases, the frequency of movement of the recognition element in the nanopore system can be at most about 1 MHz, at most about 1,000 kHz, at most about 900 kHz, at most about 800 kHz, at most about 700 kHz, at most about 600 kHz, at most about 500 kHz, at most about 400 kHz, at most about 300 kHz, at most about 200 kHz, at most about 100 kHz, at most about 90 kHz, at most about 80 kHz, at most about 70 kHz, at most about 60 kHz, at most about 50 kHz, at most about 40 kHz, at most about 30 kHz, at most about 20 kHz, at most about 10 kHz, at most about 5 kHz, at most about 1 kHz, at most about 0.1 kHz, or less than 0.1 kHz.In some cases, the frequency of movement of the recognition element in the nanopore system can be about 0.1 kHz, about 1 kHz, about 5 kHz, about 10 kHz, about 20 kHz, about 30 kHz, about 40 kHz, about 50 kHz, about 60 kHz, about 70 kHz, about 80 kHz, about 90 kHz, about 100 kHz, about 200 kHz, about 300 kHz, about 400 kHz, about 500 kHz, about 600 kHz, about 700 kHz, about 800 kHz, about 900 kHz, about 1,000 kHz, or about 1 MHz.

[0078] In some embodiments, the nanopore system can measure the change in the amplitude of the ionic current moving through the nanopore. In some cases, when the recognition element is in the inner region of the nanopore, the ionic current moving through the nanopore can be increased. In some cases, when the recognition element is in the inner region of the nanopore, the ionic current moving through the nanopore can be reduced. In some cases, when the recognition element is in the outer region of the nanopore, the ionic current moving through the nanopore can be increased. In some cases, the change in the ionic current amplitude can indicate the presence of an analyte. In some cases, the change in the ionic current amplitude can indicate the absence of an analyte. In some cases, the amplitude of the ionic current can be from about 1 picoampere (pA) to about 1,000 pA. In some cases, the amplitude of the ionic current can be from 1 pA to about 10 pA, from about 10 pA to about 100 pA, from about 1 pA to about 100 pA, or from about 100 pA to about 1,000 pA. In some cases, the amplitude of the ionic current can be at least about 1 pA, at least about 5 pA, at least about 10 pA, at least about 20 pA, at least about 30 pA, at least about 40 pA, at least about 50 pA, at least about 60 pA, at least about 70 pA, at least about 80 pA, at least about 90 pA, at least about 100 pA, at least about 200 pA, at least about 300 pA, at least about 400 pA, at least about 500 pA, at least about 600 pA, at least about 700 pA, at least about 800 pA, at least about 900 pA, at least about 1,000 pA, or greater than 1,000 pA. In some cases, the amplitude of the ionic current can be at most about 1,000 pA, at most about 900 pA, at most about 800 pA, at most about 700 pA, at most about 600 pA, at most about 500 pA, at most about 400 pA, at most about 300 pA, at most about 200 pA, at most about 100 pA, at most about 90 pA, at most about 80 pA, at most about 70 pA, at most about 60 pA, at most about 50 pA, at most about 40 pA, at most about 30 pA, at most about 20 pA, at most about 10 pA, at most about 5 pA, at most about 1 pA, or less than 1 pA. In some cases, the amplitude of the ionic current can be about 1 pA, about 5 pA, about 10 pA, about 20 pA, about 30 pA, about 40 pA, about 50 pA, about 60 pA, about 70 pA, about 80 pA, about 90 pA, about 100 pA, about 200 pA, about 300 pA, about 400 pA, about 500 pA, about 600 pA, about 700 pA, about 800 pA, about 900 pA, or about 1,000 pA.

[0079] In some embodiments, the nanopore system can measure changes in the noise of the ionic current moving through the nanopore. In some cases, the change in the noise of the ionic current can refer to fluctuations in the ionic current (e.g., statistical fluctuations). In some cases, when the recognition element is in the interior region of the nanopore, the noise of the ionic current can increase. In some cases, when the recognition element is in the interior region of the nanopore, the noise of the ionic current can decrease. In some cases, when the recognition element is in the exterior region of the nanopore, the noise of the ionic current can increase. In some cases, when the recognition element is in the exterior region of the nanopore, the noise of the ionic current can decrease. In some embodiments, the noise of the ionic current can be determined by measuring changes in the frequency of the noise. In some cases, the frequency of the noise can be 0.1 kilohertz (kHz) to about 1 megahertz (MHz). In some cases, the frequency of the nanopore system can be 0.1 kHz to about 1 kHz, about 1 kHz to about 100 kHz, or about 100 kHz to about 1 MHz. In some cases, the frequency of the noise can be at least about 0.1 kHz, at least about 1 kHz, at least about 5 kHz, at least about 10 kHz, at least about 20 kHz, at least about 30 kHz, at least about 40 kHz, at least about 50 kHz, at least about 60 kHz, at least about 70 kHz, at least about 80 kHz, at least about 90 kHz, at least about 100 kHz, at least about 200 kHz, at least about 300 kHz, at least about 400 kHz, at least about 500 kHz, at least about 600 kHz, at least about 700 kHz, at least about 800 kHz, at least about 900 kHz, at least about 1,000 kHz, at least about 1 MHz, or greater than 1 MHz. In some cases, the frequency of the noise can be at most about 1 MHz, at most about 1,000 kHz, at most about 900 kHz, at most about 800 kHz, at most about 700 kHz, at most about 600 kHz, at most about 500 kHz, at most about 400 kHz, at most about 300 kHz, at most about 200 kHz, at most about 100 kHz, at most about 90 kHz, at most about 80 kHz, at most about 70 kHz, at most about 60 kHz, at most about 50 kHz, at most about 40 kHz, at most about 30 kHz, at most about 20 kHz, at most about 10 kHz, at most about 5 kHz, at most about 1 kHz, at most about 0.1 kHz, or less than 0.1 kHz.In some cases, the frequency of the noise can be about 0.1 kHz, about 1 kHz, about 5 kHz, about 10 kHz, about 20 kHz, about 30 kHz, about 40 kHz, about 50 kHz, about 60 kHz, about 70 kHz, about 80 kHz, about 90 kHz, about 100 kHz, about 200 kHz, about 300 kHz, about 400 kHz, about 500 kHz, about 600 kHz, about 700 kHz, about 800 kHz, about 900 kHz, about 1,000 kHz, or about 1 MHz. In some embodiments, the noise of the ion current can be determined by measuring the amplitude variation (e.g., standard deviation) of the noise. In some cases, the amplitude of the noise can be about 1 picoampere (pA) to about 1,000 pA. In some cases, the amplitude of the noise can be from 1 pA to about 10 pA, from about 10 pA to about 100 pA, from about 1 pA to about 100 pA, or from about 100 pA to about 1,000 pA. In some cases, the amplitude of the noise can be at least about 1 pA, at least about 5 pA, at least about 10 pA, at least about 20 pA, at least about 30 pA, at least about 40 pA, at least about 50 pA, at least about 60 pA, at least about 70 pA, at least about 80 pA, at least about 90 pA, at least about 100 pA, at least about 200 pA, at least about 300 pA, at least about 400 pA, at least about 500 pA, at least about 600 pA, at least about 700 pA, at least about 800 pA, at least about 900 pA, at least about 1,000 pA, or greater than 1,000 pA. In some cases, the amplitude of the noise can be at most about 1,000 pA, at most about 900 pA, at most about 800 pA, at most about 700 pA, at most about 600 pA, at most about 500 pA, at most about 400 pA, at most about 300 pA, at most about 200 pA, at most about 100 pA, at most about 90 pA, at most about 80 pA, at most about 70 pA, at most about 60 pA, at most about 50 pA, at most about 40 pA, at most about 30 pA, at most about 20 pA, at most about 10 pA, at most about 5 pA, at most about 1 pA, or less than 1 pA. In some cases, the amplitude of the noise can be about 1 pA, about 5 pA, about 10 pA, about 20 pA, about 30 pA, about 40 pA, about 50 pA, about 60 pA, about 70 pA, about 80 pA, about 90 pA, about 100 pA, about 200 pA, about 300 pA, about 400 pA, about 500 pA, about 600 pA, about 700 pA, about 800 pA, about 900 pA, or about 1,000 pA.

[0080] In some embodiments, recognition element can be coupled with analyte. In some cases, recognition element can be coupled with little analyte (e.g., 0.1 nm to 10 nm). In some cases, recognition element coupled with little analyte can move between the inner region of nanopore and the outer region of nanopore. In some cases, recognition element coupled with little analyte can move into the inner region of nanopore. In some cases, recognition element can be coupled with big analyte (e.g., 10 nm or bigger). In some cases, recognition element coupled with big analyte can not move between the inner region of nanopore and the outer region of nanopore. In some cases, recognition element coupled with big analyte can not move into the inner region of nanopore.

[0081] In some embodiments, the recognition element can be coupled to the analyte. In some cases, the recognition element can be specifically coupled to the analyte. In some embodiments, the coupling of the recognition element to the analyte can enable the movement of the recognition element. In some cases, when the recognition element is coupled to the analyte, the recognition element may not be able to move into the interior area of the nanopore.

[0082] In some embodiments, the recognition element coupled to the analyte can be larger than the inner diameter (e.g., lumen diameter) of the nanopore. In some cases, the recognition element coupled to the analyte can be approximately 0.1% to approximately 500% larger than the inner diameter (e.g., lumen diameter) of the nanopore. In some cases, the recognition element coupled to the analyte can be approximately 0.1% to approximately 0.5%, approximately 0.5% to approximately 1%, approximately 1% to approximately 5%, approximately 5% to approximately 10%, approximately 10% to approximately 20%, approximately 20% to approximately 30%, approximately 30% to approximately 40%, approximately 40% to approximately 45%, approximately 45% to approximately 50%, approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% to approximately 70%, approximately 70% to approximately 75%, approximately 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 370% to about 380%, about 390% to about 400%, about 410% to about 420%, about 430% to about 440%, about 450% to about 460%, about 470% to about 480%, about 490% to about 500%, about 510% to about 520%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 600%, about 610% to about 620%, about 630% to about 640%, about 650% to about 660%, about 670% to about 680%, about 690% to about 700%, about From about 450% to about 460%, from about 460% to about 470%, from about 470% to about 480%, from about 480% to about 490%, or from about 490% to about 500%.In some cases, the recognition element coupled to the analyte can be at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 201%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 251%, at least about 261%, at least about 271%, at least about 280%, at least about 290%, at least about 301%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 351%, at least about 361%, at least about 371%, at least about 380%, at least about 390%, at least about 401%, at least about At least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or more than 500%.In some cases, the recognition element coupled to the analyte can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 340%, at most about 350%, at most about 360%, at most about 370%, at most about 380%, at most about 390%, at most about 390%, at most about 390%, at most about 380%, at most about 390%, at most about 310%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 3 At most, about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1%. In some cases, the recognition element coupled to the analyte can be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0083] In some embodiments, the recognition element coupled to the analyte can be smaller than the inner diameter (e.g., lumen diameter) of the nanopore. In some cases, the recognition element coupled to the analyte can be smaller than the inner diameter (e.g., lumen diameter) of the nanopore by about 0.1% to about 500%. In some cases, the recognition element coupled to the analyte can be smaller than the inner diameter (e.g., lumen diameter) of the nanopore by about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 200%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350%, about 350% to about 360%, about 370% to about 380%, about 390% to about 400%, about 410% to about 420%, about 430% to about 440%, about 450% to about 460%, about 470% to about 480%, about 490% to about 500%, about 510% to about 520%, about 530% to about 540%, about 550% to about 560%, about 570% to about 580%, about 590% to about 600%, about 610% to about 620%, about 630% to about 640%, about 650% to about 660%, about 670% to about 680%, about 690% to about 700%, about From about 450% to about 460%, from about 460% to about 470%, from about 470% to about 480%, from about 480% to about 490%, or from about 490% to about 500%.In some cases, the recognition element coupled to the analyte can be at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 201%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 251%, at least about 261%, at least about 271%, at least about 280%, at least about 290%, at least about 301%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 351%, at least about At least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or more than 500%.In some cases, the recognition element coupled to the analyte can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 340%, at most about 350%, at most about 360%, at most about 370%, at most about 380%, at most about 390%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about At most, about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1%. In some cases, the recognition element coupled to the analyte can be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0084] In some cases, when the recognition element is coupled to the analyte, movement of the recognition element into the interior region of the nanopore can be reduced. In some cases, movement of the recognition element into the interior region of the nanopore can be reduced by about 0.1% to about 500% when the recognition element is coupled to the analyte as compared to when the recognition element is not coupled to the analyte. In some cases, movement of the recognition element into the interior region of the nanopore can be reduced by about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75 ... 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 20 0%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350% , about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490%, or about 490% to 500%.In some cases, movement of the recognition element into the interior region of the nanopore can be reduced by at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about %, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or more than 500%.In some cases, movement of the recognition element into the interior region of the nanopore can be reduced by at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 260%, at most about 270%, at most about 280%, at most about 29 ... At most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1%. In some cases, movement of the recognition element into the interior region of the nanopore can be reduced by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 0%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0085] In some cases, movement of the recognition element into the interior region of the nanopore can be increased when the recognition element is coupled to the analyte. In some cases, movement of the recognition element into the interior region of the nanopore can be increased by about 0.1% to about 500% when the recognition element is coupled to the analyte compared to when the recognition element is not coupled to the analyte. In some cases, movement of the recognition element into the interior region of the nanopore can be increased by about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 75% to about 80%, about 75% to about 90%, about 80% to about 95%, about 80% to about 90%, about 85% to about 8 ... 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 130% to about 140%, about 140% to about 150%, about 150% to about 160%, about 160% to about 170%, about 170% to about 180%, about 180% to about 190%, about 190% to about 20 0%, about 200% to about 210%, about 210% to about 220%, about 220% to about 230%, about 230% to about 240%, about 240% to about 250%, about 250% to about 260%, about 260% to about 270%, about 270% to about 280%, about 280% to about 290%, about 290% to about 300%, about 300% to about 310%, about 310% to about 320%, about 320% to about 330%, about 330% to about 340%, about 340% to about 350% , about 350% to about 360%, about 360% to about 370%, about 370% to about 380%, about 380% to about 390%, about 390% to about 400%, about 400% to about 410%, about 410% to about 420%, about 420% to about 430%, about 430% to about 440%, about 440% to about 450%, about 450% to about 460%, about 460% to about 470%, about 470% to about 480%, about 480% to about 490%, or about 490% to 500%.In some cases, movement of the recognition element into the interior region of the nanopore can be increased by at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about %, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or more than 500%.In some cases, movement of the recognition element into the interior region of the nanopore can be increased by at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 260%, at most about 270%, at most about 280%, at most about 29 ... At most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1%. In some cases, movement of the recognition element into the interior region of the nanopore can be increased by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 0%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0086] In some embodiments, when recognition element is not coupled with analyte, recognition element can move between the inner region of nanopore and the outer region of nanopore. In some cases, when recognition element is in the inner region of nanopore, recognition element can reduce the ionic current (for example, the amplitude or noise of ionic current) through nanopore. In some cases, when recognition element is in the inner region of nanopore, recognition element can increase the ionic current through nanopore. In some cases, the increase and / or reduction of ionic current can be measured. In some cases, the increase and / or reduction of ionic current can indicate the absence of analyte.

[0087] In some embodiments, when the recognition element is coupled to the analyte, the interior region of the nanopore can be open. In some cases, when the interior region of the nanopore is open, an ion current can pass through the nanopore. In some cases, the ion current passing through the nanopore can be measured. In some cases, measuring the ion current passing through the nanopore can indicate the presence of the analyte.

[0088] In some embodiments, when the recognition element is coupled to the analyte, the movement of the recognition element into the interior region of the nanopore can be reduced. In some cases, when the movement of the recognition element into the interior region of the nanopore is reduced, the ionic current passing through the nanopore can increase. In some cases, the increase in ionic current passing through the nanopore can be measured. In some cases, measuring the increase in ionic current passing through the nanopore can indicate the presence of the analyte. In some cases, measuring the increase in ionic current passing through the nanopore can indicate the absence of the analyte. In some cases, measuring the decrease in ionic current passing through the nanopore can indicate the presence of the analyte. In some cases, measuring the decrease in ionic current passing through the nanopore can indicate the absence of the analyte.

[0089] The present disclosure provides a method for detecting the presence of at least one analyte in a sample using a nanopore system, the nanopore system comprising a first side of a fluid chamber, the first side of the fluid chamber being in liquid communication with a second side of the fluid chamber via a modified nanopore, the first side of the fluid chamber comprising a first conductive liquid medium, the second side of the fluid chamber comprising a second conductive liquid medium, the method comprising: (a) adding a sample to be analyzed for the presence of a target analyte to the first side; (b) optionally applying an electric potential across the modified nanopore; and (c) measuring an ionic current passing through the modified nanopore, wherein the modified nanopore is a nanopore (e.g., a biological nanopore) functionalized with a recognition element (e.g., a protein recognition element) R of 5 kDa to 50 kDa (e.g., 10 kDa to 40 kDa), the recognition element R being capable of specifically binding to an analyte as defined above.

[0090] The present disclosure also provides a method for detecting the presence of at least one target analyte in a sample using a nanopore system, the nanopore system comprising a cis chamber in liquid communication with a trans chamber via a modified nanopore, the cis chamber comprising a first conductive liquid medium, the trans chamber comprising a second conductive liquid medium, the method comprising: (a) adding a sample to be analyzed for the presence of a target analyte to the cis chamber; (b) optionally applying an electric potential across the modified nanopore; and (c) measuring the ionic current passing through the modified nanopore; wherein the modified nanopore is a biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, capable of specifically binding to a target analyte as defined above, preferably wherein R dynamically moves in and out of the nanopore to cause transient current blockade events, and wherein binding of R to the target analyte modulates its dynamic movement, thereby causing a change in the frequency and / or amplitude of the current blockade events, and wherein a change in the frequency and / or amplitude of the current blockade events indicates the presence of the target analyte in the sample.

[0091] WO2016 / 166232 relates to a nanopore-based sensor system comprising a nanopore and a protein adaptor internalized in the cavity of the nanopore. The present disclosure provides a nanopore with a recognition element (e.g., a nanobody) that is coupled to the top of the nanopore and moves freely in and out of the cavity.

[0092] In some embodiments, the recognition element (e.g., R) can be a protein recognition element. In some cases, the protein recognition element can be an antibody-based protein molecule. In some cases, the antibody-based protein molecule can be an IgM molecule, an IgG molecule, an IgA molecule, an IgE molecule, an IgD molecule, an IgY molecule, an IgW molecule, an IgT molecule, an IgZ molecule, a nanobody, a scFv, a Fab fragment, or any combination thereof. In some embodiments, the protein recognition element can be a single domain antibody, also referred to as a nanobody. For example, a nanobody (also referred to as a V antibody) derived from a heavy chain antibody found in camelids can be a single domain antibody. H H fragment), or nanobodies derived from heavy chain antibodies of cartilaginous fish (also known as variable neoantigen receptor V NAR Alternatively, R can be a Fab fragment, such as an IgG-based portion, such as a single-chain variable fragment (scFv).

[0093] Alternatively, in some embodiments, the protein recognition element can be a non-antibody-based protein molecule. In some cases, the non-antibody-based protein molecule can be an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, fynomer, gastrobody, Kunitz domain peptide, single antibody, nanoCLAMP, optimizer, repebody, probent, centyrin, obody or any combination thereof. In some cases, the non-antibody-based protein molecule (e.g., R) can be a non-IgG-based part, such as based on affimer, affibody (based on the Z domain of protein A from Staphylococcus aureus (Staphylococcus aureus)), single antibody and Adnectin (based on type III fibronectin domain), DARPin (designed ankyrin repeat protein) or anticalin (based on lipocalin) those. In some embodiments, the protein recognition element can be an antibody, a nanobody, a scFv, a Fab fragment, an affibody, an affilin, an affimer, an affitin, an alphabody, anticalin, avimer, DARPin, fynomer, gastrobody, Kunitz domain peptide, monoclonal antibody, nanoCLAMP, optimizer, repebody, probetin, centyrin, obody, or any combination thereof.

[0094] In some embodiments, the nanopore is at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, at least about 10 kDa, at least about 11 kDa, at least about 12 kDa, at least about 13 kDa, at least about 14 kDa, at least about 15 kDa, at least about 16 kDa, at least about 17 kDa, at least about 18 kDa, at least about 19 kDa, at least about 20 kDa, at least about 21 kDa, at least about 22 kDa, at least about 23 kDa, at least about 24 kDa, at least about 25 kDa, at least about 26 kDa, at least about 27 kDa, at least about 28 kDa, at least about 29 kDa, at least about 30 kDa, at least about 31 kDa, at least about 32 kDa, at least about 33 kDa, at least about 34 kDa, at least about 35 kDa, at least about 36 kDa, kDa, at least about 37 kDa, at least about 38 kDa, at least about 39 kDa, at least about 40 kDa, at least about 41 kDa, at least about 42 kDa, at least about 43 kDa, at least about 44 kDa, at least about 45 kDa, at least about 46 kDa, up to about 47 kDa, at least about 48 kDa, at least about 49 kDa, at least about 50 kDa, or more than about 50 kDa, which recognition element is capable of specifically binding to the analyte.In some embodiments, the nanopore is at most about 50 kDa, at most about 49 kDa, at most about 48 kDa, at most about 47 kDa, at most about 46 kDa, at most about 45 kDa, at most about 44 kDa, at most about 43 kDa, at most about 42 kDa, at most about 41 kDa, at most about 40 kDa, at most about 39 kDa, at most about 38 kDa, at most about 37 kDa, at most about 36 kDa, at most about 35 kDa, at most about 34 kDa, at most about 33 kDa, at most about 32 kDa, at most about 31 kDa, at most about 30 kDa, at most about 29 kDa, at most about 28 kDa, at most about 27 kDa, at most about 26 kDa, at most about 25 kDa, at most about 24 kDa, at most about 23 kDa, at most about 22 kDa, at most about 21

[0015] The present invention relates to a method for producing an analyte that is conjugated to a recognition element of at most about 10 kDa, at most about 20 kDa, at most about 19 kDa, at most about 18 kDa, at most about 17 kDa, at most about 16 kDa, at most about 15 kDa, at most about 14 kDa, at most about 13 kDa, at most about 12 kDa, at most about 11 kDa, at most about 10 kDa, at most about 9 kDa, at most about 8 kDa, at most about 7 kDa, at most about 6 kDa, at most about 5 kDa, at most about 4 kDa, at most about 3 kDa, at most about 2 kDa, at most about 1 kDa, or less than about 1 kDa that is capable of specifically binding to the analyte.

[0095] In some embodiments, the nanopore is coupled to a recognition element of about 5 kDa to about 60 kDa that is capable of specifically binding to an analyte. In some embodiments, the nanopore is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 25 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 35 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 45 kDa, about 5 kDa to about 50 kDa, about 5 kDa to about 55 kDa, about 5 kDa to about 60 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 35 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 45 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 55 kDa, about 10 kDa to about 60 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 35 kDa, about 15 kDa to about 40 kDa, about 15 kDa to about 45 kDa, about 15 kDa to about 50 kDa, about 15 kDa to about 55 kDa, about 15 kDa to about 60 kDa, about 20 kDa to about 25 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 35 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 45 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 55 kDa, about 20 kDa to about 60 kDa, about 25 kDa to about 30 kDa, about 25 kDa to about 35 kDa, about 25 kDa to about 40 kDa, about 25 kDa to about 45 kDa, about 25 kDa to about 50 kDa, about 25 kDa to about 55 kDa, about 25 kDa to about 60 kDa, about 30 kDa to about 35 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 45 kDa, about 30 kDa to about 50 kDa, about 30 kDa to about 55 kDa, about 30 kDa to about 60 kDa, about 35 kDa to about 40 kDa, about 35 kDa to about 45 kDa, about 35 kDa to about 50 kDa, about 35 kDa to about 55 kDa, about 35 kDa to about 60 kDa, about 40 kDa to about 45 kDa, about 40 kDa to about 50 kDa, about 40 kDa to about 55kDa, about 40 kDa to about 60 kDa, about 45 kDa to about 50 kDa, about 45 kDa to about 55 kDa, about 45 kDa to about 60 kDa, about 50 kDa to about 55 kDa, about 50 kDa to about 60 kDa, or about 55 kDa to about 60 kDa, that is coupled to a recognition element that is capable of specifically binding to the analyte.

[0096] In some embodiments, the nanopore is about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 The analyte is coupled to a recognition element of about 40 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa or about 50 kDa, which is capable of specifically binding to the analyte.

[0097] Alternatively, in some embodiments, the recognition element can be a nucleic acid recognition element. In some cases, the nucleic acid recognition element can be an aptamer. In some cases, the nucleic acid recognition element can be a riboswitch. In some cases, the nucleic acid recognition element can be DNA, RNA, xeno nucleic acid (xeno nucleic acid, XNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (bridged nucleic acid, BNA), glycol nucleic acid (glycol nucleic acid, GNA), threose nucleic acid (threose nucleic acid, TNA), hexitol nucleic acid (hexitol nucleic acid, HNA) or any combination thereof. In some cases, the nucleic acid recognition element can be a naturally occurring nucleic acid molecule. In some cases, the nucleic acid recognition element can be a synthetic (e.g., produced in a laboratory) nucleic acid molecule. In some cases, the nucleic acid recognition element can be a recombinant nucleic acid molecule.

[0098] In some embodiments, the size of the nucleic acid recognition element can be from about 1 kDa to about 50 kDa. In some cases, the size of the nucleic acid recognition element can be from about 1 kDa to about 5 kDa, from about 5 kDa to about 10 kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20 kDa, from about 20 kDa to about 25 kDa, from about 25 kDa to about 30 kDa, from about 30 kDa to about 35 kDa, from about 35 kDa to about 40 kDa, from about 40 kDa to about 45 kDa, or from about 45 kDa to about 50 kDa. In some cases, the size of the nucleic acid recognition element can be at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, at least about 10 kDa, at least about 11 kDa, at least about 12 kDa, at least about 13 kDa, at least about 14 kDa, at least about 15 kDa, at least about 16 kDa, at least about 17 kDa, at least about 18 kDa, at least about 19 kDa, at least about 20 kDa, at least about 21 kDa, at least about 22 kDa, at least about 23 kDa, at least about 24 kDa, at least about 25 kDa, at least about 26 kDa, at least about 27 kDa, at least about 28 kDa, at least about 29 kDa, at least about 30 kDa, at least about 31 kDa, at least about 32 kDa, at least about 33 kDa, at least about 34 kDa, kDa, at least about 35 kDa, at least about 36 kDa, at least about 37 kDa, at least about 38 kDa, at least about 39 kDa, at least about 40 kDa, at least about 41 kDa, at least about 42 kDa, at least about 43 kDa, at least about 44 kDa, at least about 45 kDa, at least about 46 kDa, at least about 47 kDa, at least about 48 kDa, at least about 49 kDa, at least about 50 kDa, or more.In some cases, the size of the nucleic acid recognition element can be at most about 50 kDa, at most about 49 kDa, at most about 48 kDa, at most about 47 kDa, at most about 46 kDa, at most about 45 kDa, at most about 44 kDa, at most about 43 kDa, at most about 42 kDa, at most about 41 kDa, at most about 40 kDa, at most about 39 kDa, at most about 38 kDa, at most about 37 kDa, at most about 36 kDa, at most about 35 kDa, at most about 34 kDa, at most about 33 kDa, at most about 32 kDa, at most about 31 kDa, at most about 30 kDa, at most about 29 kDa, at most about 28 kDa, at most about 27 kDa, at most about 26 kDa, at most about 25 kDa, at most about 24 kDa, at most about 23 kDa, at most about 22 kDa, at most about 21 kDa, at most about 20 kDa, at most about 19 kDa, at most about 18 kDa, at most about 17 kDa, at most about 16 kDa, at most about 15 kDa, at most about 14 kDa, at most about 13 kDa, at most about 12 kDa, at most about 11 kDa, at most about 10 kDa, at most about 9 kDa, at most about 8 kDa, at most about 7 kDa, at most about 6 kDa, at most about 5 kDa, at most about 4 kDa, at most about 3 kDa, at most about 2 kDa, at most about 1 kDa or less. In some cases, the size of the nucleic acid recognition element can be about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa or about 50 kDa.

[0099] In some embodiments, a nanopore (e.g., a biological nanopore) can be functionalized with at least two different recognition elements (e.g., protein recognition elements) R' and R", for example, where R' and R" bind to different sites (epitopes) of a given analyte (e.g., a target analyte), or where R' and R" bind to different analytes (e.g., a target analyte).

[0100] In some embodiments, the analyte (e.g., target analyte) can be a protein, a protein assembly, a nucleic acid molecule, a peptide, a small molecule, a protein / DNA assembly, a protein / RNA assembly, a lipid, a lipid membrane, a carbohydrate, a vitamin, a lipid particle, an oligosaccharide, a bacterium, a bacterial membrane, a bacterial membrane protein, a bacterial nucleic acid, a viral nucleic acid, a viral membrane, a viral membrane protein, a viral capsid, a viral particle, a pathogen protein, a pathogen nucleic acid, a dendrimer, a polymer, or any combination thereof. In one aspect, the analyte (e.g., target analyte) is a protein, e.g., selected from a folded / native protein, a peptide, a digested folded protein, a clinically relevant protein, a biomarker, a pathogenic protein, a bacterial protein, a viral protein, a prokaryotic protein, a eukaryotic protein, a parasitic protein, an antibody, a contractile protein, an enzyme, a hormonal protein, a structural protein, a storage protein, a transport protein, a cell surface protein, or any combination thereof.

[0101] The sample can be any type. It can be an aqueous solution comprising one or more (biological) components. On the one hand, it is a complex sample comprising a protein mixture, preferably wherein the sample comprises a clinical sample, more preferably a body fluid, such as whole blood, blood plasma, serum, semen, amniotic fluid, mucus, ascites, peritoneum, peritoneal fluid, extracellular fluid, interstitial fluid, transcellular fluid, lymph, synovial joint fluid, synovial fluid, tears, breast milk, bile, pericardial fluid, gastric acid, pleural fluid, sputum, urine, feces, saliva, cerebrospinal fluid, their aqueous dilutions or any combination thereof.

[0102] The nanopore (e.g., a biological nanopore) can be a pore-forming toxin, preferably having a maximum inner diameter (e.g., lumen diameter) of 5 nm to 20 nm. In some cases, the pore is suitably selected from cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2 (PFN2, PDB_ID 6SB3), tripartite α-pore-forming toxin (AhlB, PDB_ID 6GRJ), C9 (PDB_ID6DLW), GspD secretin (PDB_ID 5WQ7), Helicobacter pylori OMC (PDB_ID 6X6S), SpoIIIAG (PDB_ID5WC3), Gasdermin-A3 (PDB_ID 6CB8), or mutants thereof that are site-specifically functionalized with a recognition element (e.g., a protein recognition element).

[0103] In some embodiments, the inner diameter (e.g., lumen diameter) can be at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, or greater than about 20 nm. In some embodiments, the inner diameter (e.g., lumen diameter) can be at most about 20 nm, at most about 19 nm, at most about 18 nm, at most about 17 nm, at most about 16 nm, at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, or less than about 5 nm.

[0104] In some embodiments, the inner diameter (eg, lumen diameter) can be from about 5 nm to about 25 nm. In some embodiments, the inner diameter (e.g., lumen diameter) can be from about 5 nm to about 6 nm, from about 5 nm to about 7 nm, from about 5 nm to about 8 nm, from about 5 nm to about 9 nm, from about 5 nm to about 10 nm, from about 5 nm to about 12 nm, from about 5 nm to about 14 nm, from about 5 nm to about 16 nm, from about 5 nm to about 18 nm, from about 5 nm to about 20 nm, from about 5 nm to about 25 nm, from about 6 nm to about 7 nm, from about 6 nm to about 8 nm, from about 6 nm to about 9 nm, from about 6 nm to about 10 nm, from about 6 nm to about 12 nm, from about 6 nm to about 14 nm, from about 6 nm to about 16 nm, from about 6 nm to about 18 nm, from about 6 nm to about 20 nm, from about 6 nm to about 25 nm, nm, about 8 nm to about 9 nm, about 8 nm to about 10 nm, about 8 nm to about 12 nm, about 8 nm to about 14 nm, about 8 nm to about 16 nm, about 8 nm to about 18 nm, about 8 nm to about 20 nm, about 8 nm to about 25 nm, about 9 nm to about 10 nm, about 9 nm to about 12 nm, about 9 nm to about 14 nm, about 9 nm to about 16 nm, about 9 nm to about 18 nm, about 9 nm to about 20 nm, about 9 nm to about 25 nm, about 10 nm to about 12 nm, about 10 nm to about 14 nm, about 10 nm to about 16 nm, about 10 nm to about 18 nm, about 10 nm to about 20 nm, about 10 nm to about 25 nm, about 12 nm to about 14 nm, about 12 nm to about 16 nm, about 12 nm to about 18 nm, about 12 nm to about 20 nm, about 12 nm to about 25 nm, about 14 nm to about 16 nm, about 14 nm to about 18 nm nm, about 14 nm to about 25 nm, about 16 nm to about 18 nm, about 16 nm to about 20 nm, about 16 nm to about 25 nm, about 18 nm to about 20 nm, about 18 nm to about 25 nm, or about 20 nm to about 25 nm.

[0105] In some embodiments, the inner diameter (e.g., lumen diameter) can be about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm.

[0106] In some embodiments, the modified nanopore can be an oligomeric assembly comprising or consisting of monomers of the general formula NLR, wherein N is a monomer of a pore-forming toxin having a maximum inner diameter (e.g., lumen diameter) of 5 nm to 20 nm, L is a flexible linker attached to the wide (e.g., cis) entrance of the pore, and R is a recognition element (e.g., a protein recognition element) capable of specific binding to an analyte (e.g., a target analyte). The flexible linker can be of any size as long as it allows functional positioning of R relative to the entrance / opening of the pore. In one embodiment, L has a length of about 4-8 nm, preferably 5-6 nm. L can be an oligonucleotide, preferably DNA, or chemically modified RNA (e.g., locked nucleic acid or RNA chemically modified with -F, -OMe at the 2' position to enhance stability). In one embodiment, L contains 8-20 nucleotides, e.g., 10-18, 12-20, 8-14, or 16-20 nucleotides.

[0107] In some embodiments, the recognition element can be coupled to a nanopore in a first side of a nanopore system. In some embodiments, the recognition element can be coupled to a nanopore in a second side of a nanopore system. In some embodiments, the recognition element can be coupled to a nanopore in both a first side of a nanopore system and a second side of a nanopore system.

[0108] In some embodiments, the analyte can be added to a first side of the nanopore system. In some embodiments, the analyte can be added to a second side of the nanopore system. In some embodiments, the analyte can be added to both a first side of the nanopore system and a second side of the nanopore system.

[0109] In some embodiments, the recognition element can be coupled to a nanopore in a first side of a nanopore system, and the analyte can be added to the first side of the nanopore system. In some embodiments, the recognition element can be coupled to a nanopore in a second side of a nanopore system, and the analyte can be added to the second side of the nanopore system. In some embodiments, the recognition element can be coupled to nanopores in both the first side of the nanopore and the second side of the nanopore, and the analyte can be added to both the first side of the nanopore system and the second side of the nanopore system.

[0110] In some embodiments, the recognition element can be coupled to the nanopore. In some cases, the recognition element can be reversibly coupled to the nanopore. In some cases, the recognition element can be irreversibly coupled to the nanopore.

[0111] In some embodiments, the recognition element can be directly coupled to the nanopore. In some cases, the recognition element can be directly coupled to the nanopore via a covalent bond. In some cases, the covalent bond can be a non-polar covalent bond. In some cases, the covalent bond can be a polar covalent bond. In some cases, the recognition element can be directly coupled to the nanopore via a non-covalent bond. In some cases, the non-covalent bond can be a hydrophobic interaction, a van der Waals interaction, an electrostatic interaction, a hydrogen bond, or any combination thereof.

[0112] In some embodiments, the recognition element can be indirectly coupled to the nanopore. In some cases, the recognition element can be indirectly coupled to the nanopore via a connexon. In some cases, the connexon can be a flexible connexon. In some cases, the nanopore can be coupled to the connexon via a conjugation reaction. In some cases, the conjugation reaction can be a sulfide-based conjugation reaction, an ester reaction, a thioester reaction, an amide reaction, a native chemical connection reaction, or any combination thereof. In some cases, the nanopore can be coupled to the connexon via a bioconjugation reaction. In some cases, the bioconjugation reaction can include reacting lysine with N-hydroxysuccinimide (NHS) ester, acylation of lysine, reacting lysine with isocyanate, reacting lysine with isothiocyanate, reacting lysine with benzoyl fluoride, reacting cysteine with maleimide, reacting cysteine with iodoacetamide, reacting cysteine with 2-thiopyridine, reacting cysteine with 3-arylpropiolonitrile, aromatic electrophilic substitution, reacting tyrosine with a diazonium salt, reacting tyrosine with 4-phenyl-1,2,4-triazole-3,5-dione (PTAD), a Mannich reaction, reacting an N-terminal serine or threonine with NaIO4, reacting an N-terminal cysteine with iodoacetamide, reacting the N-terminus of the analyte with pyridoxal phosphate, Staudinger ligation with an azide. ligation), huisgen cyclization of azides, strain-promoted huisgen cyclization of azides, cysteine or tryptophan RH-catalyzed alkylation, lysine or N-terminal Ir-catalyzed alkylation, tyrosine Pd-catalyzed O-alkylation, cysteine Au-catalyzed alkylation, tryptophan arylation, cysteine arylation, lysine arylation, or any combination thereof.

[0113] In some cases, the linker can be an amino acid linker. In some cases, the amino acid linker can include any combination of amino acids. In some cases, classical amino acids can 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, amino acids can be non-natural amino acids. In some cases, the unnatural amino acid can include hydroproline, β-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, pyroglutamate, naphthylalanine, Abu, DAB, methionine sulfoxide, methionine sulfone, α-amino-n-butyric acid, norvaline, alloisoleucine, tert-leucine, α-amino-n-heptanoic acid, pipecolic acid, allothreonine, homocysteine, homoserine, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, β-alanine, β-amino-n-butyric acid, β-aminoisobutyric acid, β-aminoisobutyric acid, γ-aminobutyric acid, α-aminoisobutyric acid, isovaline, sarcosine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, N-methyl-β-alanine, N-ethyl-β-alanine, isoserine, α-hydroxy-γ-aminobutyric acid, or any combination thereof. In some cases, the connexon can include any combination of classical amino acids and non-natural amino acids. In some cases, the amino acid connexon can be a combination of glycine and serine amino acids. In some cases, the amino acid connexon can be a combination of aspartic acid and serine amino acids. In some cases, the amino acid connexon can include about one amino acid to about 10 amino acids. In some cases, the amino acid connexon can include at least one amino acid, at least about two amino acids, at least about three amino acids, at least about four amino acids, at least about five amino acids, at least about six amino acids, at least about seven amino acids, at least about eight amino acids, at least about nine amino acids, at least about ten amino acids, or more than ten amino acids. In some cases, the amino acid connexon can include up to about ten amino acids, up to about nine amino acids, up to about eight amino acids, up to about seven amino acids, up to about six amino acids, up to about five amino acids, up to about four amino acids, up to about three amino acids, up to about two amino acids, up to about one amino acid, or less than one amino acid. In some cases, the amino acid linker can comprise about one amino acid, about two amino acids, about three amino acids, about four amino acids, about five amino acids, about six amino acids, about seven amino acids, about eight amino acids, about nine amino acids, or about ten amino acids.

[0114] In some cases, the linker can be a polymer linker. In some cases, the polymer linker can be ethylene glycol, polyethylene glycol, or a combination thereof.

[0115] In some cases, the linker can be a peptide linker. In some cases, the peptide linker can be a biotin linker. In some cases, the peptide linker can be a streptavidin linker.

[0116] In some cases, the linker can be a chemical linker. In some cases, the chemical linker can be a disulfide linker. In some cases, the chemical linker can be a cysteine interaction linker. In some cases, the chemical linker can be a click chemistry linker. In some cases, the click chemistry linker can involve one or more click reagents. In some cases, one or more click reagents can include 1,3-dipole family, epoxides, aziridines, cyclic sulfates, epoxides, aziridines, cyclic sulfates, oxine ethers, hydrazones, aromatic heterocycles, or any combination thereof.

[0117] In some cases, the linker can be a nucleic acid linker. In some cases, the nucleic acid linker can be a polynucleic acid linker.

[0118] In some embodiments, the linker contains at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, or more than about 20 nucleotides. In some embodiments, the linker contains at most about 20 nucleotides, at most about 19 nucleotides, at most about 18 nucleotides, at most about 17 nucleotides, at most about 16 nucleotides, at most about 15 nucleotides, at most about 14 nucleotides, at most about 13 nucleotides, at most about 12 nucleotides, at most about 11 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, at most about 4 nucleotides, at most about 3 nucleotides, at most about 2 nucleotides, at most about 1 nucleotide, or less than about 1 nucleotide.

[0119] In some embodiments, the linker contains about 1 nucleotide to about 20 nucleotides. In some embodiments, the linker contains about 1 nucleotide to about 2 nucleotides, about 2 nucleotides to about 3 nucleotides, about 3 nucleotides to about 4 nucleotides, about 4 nucleotides to about 5 nucleotides, about 5 nucleotides to about 6 nucleotides, about 6 nucleotides to about 7 nucleotides, about 7 nucleotides to about 8 nucleotides, about 8 nucleotides to about 9 nucleotides, about 8 nucleotides to about 10 nucleotides, about 8 nucleotides to about 12 nucleotides, about 8 nucleotides to about 13 nucleotides, about 8 nucleotides to about 14 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotides to about 16 nucleotides, about 8 nucleotides to about 17 nucleotides, about 8 nucleotides to about 18 nucleotides. nucleotides, about 8 nucleotides to about 19 nucleotides, about 8 nucleotides to about 20 nucleotides, about 9 nucleotides to about 10 nucleotides, about 9 nucleotides to about 12 nucleotides, about 9 nucleotides to about 13 nucleotides, about 9 nucleotides to about 14 nucleotides, about 9 nucleotides to about 15 nucleotides, about 9 nucleotides to about 16 nucleotides, about 9 nucleotides to about 17 nucleotides, about 9 nucleotides to about 18 nucleotides, about 9 nucleotides to about 19 nucleotides, about 9 nucleotides to about 20 nucleotides, about 10 nucleotides to about 12 nucleotides, about 10 nucleotides to about 13 nucleotides, about 10 nucleotides to about 14 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 16 nucleotides, about 10 nucleotides to about 17 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 19 nucleotides, about 10 nucleotides to about 20 nucleotides, about 12 nucleotides to about 13 nucleotides, about 12 nucleotides to about 14 nucleotides, about 12 nucleotides to about 15 nucleotides, about 12 nucleotides to about 16 nucleotides, about 12 nucleotides to about 17 nucleotides, about 12 nucleotides to about 18 nucleotides, about 12 nucleotides to about 19 nucleotides, about 12 nucleotides to about 20 nucleotides, about 13 nucleotides to about 14 nucleotides, about 13 nucleotides to about 15 nucleotides nucleotides, about 13 nucleotides to about 16 nucleotides, about 13 nucleotides to about 17 nucleotides, about 13 nucleotides to about 18 nucleotides, about 13 nucleotides to about 19 nucleotides, about 13 nucleotides to about 20 nucleotides, about 14 nucleotides to about 15 nucleotides, about 14 nucleotides to about 16 nucleotides, about 14 nucleotides to about 17 nucleotides, about 14 nucleotides to about 18 nucleotides, about 14 nucleotides to about 19 nucleotides, about 14 nucleotides to about 20 nucleotides, about 15 nucleotides to about 16 nucleotides, about 15 nucleotides to about 17 nucleotides, about 15 nucleotides to about 18 nucleotides, about 15 nucleotides to about 19 nucleotides,about 15 nucleotides to about 20 nucleotides, about 16 nucleotides to about 17 nucleotides, about 16 nucleotides to about 18 nucleotides, about 16 nucleotides to about 19 nucleotides, about 16 nucleotides to about 20 nucleotides, about 17 nucleotides to about 18 nucleotides, about 17 nucleotides to about 19 nucleotides, about 17 nucleotides to about 20 nucleotides, about 18 nucleotides to about 19 nucleotides, about 18 nucleotides to about 20 nucleotides, or about 19 nucleotides to about 20 nucleotides.

[0120] In some embodiments, the linker contains about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, or about 20 nucleotides.

[0121] In some embodiments, the nanopore is (reversibly) functionalized with R via a linker L, preferably wherein L is formed by nucleic acid hybridization between a first oligonucleotide, which is conjugated to the nanopore, and a second oligonucleotide, which is conjugated to R, which is complementary to the first oligonucleotide.

[0122] In one embodiment, the present invention provides a nanopore (e.g., a modified nanopore, e.g., a protein nanopore) having a minimum pore diameter of 5 nm that is functionalized via a flexible linker having a 5 kDa to 50 kDa (e.g., 10 kDa to 40 kDa) recognition element (e.g., a protein recognition element) R that specifically reacts with an analyte (e.g., a target analyte) (e.g., a protein (e.g., a target protein)). In some cases, R can move in and out of the pore to cause a blockade current. In some cases, the recognition element R is tethered to the top of the nanopore.

[0123] In one aspect, the present invention provides a modified protein nanopore having a minimum pore diameter of 5 nm, which is functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, via a flexible linker that specifically reacts with a target analyte, preferably a target protein. In a preferred embodiment, R can move in and out of the pore to induce a blockade current. In a preferred embodiment, the recognition element R is tethered to the top of the nanopore.

[0124] In one embodiment, the present invention provides a sensor system for protein analysis, comprising a fluid-filled compartment separated by a membrane into a first chamber and a second chamber, an electrode capable of applying a potential across the membrane, and at least one nanopore (e.g., a biological nanopore), wherein the nanopore is functionalized with a recognition element (e.g., a protein recognition element) R of 5 kDa to 50 kDa, preferably, for example, 10 kDa to 40 kDa, which is capable of specifically binding to an analyte, and wherein R is positioned at the top of the nanopore, for example, via a flexible linker, to allow movement in and out of the nanopore to cause a transient current blockade event.

[0125] In one aspect, the present invention provides a sensor system for protein analysis, comprising a fluid-filled compartment separated by a membrane into a first chamber and a second chamber, an electrode capable of applying a potential across the membrane, and at least one biological nanopore, wherein the biological nanopore is functionalized with a recognition element (e.g., a protein recognition element) R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, which is capable of specifically binding to a target analyte, and wherein R is preferably positioned at the top of the nanopore via a flexible link to allow movement in and out of the nanopore to cause a transient current blockade event.

[0126] In one embodiment, the present invention provides an array comprising a plurality of sensor systems according to the present invention, as well as methods and kits for preparing such arrays. Preferably, the array comprises a plurality of discrete reservoirs, each reservoir comprising a nanopore modified with a different R element to allow detection of a different analyte.

[0127] In one aspect, the present disclosure provides a kit for preparing an array according to the present invention comprising nanopores pre-modified with a linker moiety, preferably as part of a double-stranded DNA complex consisting of an original strand and a complementary protection strand.

[0128] Also provided herein are methods, nanopore or sensor systems, arrays or kits for use in single protein detection, preferably in combination with high throughput analysis.

[0129] definition

[0130] Identification element R

[0131] In some embodiments, a recognition element (e.g., a protein recognition element) R is tethered to the top of the nanopore and dynamically moves in and out of the nanopore cavity (vestibule) to cause transient current blockade events. Binding of R to an analyte (e.g., a target analyte) modulates this dynamic movement, thereby causing a change in the frequency and / or amplitude of the current blockade events, wherein the change in the frequency and / or amplitude of the current blockade events indicates the presence of the analyte (e.g., a target analyte) in the sample. Typically, binding of R to an analyte (e.g., a target analyte) increases the time that R stays outside the pore, thereby reducing the frequency of the current blockade events.

[0132] In some embodiments, to allow for dynamic movement in and out of the vestibule of the nanopore, the R moiety used in the present invention is much smaller than a conventional IgG antibody composed of two different polypeptide chains with a molecular weight of approximately 150 kDa. The typical dimensions of an IgG are approximately 14.5 nm × 8.5 nm × 4.0 nm, with antigen binding sites separated by 13.7 nm. The molecular weight of R is in the range of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, 10 kDa to 35 kDa, 10 kDa to 30 kDa, and more preferably 12-15 kDa. Preferred R moieties have dimensions in the single-digit nanometer range, for example 1-5 nm × 1-5 nm.

[0133] In some embodiments, the molecular weight of the recognition element can be at least about 5 kDa, at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, at least about 25 kDa, at least about 30 kDa, at least about 35 kDa, at least about 40 kDa, at least about 45 kDa, at least about 50 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight of the recognition element can be at most about 50 kDa, at most about 45 kDa, at most about 40 kDa, at most about 35 kDa, at most about 30 kDa, at most about 25 kDa, at most about 20 kDa, at most about 15 kDa, at most about 10 kDa, at most about 5 kDa, or less than about 5 kDa.

[0134] In some embodiments, the recognition element can have a molecular weight of about 5 kDa to about 60 kDa. In some embodiments, the molecular weight of the recognition element can be about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 25 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 35 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 45 kDa, about 5 kDa to about 50 kDa, about 5 kDa to about 55 kDa, about 5 kDa to about 60 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 35 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 45 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 55 kDa, about 10 kDa to about 60 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 35 kDa, about 15 kDa to about 40 kDa, about 15 kDa to about 45 kDa, about 15 kDa to about 50 kDa, about 15 kDa to about 55 kDa, about 15 kDa to about 60 kDa, about 20 kDa to about 25 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 35 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 45 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 55 kDa, about 25 kDa to about 60 kDa, about 25 kDa to about 30 kDa, about 25 kDa to about 35 kDa, about 25 kDa to about 40 kDa, about 25 kDa to about 45 kDa, about 25 kDa to about 50 kDa, about 25 kDa to about 55 kDa, about 25 kDa to about 60 kDa, about 30 kDa to about 35 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 45 kDa, about 30 kDa to about 50 kDa, about 30 kDa to about 55 kDa, about 30 kDa to about 60 kDa, about 35 kDa to about 40 kDa, about 35 kDa to about 45 kDa, about 35 kDa to about 50 kDa, about 35 kDa to about 55 kDa, about 35 kDa to about 60 kDa, about 40 kDa to about 45 kDa, about 40 kDa to about 50 kDa, about 40 kDa to about 55 kDa, about 40 kDa to about 60 kDa, about 45kDa to about 50 kDa, about 45 kDa to about 55 kDa, about 45 kDa to about 60 kDa, about 50 kDa to about 55 kDa, about 50 kDa to about 60 kDa, or about 55 kDa to about 60 kDa.

[0135] In some embodiments, the molecular weight of the recognition element can be about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, or about 50 kDa.

[0136] In some embodiments, the recognition element (e.g., R) can be a single domain antibody, also known as a nanobody. For example, a nanobody (also known as a V domain antibody) derived from a heavy chain antibody found in camelids H H fragment), or nanobodies derived from cartilaginous fish heavy chain antibodies (also known as variable neoantigen receptor V NAR Alternatively, R may be a Fab fragment, such as an IgG-based portion, such as a single-chain variable fragment (scFv). Alternatively, R may be a non-IgG-based portion, such as those based on an affimer, an affibody (based on the Z domain of protein A from Staphylococcus aureus), a monoclonal antibody, and an Adnectin (based on a type III fibronectin domain), a DARPin (designed ankyrin repeat protein), or an anti-anticalin (based on lipocalin).

[0137] On the one hand, R is a Fab fragment. In some embodiments, the fragment antigen binding region (Fab region) is the region on the antibody that binds to the antigen. It is composed of a constant region and a variable region of each of the heavy chain and the light chain. Fab fragment antibodies can be produced by papain digestion of the entire IgG antibody to remove the entire Fc fragment including the hinge region. These antibodies are monovalent and contain only a single antigen binding site. The molecular weight of the Fab fragment is about 50 kDa. At the amino terminal end of the monomer, the variable domain contains a paratope (antigen binding site) comprising a set of complementary determining regions. Therefore, each arm of the Y binds to an epitope on the antigen.

[0138] In another embodiment, R is based on a single-chain variable fragment (scFv), which is a fusion protein of approximately 30-35 kDa and 2 × 3 nm connecting the variable regions of the immunoglobulin heavy chain (VH) and light chain (VL). See Asaadi et al. (Biomarker Research volume 9, Article number: 87 (2021), which is incorporated herein by reference in its entirety).

[0139] In some embodiments, R is a nanobody or a so-called VHH antibody, originally called a heavy chain antibody (HCAb), also known as a single domain antibody. Nanobodies, consisting of only the variable domains of heavy chain antibodies of camelid origin, have emerged as a rapidly growing family of strong protein binders. 31 32 33 The nomenclature of "nanobodies", originally adopted by the Belgian company Ablynx®, stems from their nanometer dimensions, i.e., 4 nm in length, 2.5 nm in width, and a molecular weight of only 12-14 kD. Nanobodies are nuclease-resistant, which makes them more advantageous for indirect protein sensing compared to aptamers. In addition to this, nanobodies can be easily produced as recombinant proteins in bacterial expression systems and can be easily equipped with customized tags without affecting their functionality. 35 36 37 38 In addition, it has been reported that nanobody multimerization can improve its binding affinity 39 and enhance detection sensitivity 40 .

[0140] In some embodiments, R is a non-IgG-based moiety, such as an affimer. Affimer® molecules are small proteins that bind to analytes (e.g., target analytes) with an affinity in the nanomolar range. At 12-14 kDa, Affimer reagents are small non-antibody binding proteins that are approximately 10 times smaller than IgG antibodies, and their length is less than 4 nm. These engineered non-antibody binding proteins are designed to mimic the molecular recognition properties of monoclonal antibodies in different applications.

[0141] In preferred aspects, R is a nanobody or so-called VHH antibody, originally known as a heavy chain antibody (HCAb), also known as a single domain antibody, as described above. In some embodiments, R is a non-IgG based portion, such as an affimer. Affimer® molecules are small proteins that bind to a target protein with an affinity in the nanomolar range, as described above.

[0142] See also Bedford et al. (Biophysical Reviews volume 9, pg. 299-308; 2017, which is incorporated herein by reference in its entirety), which provides examples of smaller-sized immunoglobulin G (IgG) and non-IgG-based binding reagents suitable for nanopore functionalization.

[0143] In some embodiments, one nanopore may be conjugated to R moieties of the same type (e.g. all Nanobodies, scFvs or affimers), or to mixed types of R moieties (e.g. a combination of two or more types of those listed above, such as an scFv and an affimer or an scFv and a Nanobody).

[0144] In some embodiments, the nanopore can be coupled to one or more recognition elements. In some embodiments, the nanopore can be coupled to at least about 1 recognition element, at least about 2 recognition elements, at least about 3 recognition elements, at least about 4 recognition elements, at least about 5 recognition elements, at least about 10 recognition elements, at least about 12 recognition elements, at least about 15 recognition elements, at least about 18 recognition elements, at least about 20 recognition elements, at least about 25 recognition elements, at least about 30 recognition elements, at least about 35 recognition elements, at least about 40 recognition elements, at least about 45 recognition elements, at least about 50 recognition elements, or more than about 50 recognition elements. In some embodiments, the nanopore can be coupled to at most about 50 recognition elements, at most about 45 recognition elements, at most about 40 recognition elements, at most about 35 recognition elements, at most about 30 recognition elements, at most about 25 recognition elements, at most about 20 recognition elements, at most about 18 recognition elements, at most about 15 recognition elements, at most about 12 recognition elements, at most about 10 recognition elements, at most about 5 recognition elements, at most about 4 recognition elements, at most about 3 recognition elements, at most about 2 recognition elements, at most about 1 recognition element, or less than 1 recognition element.

[0145] In some embodiments, the nanopore can be coupled to about 1 recognition element to about 50 recognition elements. In some embodiments, the nanopore can be coupled to about 1 recognition element to about 2 recognition elements, about 1 recognition element to about 3 recognition elements, about 1 recognition element to about 4 recognition elements, about 1 recognition element to about 5 recognition elements, about 1 recognition element to about 10 recognition elements, about 1 recognition element to about 15 recognition elements, about 1 recognition element to about 20 recognition elements, about 1 recognition element to about 25 recognition elements, about 1 recognition element to about 30 recognition elements, about 1 recognition element to about 40 recognition elements, about 1 recognition element to about 50 recognition elements, about 2 recognition elements to about 3 recognition elements, about 2 recognition elements to about 4 recognition elements, about 2 recognition elements to about 50 recognition elements, about 2 recognition elements to about 3 recognition elements, about 2 recognition elements to about 4 recognition elements, about 2 recognition elements to about 5 recognition elements. to about 5 recognition elements, about 2 recognition elements to about 10 recognition elements, about 2 recognition elements to about 15 recognition elements, about 2 recognition elements to about 20 recognition elements, about 2 recognition elements to about 25 recognition elements, about 2 recognition elements to about 30 recognition elements, about 2 recognition elements to about 40 recognition elements, about 2 recognition elements to about 50 recognition elements, about 3 recognition elements to about 4 recognition elements, about 3 recognition elements to about 5 recognition elements, about 3 recognition elements to about 10 recognition elements, about 3 recognition elements to about 15 recognition elements, about 3 recognition elements to about 20 recognition elements, about 3 recognition elements to about 25 recognition elements, about 3 recognition elements to about 30 recognition elements, about 2 recognition elements to about 40 recognition elements, about 2 recognition elements to about 50 recognition elements, about 3 recognition elements to about 4 recognition elements, about 3 recognition elements to about 5 recognition elements, about 3 recognition elements to about 10 recognition elements, about 3 recognition elements to about 15 recognition elements, about 3 recognition elements to about 20 recognition elements, about 3 recognition elements to about 25 recognition elements, about 3 recognition elements to about 3 0 recognition elements, about 3 recognition elements to about 40 recognition elements, about 3 recognition elements to about 50 recognition elements, about 4 recognition elements to about 5 recognition elements, about 4 recognition elements to about 10 recognition elements, about 4 recognition elements to about 15 recognition elements, about 4 recognition elements to about 20 recognition elements, about 4 recognition elements to about 25 recognition elements, about 4 recognition elements to about 30 recognition elements, about 4 recognition elements to about 40 recognition elements, about 4 recognition elements to about 50 recognition elements, about 5 recognition elements to about 10 recognition elements, about 5 recognition elements to about 15 recognition elements, about 5 recognition elements to about 20 recognition elements, about 5 recognition elements to about 25 recognition elements, about 4 recognition elements to about 30 recognition elements, about 4 recognition elements to about 40 recognition elements, about 4 recognition elements to about 50 recognition elements, about 5 recognition elements to about 10 recognition elements, about 5 recognition elements to about 15 recognition elements, about 5 recognition elements to about 20 recognition elements, about 5 recognition elements to about 2 5 recognition elements, about 5 recognition elements to about 30 recognition elements, about 5 recognition elements to about 40 recognition elements, about 5 recognition elements to about 50 recognition elements, about 10 recognition elements to about 15 recognition elements, about 10 recognition elements to about 20 recognition elements, about 10 recognition elements to about 25 recognition elements, about 10 recognition elements to about 30 recognition elements, about 10 recognition elements to about 40 recognition elements, about 10 recognition elements to about 50 recognition elements, about 15 recognition elements to about 20 recognition elements, about 15 recognition elements to about 25 recognition elements, about 15 recognition elements to about 30 recognition elements, about 15 recognition elements to about 40 recognition elements,About 15 recognition elements to about 50 recognition elements, about 20 recognition elements to about 25 recognition elements, about 20 recognition elements to about 30 recognition elements, about 20 recognition elements to about 40 recognition elements, about 20 recognition elements to about 50 recognition elements, about 25 recognition elements to about 30 recognition elements, about 25 recognition elements to about 40 recognition elements, about 25 recognition elements to about 50 recognition elements, about 30 recognition elements to about 40 recognition elements, about 30 recognition elements to about 50 recognition elements, or about 40 recognition elements to about 50 recognition elements are coupled.

[0146] In some embodiments, the nanopore can be coupled to about 1 recognition element, about 2 recognition elements, about 3 recognition elements, about 4 recognition elements, about 5 recognition elements, about 10 recognition elements, about 12 recognition elements, about 15 recognition elements, about 18 recognition elements, about 20 recognition elements, about 25 recognition elements, about 30 recognition elements, about 35 recognition elements, about 40 recognition elements, about 45 recognition elements, or about 50 recognition elements.

[0147] In some embodiments, one or more recognition elements can be coupled to the same region of an analyte. In some embodiments, one or more recognition elements can be coupled to different regions of an analyte. In some embodiments, one or more recognition elements can be coupled to different analytes.

[0148] In some embodiments, R is coupled to or positioned at the top of the nanopore (e.g., at the top cis side of the nanopore) via a flexible tether to allow access to analytes added to the first side (e.g., cis chamber). The site of the linker coupled to R is selected on the surface, loop, or terminus of the protein so that it leaves the binding domain motif of R free and unencumbered by space. Common conjugation sites (e.g., for binding R to beads or surfaces) are well known for many suitable R binders. The site for modifying the nanopore with R is selected so that it allows R to dynamically move in and out of the interior of the nanopore, or at least cause transient current blockade events in the absence of an analyte (e.g., a target analyte), and wherein the binding of the analyte (e.g., target) to R modulates its dynamic movement, thereby causing changes in the frequency and / or amplitude of the current blockade events.

[0149] In some embodiments, binding of R to an analyte (e.g., a target analyte) increases the time R remains outside the pore (e.g., through steric or electrostatic effects that reduce the ability of the R-analyte complex to enter the nanopore cavity), thereby reducing the frequency of current blockade events. Alternatively, binding of R to an analyte (e.g., a target analyte) reduces the time R remains outside the pore, thereby increasing the frequency of current blockade events. For example, binding to a highly charged analyte may aid internalization by altering the electrophoretic forces acting on the R-analyte complex.

[0150] In other embodiments, when the R-analyte complex is within the nanopore, the binding of R to the analyte (e.g., target analyte) changes the ionic current passing through the nanopore. For example, in embodiments where the R-analyte complex is able to enter the nanopore cavity, the presence of the analyte increases or decreases the ionic current flowing through the nanopore relative to the unbound R current level due to changes in excluded volume or electrostatics. In some embodiments, the R-analyte complex exhibits multiple current levels due to the complex being located at different positions within the nanopore. Changes in current levels can be used to detect the presence of the analyte. Changes and absolute values of current levels associated with the R-analyte complex within the nanopore can also be used to determine other properties of the analyte, such as the presence and type of one or more post-translational modifications (e.g., phosphorylation, glycosylation, etc.). For example, R can be designed to universally bind to a specific analyte (e.g., a specific target protein analyte) (e.g., bind to an unmodified epitope region of the protein), which exists in a mixture in a variety of post-translational or other modified forms, such that the modified region of the protein analyte facing the nanopore changes the ionic current in different ways.

[0151] In some embodiments, a nanopore (e.g., a biological nanopore) can be functionalized with one type of R to allow sensing of one analyte (e.g., a target analyte), or it can be functionalized with at least two different recognition elements (e.g., protein recognition elements), R' and R". In one embodiment, the nanopore is functionalized with at least R' and R", wherein each of the R' and R" specifically binds to a different analyte (e.g., a target analyte), thereby enabling a single nanopore to detect multiple different analytes (e.g., target analytes). In a preferred embodiment, the nanopore is functionalized with at least R' and R", wherein each of the R' and R" specifically binds to a different site (epitope) on the same analyte (e.g., a target analyte). In this way, the binding strength and duration of the analyte-bound state can be increased, as well as the specificity of binding to a given analyte (e.g., a target analyte) relative to other background analytes.

[0152] In some embodiments, R is preferably positioned on a first side (or cis side) of the top of the nanopore via a flexible tether. This flexible tether allows R to move in and out of the pore as described herein.

[0153] On the one hand, recognition element R is directly coupled to nanopore. On the one hand, flexibility (such as allowing R to rotate or bend so that it can move in and out of hole as described herein) can be achieved by a key, and R is attached to nanopore via the key. On the one hand, such flexibility can be achieved by the flexibility in R or in nanopore. In some instances, nanopore can be conjugated to the flexible region of R (such as the flexible N or C end on the outer surface of R, or a flexible ring). Selectively or in combination, R can be coupled to the flexible region of nanopore (such as the flexible N or C end on the outer surface of nanopore, or a flexible ring).

[0154] In one aspect, the modified nanopore is an oligomeric assembly comprising or consisting of monomers of the general formula NLR, wherein N is a monomer of a pore-forming toxin having a maximum inner diameter (e.g., lumen diameter) of 5 nm to 20 nm, L is a flexible linker attached to the wide entrance (e.g., wide cis entrance) of the pore, and R is a recognition element (e.g., a protein recognition element) capable of specifically binding to an analyte (e.g., a target analyte).

[0155] In some embodiments, the nanopore is a monomeric protein. In some cases, the nanopore can be formed by a single β-barrel similar to the structure of outer membrane porins. In some embodiments, the nanopore can be a monomer formed by genetic fusion or chemical conjugation of multiple monomeric protein units.

[0156] In some embodiments, the nanopore may include an oligomeric assembly. In some cases, at least one subunit of the oligomeric assembly includes a subunit of a nanopore coupled to a recognition element. In some cases, at least one subunit may be directly coupled to a recognition element. In some cases, at least one subunit may be indirectly coupled to a recognition element. In some cases, at least one subunit may be coupled to at least one subunit of a nanopore via a connector. In some cases, at least one subunit of a nanopore includes a monomer of a pore-forming toxin. In some cases, the pore-forming toxin may be cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2, a triple α-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof. In some cases, the pore-forming toxin may include one or more mutations. In some cases, the pore-forming toxin is ClyA. In some cases, the ClyA pore-forming toxin may have an S110C mutation.

[0157] In some embodiments, the inner diameter (e.g., lumen diameter) of a nanopore can be at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, or greater than about 20 nm. In some embodiments, the inner diameter (e.g., lumen diameter) of a nanopore can be at most about 20 nm, at most about 19 nm, at most about 18 nm, at most about 17 nm, at most about 16 nm, at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, or less than about 5 nm.

[0158] In some embodiments, the inner diameter (eg, lumen diameter) of the nanopore can be from about 5 nm to about 25 nm. In some embodiments, the inner diameter (e.g., lumen diameter) of the nanopore can be from about 5 nm to about 6 nm, from about 5 nm to about 7 nm, from about 5 nm to about 8 nm, from about 5 nm to about 9 nm, from about 5 nm to about 10 nm, from about 5 nm to about 12 nm, from about 5 nm to about 14 nm, from about 5 nm to about 16 nm, from about 5 nm to about 18 nm, from about 5 nm to about 20 nm, from about 5 nm to about 25 nm, from about 6 nm to about 7 nm, from about 6 nm to about 8 nm, from about 6 nm to about 9 nm, from about 6 nm to about 10 nm, from about 6 nm to about 12 nm, from about 6 nm to about 14 nm, from about 6 nm to about 16 nm, from about 6 nm to about 18 nm, from about 6 nm to about 20 nm, from about 6 nm to about 25 nm, from about 7 nm to about 8 nm, from about 7 nm to about 9 nm, from about 7 nm to about 10 nm, from about 7 nm to about 12 nm, from about 7 nm to about 14 nm, nm, about 8 nm to about 9 nm, about 8 nm to about 10 nm, about 8 nm to about 12 nm, about 8 nm to about 14 nm, about 8 nm to about 16 nm, about 8 nm to about 18 nm, about 8 nm to about 20 nm, about 8 nm to about 25 nm, about 9 nm to about 10 nm, about 9 nm to about 12 nm, about 9 nm to about 14 nm, about 9 nm to about 16 nm, about 9 nm to about 18 nm, about 9 nm to about 20 nm, about 9 nm to about 25 nm, about 10 nm to about 12 nm, about 10 nm to about 14 nm, about 10 nm to about 16 nm, about 10 nm to about 18 nm, about 10 nm to about 20 nm, about 10 nm to about 25 nm, about 12 nm to about 14 nm, about 12 nm to about 16 nm, about 12 nm to about 18 nm, about 12 nm to about 20 nm, about 12 nm to about 25 nm, about 14 nm to about 16 nm, about 14 nm to about 18 nm, nm to about 20 nm, about 14 nm to about 25 nm, about 16 nm to about 18 nm, about 16 nm to about 20 nm, about 16 nm to about 25 nm, about 18 nm to about 20 nm, about 18 nm to about 25 nm, or about 20 nm to about 25 nm.

[0159] In some embodiments, the inner diameter (e.g., lumen diameter) of the nanopore can be about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm.

[0160] In some embodiments, the linker size is variable and may depend on the site of attachment of R, the pore size and / or pore geometry, etc. One skilled in the art can readily select an appropriate linker length and linker geometry. In some embodiments, the appropriate linker length and linker geometry may be such that the linker, in combination with the location of the linker attachment point on the nanopore, provides an appropriate distance for R to the pore entrance.

[0161] In some embodiments, the flexible linker can have any size as long as it allows for functional positioning of R relative to the entrance / opening of the pore. In one embodiment, L has a length of about 2-8 nm, preferably 4-6 nm. It should be understood that the length of L is the shortest distance between the nanopore attachment point and the R attachment point; the remainder of the linker portion can be of virtually any length.

[0162] In some embodiments, the linker has a length of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2.0 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 6.5 nm, at least about 7 nm, at least about 7.5 nm, at least about 8 nm, or greater than about 8 nm. In some embodiments, the linker has a length of at most about 8 nm, at most about 7.5 nm, at most about 7 nm, at most about 6.5 nm, at most about 6 nm, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.5 nm, or less than about 0.5 nm.

[0163] In some embodiments, the linker has a length of about 0.5 nm to about 8 nm. In some embodiments, the linker has a length of about 0.5 nm to about 1.0 nm, about 1.0 nm to about 1.5 nm, about 1.5 nm to about 2.0 nm, about 2 nm to about 2.5 nm, about 2 nm to about 3 nm, about 2 nm to about 3.5 nm, about 2 nm to about 4 nm, about 2 nm to about 4.5 nm, about 2 nm to about 5 nm, about 2 nm to about 5.5 nm, about 2 nm to about 6 nm, about 2 nm to about 7 nm, about 2 nm to about 8 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 3.5 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about 5.5 nm, about 2 nm to about 6 nm, about 2 nm to about 7 nm, about 2 nm to about 8 nm, nm to about 5.5 nm, about 3 nm to about 6 nm, about 3 nm to about 7 nm, about 3 nm to about 8 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 4.5 nm, about 3.5 nm to about 5 nm, about 3.5 nm to about 5.5 nm, about 3.5 nm to about 6 nm, about 3.5 nm to about 7 nm, about 3.5 nm to about 8 nm, about 4 nm to about 4.5 nm, about 4 nm to about 5 nm, about 4 nm to about 5.5 nm, about 4 nm to about 6 nm, about 4 nm to about 7 nm, about 4 nm to about 8 nm, about 4.5 nm to about 5 nm, about 4.5 nm to about 5.5 nm, about 4.5 nm to about 6 nm, about 4.5 nm to about 7 nm, about 4.5 nm to about 8 nm, about 5 nm to about 5.5 nm, about 5 nm to about 6 nm, about 5 nm to about 7 nm, about 5 nm to about 8 nm, nm to about 8 nm, about 6 nm to about 7 nm, about 6 nm to about 8 nm, or about 7 nm to about 8 nm in length.

[0164] In some embodiments, the linker has a length of about 0.5 nm, about 1.0 nm, about 1.5 nm, about 2 nm, about 2.5 nm, about 3 nm, about 3.5 nm, about 4 nm, about 4.5 nm, about 5 nm, about 5.5 nm, about 6 nm, about 6.5 nm, about 7 nm, about 7.5 nm, or about 8 nm.

[0165] In some embodiments, connexon can be made up of many well-known types, including polymers such as PEG, DNA, RNA, LNA, PNA or any combination thereof. In some cases, one or more polymeric molecules can include polyethylene glycol, ethylene, polystyrene, vinyl chloride, polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene, polyamide, silicone-based polymers, PMOXA polymers, polysaccharides, polyacrylamide polymers, polyacrylic acid polymers, polyamines, polyethyleneimine, quaternary ammonium polymers, polyvinyl alcohol polymers, polyether polymers, ethylene oxide polymers, propylene oxide polymers, polyvinyl pyrrolidone polymers, carboxyl polymethylene polymers or any combination thereof. Conjugation can adopt any suitable well-known chemical attachment method, such as relating to those reacted with cysteine (such as maleimide coupling), lysine, click chemistry etc. Conjugation chemistry is preferably located at the end of connexon, but can be partially positioned along the molecule as needed to position R relative to N. The linker can be composed of a single unit (e.g., a single polymer chain that directly couples N to R) or multiple units (e.g., a hybridized oligonucleotide in which R and N are each bound to one of the double strands). R is suitably directly coupled to N, as long as the N attachment point is provided with sufficient length and flexibility to allow R to move in and out of the nanopore. This can be achieved, for example, by attaching R to a flexible loop on N that is present at an appropriate position in the N sequence or is introduced at an appropriate position in the N sequence. Alternatively, R can be engineered with additional sequences (e.g., internal loops or extensions of the N or C termini, in the case of attachment) to create a flexible linker, which is then directly attached to a surface residue on N.

[0166] In one embodiment, L is an oligonucleotide, preferably a duplex made from the complementary strand of DNA or chemically modified RNA (e.g., locked nucleic acid or RNA chemically modified with -F, -OMe at the 2' position to enhance stability). It may comprise a stretch of at least 8, at least 10, preferably at least 14, and more preferably at least 18 nucleotides.

[0167] In some embodiments, the nanopore is (reversibly) functionalized with R via a linker L, preferably wherein L is an oligonucleotide duplex formed by nucleic acid hybridization between a first oligonucleotide, which is conjugated to the nanopore, and a second oligonucleotide, which is conjugated to R, which is complementary to the first oligonucleotide.

[0168] In some embodiments, different orientations can advantageously position R relative to the point of attachment of L on the nanopore and relative to the nanopore entrance (see Figure 14). For example, N and R can be coupled to the same end of the oligonucleotide duplex linker L. Alternatively, N and R can be coupled to the midpoint or distal end of the oligonucleotide duplex linker L.

[0169] In some embodiments, duplex formation and exchange of components attached to the nanopore are suitably achieved by a toehold-mediated strand displacement (TMSD) reaction, which involves a process in which an invading strand displaces an incumbent strand from a gate strand by initiating at an exposed toehold domain. For example, TMSD can be used to exchange strands that form a duplex with the nanopore. For example, for a nanopore comprising N-L1 (where L1 is one strand of the duplex linker), initially duplexed with L2-R1, TMSD can be used to exchange the bound entity to L3-R2 to change the coupled binder R, and thus change the analyte (e.g., target) that the nanopore is capable of detecting.

[0170] In another embodiment, the linker L on the nanopore is first protected so that it is activated only when desired. For example, the nanopore is initially double-stranded with a blank protection chain, which is removed channel by channel on a chip array containing multiple nanopores. For example, by applying a voltage to a selected channel containing a nanopore, the blank protection oligonucleotide chain can be removed from the desired nanopore channel by channel, thereby capturing and electrophoretically stripping the protection chain from the nanopore (Figure 15).

[0171] Nanopore

[0172] In some embodiments, the nanopore suitably has a maximum inner diameter (e.g., lumen diameter) of 5 nm to 20 nm (e.g., 5 nm to 10 nm). The pore can have a wide entrance (e.g., a wide cis entrance) and a narrow exit (e.g., a narrow trans exit). A constriction is typically a narrowing in the channel that passes through the nanopore, which can determine or control the signal obtained when a substrate (e.g., a target substrate) moves relative to the nanopore.

[0173] In some embodiments, the inner diameter (e.g., lumen diameter) can be at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, or greater than about 20 nm. In some embodiments, the inner diameter (e.g., lumen diameter) can be at most about 20 nm, at most about 19 nm, at most about 18 nm, at most about 17 nm, at most about 16 nm, at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, or less than about 5 nm.

[0174] In some embodiments, the inner diameter (eg, lumen diameter) can be from about 5 nm to about 25 nm. In some embodiments, the inner diameter (e.g., lumen diameter) can be from about 5 nm to about 6 nm, from about 5 nm to about 7 nm, from about 5 nm to about 8 nm, from about 5 nm to about 9 nm, from about 5 nm to about 10 nm, from about 5 nm to about 12 nm, from about 5 nm to about 14 nm, from about 5 nm to about 16 nm, from about 5 nm to about 18 nm, from about 5 nm to about 20 nm, from about 5 nm to about 25 nm, from about 6 nm to about 7 nm, from about 6 nm to about 8 nm, from about 6 nm to about 9 nm, from about 6 nm to about 10 nm, from about 6 nm to about 12 nm, from about 6 nm to about 14 nm, from about 6 nm to about 16 nm, from about 6 nm to about 18 nm, from about 6 nm to about 20 nm, from about 6 nm to about 25 nm, nm, about 8 nm to about 9 nm, about 8 nm to about 10 nm, about 8 nm to about 12 nm, about 8 nm to about 14 nm, about 8 nm to about 16 nm, about 8 nm to about 18 nm, about 8 nm to about 20 nm, about 8 nm to about 25 nm, about 9 nm to about 10 nm, about 9 nm to about 12 nm, about 9 nm to about 14 nm, about 9 nm to about 16 nm, about 9 nm to about 18 nm, about 9 nm to about 20 nm, about 9 nm to about 25 nm, about 10 nm to about 12 nm, about 10 nm to about 14 nm, about 10 nm to about 16 nm, about 10 nm to about 18 nm, about 10 nm to about 20 nm, about 10 nm to about 25 nm, about 12 nm to about 14 nm, about 12 nm to about 16 nm, about 12 nm to about 18 nm, about 12 nm to about 20 nm, about 12 nm to about 25 nm, about 14 nm to about 16 nm, about 14 nm to about 18 nm nm, about 14 nm to about 25 nm, about 16 nm to about 18 nm, about 16 nm to about 20 nm, about 16 nm to about 25 nm, about 18 nm to about 20 nm, about 18 nm to about 25 nm, or about 20 nm to about 25 nm.

[0175] In some embodiments, the inner diameter (e.g., lumen diameter) can be about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm.

[0176] In some embodiments, the nanopore (e.g., a biological nanopore) can be a pore-forming toxin. Pathogenic pore-forming toxins (PFTs) are well-characterized virulence factors. They belong to an ancient and widely diverse family of proteins. PFTs are found in both Gram-negative and Gram-positive bacterial branches, with members in human, insect, and plant pathogens. PFTs are divided into two families based on the secondary structural properties of the membrane-penetrating channel: α-PFTs form α-helical pores, while β-PFTs produce β-barrel pores.

[0177] In one aspect, the nanopore is a member of the cytolysin A (ClyA) toxin family or a mutant thereof that is site-specifically functionalized with a recognition element (e.g., a protein recognition element). ClyA-like toxins include PDB IDs: 1QOY (soluble ClyA), 2WCD (protomer ClyA), 6EK7 (soluble YaxA), 6EL1 (protomer YaxA, protomer YaxB), 6EK4 (soluble PaxB), 4K1P (soluble NheA), 5KUC (Cry6AA), 2NRJ (Hb1-B), see Bräuning et al. (which is incorporated herein by reference in its entirety) (Toxins (Basel). 2018 Sep; 10(9): 343, which is incorporated herein by reference in its entirety) and references cited therein (which are incorporated herein by reference in their entirety).

[0178] In some embodiments, the nanopore is ClyA, preferably a mutant ClyA, functionalized in the region comprising amino acids F101 to S110 as found in GenBank sequence AJ313032.1 with R. ClyA is an α-helical PFT with a relatively large diameter (3-6 nm, depending on the entrance).

[0179] In one aspect, the modified nanopore is based on the ClyA variant ClyA-AS, which has the sequence

[0180] MTGIFAEQTVEVVKSAIETADGALDLYNKYLDQVIPWKTFDETIKELSRFKQEYSQEASVLVGDIKVLLMDSQDKYFEATQTVYEWAGVVTQLLSAYIQLFDGYNEKKASAQKDILIRILDDGVKKLNEAQKSLLTSSQSFNNASGKLLALDSQLT NDFSEKSSYYQSQVDRIRKEAYAGAAAGIVAGPFGLIISYSIAAGVVEGKLIPELNNRLKTVQNFFTSLSATVKQANKDIDAAKLKLATEIAAIGEIKTETETTRFYVDYDDLMLSLLKGAAKKMINTSNEYQQRHGRKTLFEVPDVGSSYHHHHH. This variant contains the following mutations relative to the wild-type ClyA protein: C87A, L99Q, E103G, F166Y, I203V, C285S, K294R. To allow R functionalization, an additional mutation S110C was introduced.

[0181] In some embodiments, suitable amino acid facing water near the entrance of nanopore (for example, cis entrance) can be modified by structural model.In some instances, other useful areas for ClyA functionalization include residue D267-S272, including the residue exposed in the spiral cavity of A111-Q139, and the spiral including D71-D64.Preferably, the limiting examples of the residue for modification include D114, E129, K132, S133, V136, Q139, E78, D71 and D64.The ClyA residues in the outside of ClyA nanopore to be modified include those of the end, F101 to K66, and D267-K230 from S272 until protein.

[0182] In one aspect, ClyA is suitably functionalized at position 110 by using the mutation S110C.

[0183] In one aspect, the nanopore is a modified member of the YaxAB family.The Yersinia YaxAB system represents a family of binary α-PFTs with interspecies homologs in human, insect and plant pathogens.

[0184] In some embodiments, the nanopore is Pleurotus ostreatus) Pleurolysin (PlyAB; PDB ID 4V2T) or its mutants that realize site-specific functionalization with recognition elements (e.g., protein recognition elements). PlyAB consists of two different components: Pleurolysin A (PlyA, 16 kDa) as a scaffold to recruit a second component, Pleurolysin B (PlyB, 54 kDa), across the lipid bilayer. Cryogenic electron microscopy reveals a nanopore with an entrance (e.g., cis-entrance) of approximately 10.5 nm, an entrance (e.g., trans-entrance) of approximately 7.2 nm, and a constriction of approximately 5.5 nm in diameter. Based on the residue numbering of AJ313032.1, the appropriate regions for attaching R include S49 to D71, R100 to S89, G181 to G205, D298 to E316, and V329 to P336.

[0185] In some embodiments, the concept of analyte-dependent kinetics of the movement of the recognition element R into / out of the lumen of the nanopore is also advantageously applied to de novo fabricated nanopores, such as DNA-based membrane nanopores with tunable pore shapes and lumen widths of up to tens of nanometers (Xingh et al., 2001). et al . 2022, Nature Nanotechnology vol. 17, pg. 708-713, which is incorporated herein by reference in its entirety), or DNA origami nanopores with an inner diameter as large as 30 nm (Fragrasso et al. ACS Nano 2021, 15, 8, 12768-12779, which is incorporated herein by reference in its entirety). In addition, the nanopore is a de novo nanopore based on a de novo α-helix or β-barrel transmembrane region (see, for example, Shimizu et al. 2022, Nature Nanotechnology volume 17, pg. 67-75, which is incorporated herein by reference in its entirety; or Scott et al. 2021, Nature Chemistry volume, 13, pg. 643-650, which is incorporated herein by reference in its entirety; or Vorobieva et al. 2021, Science, Vol 371, Issue 6531, which is incorporated herein by reference in its entirety).

[0186] Thus, the present invention also provides methods and sensor systems comprising an artificial non-solid-state nanopore functionalized with a 5 kDa to 50 kDa recognition element (e.g., a protein recognition element) R, which is capable of specifically binding to an analyte (e.g., a target analyte), and wherein R dynamically moves in and out of the interior of the nanopore to cause transient current blockade events, and wherein binding of R to an analyte (e.g., a target analyte, e.g., a target protein) modulates its dynamic movement, thereby causing a change in the frequency and / or amplitude of the current blockade events. Wherein a change in the frequency and / or amplitude of the current blockade events indicates the presence of an analyte (e.g., a target analyte) in a sample. In one embodiment, it comprises a nanobody-functionalized DNA-based membrane nanopore or a DNA origami nanopore.

[0187] In some embodiments, a nanopore (e.g., an artificial or non-solid-state nanopore) has a diameter of at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, at least about 10 kDa, at least about 11 kDa, at least about 12 kDa, at least about 13 kDa, at least about 14 kDa, at least about 15 kDa, at least about 16 kDa, at least about 17 kDa, at least about 18 kDa, at least about 19 kDa, at least about 20 kDa, at least about 21 kDa, at least about 22 kDa, at least about 23 kDa, at least about 24 kDa, at least about 25 kDa, at least about 26 kDa, at least about 27 kDa, at least about 28 kDa, at least about 29 kDa, at least about 30 kDa, at least about 31 kDa, at least about 32 kDa, at least about 33 kDa, at least about 34 kDa kDa, at least about 35 kDa, at least about 36 kDa, at least about 37 kDa, at least about 38 kDa, at least about 39 kDa, at least about 40 kDa, at least about 41 kDa, at least about 42 kDa, at least about 43 kDa, at least about 44 kDa, at least about 45 kDa, at least about 46 kDa, at least about 47 kDa, at least about 48 kDa, at least about 49 kDa, at least about 50 kDa, or more than about 50 kDa, which recognition element is capable of specifically binding to an analyte (e.g., an analyte of interest).In some embodiments, the nanopore (e.g., an artificial or non-solid-state nanopore) is at most about 50 kDa, at most about 49 kDa, at most about 48 kDa, at most about 47 kDa, at most about 46 kDa, at most about 45 kDa, at most about 44 kDa, at most about 43 kDa, at most about 42 kDa, at most about 41 kDa, at most about 40 kDa, at most about 39 kDa, at most about 38 kDa, at most about 37 kDa, at most about 36 kDa, at most about 35 kDa, at most about 34 kDa, at most about 33 kDa, at most about 32 kDa, at most about 31 kDa, at most about 30 kDa, at most about 29 kDa, at most about 28 kDa, at most about 27 kDa, at most about 26 kDa, at most about 25 kDa, at most about 24 kDa, at most about 23 kDa, at most about 22

[0015] The present invention relates to a method for producing an analyte that is conjugated to a recognition element of at most about 10 kDa, at most about 21 kDa, at most about 20 kDa, at most about 19 kDa, at most about 18 kDa, at most about 17 kDa, at most about 16 kDa, at most about 15 kDa, at most about 14 kDa, at most about 13 kDa, at most about 12 kDa, at most about 11 kDa, at most about 10 kDa, at most about 9 kDa, at most about 8 kDa, at most about 7 kDa, at most about 6 kDa, at most about 5 kDa, at most about 4 kDa, at most about 3 kDa, at most about 2 kDa, at most about 1 kDa, or less than about 1 kDa that is capable of specifically binding to the analyte.

[0188] In some embodiments, a nanopore (eg, an artificial or non-solid-state nanopore) is coupled to a recognition element of about 5 kDa to about 60 kDa that is capable of specifically binding to an analyte. In some embodiments, the nanopore (e.g., an artificial or non-solid-state nanopore) is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 25 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 35 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 45 kDa, about 5 kDa to about 50 kDa, about 5 kDa to about 55 kDa, about 5 kDa to about 60 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 35 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 45 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 55 kDa, about 10 kDa to about 60 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 35 kDa, about 15 kDa to about 40 kDa, about 15 kDa to about 45 kDa, about 15 kDa to about 50 kDa, about 15 kDa to about 55 kDa, about 15 kDa to about 60 kDa, about 20 kDa to about 25 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 35 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 45 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 55 kDa, about 20 kDa to about 60 kDa, about 25 kDa to about 30 kDa, about 25 kDa to about 35 kDa, about 25 kDa to about 40 kDa, about 25 kDa to about 45 kDa, about 25 kDa to about 50 kDa, about 25 kDa to about 55 kDa, about 25 kDa to about 60 kDa, about 30 kDa to about 35 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 45 kDa, about 30 kDa to about 50 kDa, about 30 kDa to about 55 kDa, about 30 kDa to about 60 kDa, about 35 kDa to about 40 kDa, about 35 kDa to about 45 kDa, about 35 kDa to about 50 kDa, about 35 kDa to about 55 kDa, about 35 kDa to about 60 kDa, about 40 kDa to about 45 kDa, about 40The invention relates to a method for preparing an analyte that is coupled to a recognition element of about 50 kDa to about 50 kDa, about 40 kDa to about 55 kDa, about 40 kDa to about 60 kDa, about 45 kDa to about 50 kDa, about 45 kDa to about 55 kDa, about 45 kDa to about 60 kDa, about 50 kDa to about 55 kDa, about 50 kDa to about 60 kDa, or about 55 kDa to about 60 kDa, that is capable of specifically binding to the analyte.

[0189] In some embodiments, a nanopore (e.g., an artificial or non-solid-state nanopore) is about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 The analyte is coupled to a recognition element of about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa or about 50 kDa, which recognition element is capable of specifically binding to the analyte.

[0190] Analytes

[0191] In some embodiments, the methods or sensor systems of the present invention can be easily designed to detect any analyte of interest (e.g., a target analyte) or multiple analytes of interest (e.g., multiple target analytes). The present invention is advantageously used to detect label-free analytes (e.g., target analytes).

[0192] In one embodiment, the present invention provides a method for detecting an analyte / antigen (e.g., an analyte / antigen of interest) selected from a protein, a polypeptide, a protein assembly, a protein / DNA assembly, a polysaccharide, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a dendrimer, a polymer, or any combination thereof.

[0193] In some cases, the analyte (e.g., target analyte) is a protein. In some cases, the protein (e.g., target protein) is selected from the group consisting of a folded / native protein, a clinically relevant protein, a protein biomarker, a pathogenic protein, and a cell surface protein.

[0194] The present invention is particularly suitable for detecting protein targets covering a very wide range of masses and sizes. In one aspect, the present invention detects proteins (e.g., protein targets) covering a very wide range of masses and sizes, from very small proteins and peptides to very large proteins and complexes. Because the recognition element R is dynamic, moving in and out of the nanopore, the systems and methods are sensitive to the binding of R to very small analytes and very large analytes that cannot fit inside the nanopore.

[0195] In some embodiments, the present invention is particularly suitable for detecting analytes or analytes (eg, protein analytes or protein analyte complexes) greater than 50 Da, preferably greater than 100 Da, and most preferably greater than 150 Da.

[0196] In some embodiments, the analyte or analyte complex is at least about 25 kDa, at least about 50 kDa, at least about 60 kDa, at least about 70 kDa, at least about 80 kDa, at least about 90 kDa, at least about 100 kDa, at least about 110 kDa, at least about 120 kDa, at least about 130 kDa, at least about 140 kDa, at least about 150 kDa, at least about 175 kDa, at least about 200 kDa, at least about 250 kDa, at least about 300 kDa, at least about 400 kDa, at least about 500 kDa, at least about 750 kDa, at least about 1,000 kDa, or greater than about 1,000 kDa. In some embodiments, the analyte or analyte complex is at most about 1,000 kDa, at most about 750 kDa, at most about 500 kDa, at most about 400 kDa, at most about 300 kDa, at most about 250 kDa, at most about 200 kDa, at most about 175 kDa, at most about 150 kDa, at most about 140 kDa, at most about 130 kDa, at most about 120 kDa, at most about 110 kDa, at most about 100 kDa, at most about 90 kDa, at most about 80 kDa, at most about 70 kDa, at most about 60 kDa, at most about 50 kDa, at most about 25 kDa, or less than about 25 kDa.

[0197] In some embodiments, the analyte or analyte complex is about 50 kDa to about 500 kDa. In some embodiments, the analyte or analyte complex is about 50 kDa to about 60 kDa, about 50 kDa to about 70 kDa, about 50 kDa to about 80 kDa, about 50 kDa to about 90 kDa, about 50 kDa to about 100 kDa, about 50 kDa to about 125 kDa, about 50 kDa to about 150 kDa, about 50 kDa to about 175 kDa, about 50 kDa to about 200 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 500 kDa, about 60 kDa to about 70 kDa, about 60 kDa to about 80 kDa, about 60 kDa to about 90 kDa, about 60 kDa to about 100 kDa, about 60 kDa to about 125 kDa, about 60 kDa to about 150 kDa, about 60 kDa to about 175 kDa, about 60 kDa to about 200 kDa, about 60 kDa to about 250 kDa, kDa, about 60 kDa to about 500 kDa, about 70 kDa to about 80 kDa, about 70 kDa to about 90 kDa, about 70 kDa to about 100 kDa, about 70 kDa to about 125 kDa, about 70 kDa to about 150 kDa, about 70 kDa to about 175 kDa, about 70 kDa to about 200 kDa, about 70 kDa to about 250 kDa, about 70 kDa to about 500 kDa, about 80 kDa to about 90 kDa, about 80 kDa to about 100 kDa, about 80 kDa to about 125 kDa, about 80 kDa to about 150 kDa, about 80 kDa to about 175 kDa, about 80 kDa to about 200 kDa, about 80 kDa to about 250 kDa, about 80 kDa to about 500 kDa, about 90 kDa to about 100 kDa, about 90 kDa to about 125 kDa, about 90 kDa to about 150 kDa, about 90 kDa to about 175 kDa, about 90 kDa to about 200 kDa, about 90 kDa to about 250 kDa, about 90 kDa to about 500 kDa, about 100 kDa to about 125 kDa, about 100 kDa to about 150 kDa, about 100 kDa to about 175 kDa, about 100 kDa to about 200 kDa, about 100 kDa to about 250 kDa, about 100 kDa to about 500 kDa, about 125 kDa to about 150 kDa, about 125 kDa to about 175 kDa, about 125 kDa to about 200 kDa, about 125 kDa to about 250 kDa, about 125kDa to about 500 kDa, about 150 kDa to about 175 kDa, about 150 kDa to about 200 kDa, about 150 kDa to about 250 kDa, about 150 kDa to about 500 kDa, about 175 kDa to about 200 kDa, about 175 kDa to about 250 kDa, about 175 kDa to about 500 kDa, about 200 kDa to about 250 kDa, about 200 kDa to about 500 kDa, or about 250 kDa to about 500 kDa.

[0198] In some embodiments, the analyte or analyte complex is about 25 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 175 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 750 kDa, or about 1,000 kDa.

[0199] The methods and systems are capable of detecting analytes that are too large to fit within even very large nanopores. In some embodiments, the present invention is capable of broadly binding and detecting a wider variety of biologically relevant biomarkers, such as large proteins, protein complexes, including intact viruses, bacteria, and cells.

[0200] In some embodiments, the disclosed methods are used to detect or characterize modifications in an analyte (e.g., an analyte, e.g., a target protein, e.g., an unlabeled target protein). In one aspect, one or more amino acids / derivatives / analogs in the target protein are post-translationally modified. Any one or more post-translational modifications may be present in a protein (e.g., a target protein).

[0201] In some embodiments, post-translational modifications include modification with hydrophobic groups, modification with cofactors, addition of chemical groups, glycosylation (non-enzymatic attachment of sugars), biotinylation, and PEGylation. Post-translational modifications can also be non-natural, such as chemical modifications introduced in the laboratory for biotechnology or biomedical purposes. This allows monitoring the level of post-translational modifications of laboratory-derived peptides, polypeptides, or proteins compared to their natural counterparts. Thus, the methods disclosed herein can be used to detect the presence, absence, degree, or number of post-translational modified positions in a polypeptide.

[0202] In some embodiments, post-translational modifications with hydrophobic groups can include myristoylation, palmitoylation, prenylation, or pentylosylation, which are attachments of isoprenoid groups; farnesylation, which is attachment of farnesol groups; geranylgeranilation, which is attachment of geranylgeraniol groups; and glycosylphosphatidylinositolization and glycosylphosphatidylinositol (GPI) anchor formation via an amide bond. Examples of post-translational modifications with cofactors include lipoylation, which is the attachment of a lipoic acid (Cs) functional group; flavination, which is the attachment of a flavin moiety (e.g., flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)); attachment of heme C, for example, via a thioether bond to cysteine; phosphopantetheinylation, which is the attachment of a 4'-phosphopantetheinyl group; retinylidene Schiff base formation; or any combination thereof.

[0203] In some embodiments, post-translational modifications by the addition of chemical groups can include acylation, such as O-acylation (ester), N-acylation (amide), or S-acylation (thioester); acetylation, which is the attachment of an acetyl group, such as to the N-terminus or to lysine; formylation; alkylation, which is the addition of an alkyl group, such as a methyl or ethyl group; methylation, which is the addition of a methyl group, such as to lysine or arginine; amidation; butyrylation; γ-carboxylation; glycosylation, which is the enzymatic attachment of a glycosyl group, such as to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan; polysialylation, which is the attachment of polysialic acid; malonylation; hydroxylation; iodination; bromination; citrullination; nucleobase; Nylide addition is the attachment of any nucleotide (e.g., any of those discussed above), ADP ribosylation; oxidation; phosphorylation is the attachment of a phosphate group, such as to serine, threonine, or tyrosine (O-linked) or histidine (N-linked); adenylylation is the attachment of an adenylyl moiety, such as to tyrosine (O-linked) or histidine or lysine (N-linked); propionylation; pyroglutamate formation; S-glutarylsulfoylation; Sumoylation; S-nitrosylation; succinylation is the attachment of a succinyl group, such as to lysine; selenoylation is the incorporation of selenium; ubiquitination is the addition of a ubiquitin subunit (N-linked); or any combination thereof.

[0204] Nanopore sensor system

[0205] Further embodiments relate to a nanopore system comprising a fluid-filled compartment separated by a membrane into a first chamber and a second chamber, electrodes capable of applying an electric potential across the membrane, one or more functionalized nanopores according to the invention inserted into the membrane.

[0206] In one aspect, the present invention provides a nanopore system and means for providing a voltage difference between a first side and a second side of a membrane, the nanopore system comprising a membrane having a modified nanopore therein, which separates a fluid chamber into a first side and a second side, wherein the modified nanopore is a biological or de novo formed non-solid-state nanopore functionalized with a recognition element of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, more preferably 12-15 kDa, capable of specifically binding to an analyte (e.g., a target analyte).

[0207] In some embodiments, the biological or de novo formed non-solid-state nanopore is coupled to a recognition element of at least about 5 kDa, at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, at least about 25 kDa, at least about 30 kDa, at least about 35 kDa, at least about 40 kDa, at least about 45 kDa, at least about 50 kDa, or greater than about 50 kDa, which is capable of specifically binding to an analyte. In some embodiments, the biological or de novo formed non-solid-state nanopore is coupled to a recognition element of at most about 50 kDa, at most about 45 kDa, at most about 40 kDa, at most about 35 kDa, at most about 30 kDa, at most about 25 kDa, at most about 20 kDa, at most about 15 kDa, at most about 10 kDa, at most about 5 kDa, or less than about 5 kDa, which is capable of specifically binding to an analyte.

[0208] In some embodiments, a biological or de novo formed non-solid-state nanopore is coupled to a recognition element of about 5 kDa to about 60 kDa that is capable of specifically binding to an analyte. In some embodiments, the biological or de novo formed non-solid-state nanopore is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 25 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 35 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 45 kDa, about 5 kDa to about 50 kDa, about 5 kDa to about 55 kDa, about 5 kDa to about 60 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 35 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 45 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 55 kDa, about 10 kDa to about 60 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 35 kDa, about 15 kDa to about 40 kDa, about 15 kDa to about 45 kDa, about 15 kDa to about 50 kDa, about 15 kDa to about 55 kDa, about 15 kDa to about 60 kDa, about 20 kDa to about 25 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 35 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 45 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 55 kDa, about 20 kDa to about 60 kDa, about 25 kDa to about 30 kDa, about 25 kDa to about 35 kDa, about 25 kDa to about 40 kDa, about 25 kDa to about 45 kDa, about 25 kDa to about 50 kDa, about 25 kDa to about 55 kDa, about 25 kDa to about 60 kDa, about 30 kDa to about 35 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 45 kDa, about 30 kDa to about 50 kDa, about 30 kDa to about 55 kDa, about 30 kDa to about 60 kDa, about 35 kDa to about 40 kDa, about 35 kDa to about 45 kDa, about 35 kDa to about 50 kDa, about 35 kDa to about 55 kDa, about 35 kDa to about 60 kDa, about 40 kDa to about 45 kDa, about 40 kDa to about 50 ...kDa to about 55 kDa, about 40 kDa to about 60 kDa, about 45 kDa to about 50 kDa, about 45 kDa to about 55 kDa, about 45 kDa to about 60 kDa, about 50 kDa to about 55 kDa, about 50 kDa to about 60 kDa, or about 55 kDa to about 60 kDa, that is coupled to a recognition element that is capable of specifically binding to the analyte.

[0209] In some embodiments, the biological or de novo formed non-solid-state nanopore is coupled to a recognition element of about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, or about 50 kDa that is capable of specifically binding to an analyte.

[0210] In some embodiments, the term "membrane" is used herein with its conventional meaning to refer to a thin film-like structure that separates the chamber of a system into a first side (e.g., first compartment) or cis side (or cis compartment) and a second side (e.g., second compartment) or trans side (or trans compartment) of a fluid chamber. The membrane separating the first side (or cis side) and the second side (or trans side) comprises at least one R functionalized nanopore. Membrane can generally be divided into synthetic membranes and biofilms. Any membrane can be used according to the present invention. Multiple nanopores can be present in a membrane.

[0211] In some embodiments, suitable membranes are well known in the art. In some cases, the membrane is an amphiphilic layer. An amphiphilic layer is a layer formed by amphiphilic molecules (e.g., phospholipids) having at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles that form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450, which is incorporated herein by reference in its entirety).

[0212] In some embodiments, the nanopore system comprises a first side of a fluid chamber or a cis chamber in fluid communication with a second side of a fluid chamber or a trans chamber, the first side of the fluid chamber or the cis chamber comprising a first conductive liquid medium, and the second side of the fluid chamber or the trans chamber comprising a second conductive liquid medium. The conductive liquid medium in the chambers of the nanopore system can have an ion content within a wide range well known in the art, typically ranging from 0.05 M to >3 M. In some embodiments, the conductive liquid medium can have an ion content of at least about 0.01 M, at least about 0.05 M, at least about 0.1 M, at least about 0.5 M, at least about 1.0 M, at least about 1.5 M, at least about 2.0 M, at least about 2.5 M, at least about 3.0 M, at least about 3.5 M, at least about 4.0 M, at least about 4.5 M, at least about 5.0 M, or greater than about 5.0 M. In some embodiments, the conductive liquid medium can have an ion content of at most about 5.0 M, at most about 4.5 M, at most about 4.0 M, at most about 3.5 M, at most about 3.0 M, at most about 2.5 M, at most about 2.0 M, at most about 1.5 M, at most about 1.0 M, at most about 0.5 M, at most about 0.1 M, at most about 0.05 M, at most about 0.01 M, or less than about 0.01 M.

[0213] In some embodiments, the conductive liquid medium can have an ion content of about 0.01 M to about 5 M. In some embodiments, the conductive liquid medium may have a range of from about 0.01 M to about 0.05 M, from about 0.01 M to about 0.1 M, from about 0.01 M to about 0.5 M, from about 0.01 M to about 1 M, from about 0.01 M to about 1.5 M, from about 0.01 M to about 2 M, from about 0.01 M to about 2.5 M, from about 0.01 M to about 3 M, from about 0.01 M to about 3.5 M, from about 0.01 M to about 4 M, from about 0.01 M to about 5 M, from about 0.05 M to about 0.1 M, from about 0.05 M to about 0.5 M, from about 0.05 M to about 1 M, from about 0.05 M to about 1.5 M, from about 0.05 M to about 2 M, from about 0.05 M to about 2.5 M, M to about 0.5 M, about 0.1 M to about 1 M, about 0.1 M to about 1.5 M, about 0.1 M to about 2 M, about 0.1 M to about 2.5 M, about 0.1 M to about 3 M, about 0.1 M to about 3.5 M, about 0.1 M to about 4 M, about 0.1 M to about 5 M, about 0.5 M to about 1 M, about 0.5 M to about 1.5 M, about 0.5 M to about 2 M, about 0.5 M to about 2.5 M, about 0.5 M to about 3 M, about 0.5 M to about 3.5 M, about 0.5 M to about 4 M, about 0.5 M to about 5 M, about 1 M to about 1.5 M, about 1 M to about 2 M, about 1 M to about 2.5 M, about 1 M to about 3 M, about 1 M to about 3.5 M, about 1 M to about 4 M, about 1 M to about 5 M, about 1.5 M to about 2 M, about 1.5 M to about 2.5 M, about 1.5 M to about 3 The invention also provides an ionic content of about 1.5 M to about 3.5 M, about 1.5 M to about 4 M, about 1.5 M to about 5 M, about 2 M to about 2.5 M, about 2 M to about 3 M, about 2 M to about 3.5 M, about 2 M to about 4 M, about 2 M to about 5 M, about 2.5 M to about 3 M, about 2.5 M to about 3.5 M, about 2.5 M to about 4 M, about 2.5 M to about 5 M, about 3 M to about 3.5 M, about 3 M to about 4 M, about 3 M to about 5 M, about 3 M to about 3.5 M, about 3 M to about 4 M, about 3 M to about 5 M, or about 4 M to about 5 M.

[0214] In some embodiments, the conductive liquid medium can have an ion content of about 0.01 M, about 0.05 M, about 0.1 M, about 0.5 M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M, about 3.0 M, about 3.5 M, about 4.0 M, about 4.5 M, or about 5.0 M.

[0215] A wide range of salts can be used, such as NaCl and KCl. Suitable solutions include 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. The first and second sides of the fluid chamber can be symmetrical or asymmetrical. The cis and trans chambers can be symmetrical or asymmetrical. A wide range of pH and temperature conditions can be used, for example, in the range of pH 5-9, 10-50°C, preferably at about 37°C. In some embodiments, the pH of the solution can be at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10, or greater than about 10. In some embodiments, the pH of the solution can be at most about 10, at most about 9.5, at most about 9, at most about 8.5, at most about 8, at most about 7.5, at most about 7, at most about 6.5, at most about 6, at most about 5.5, at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, or less than about 3.

[0216] In some embodiments, the pH of the solution can be from about 3 to about 10. In some embodiments, the pH of the solution can be from about 3 to about 4, from about 3 to about 5, from about 3 to about 5.5, from about 3 to about 6, from about 3 to about 6.5, from about 3 to about 7, from about 3 to about 7.5, from about 3 to about 8, from about 3 to about 8.5, from about 3 to about 9, from about 3 to about 10, from about 4 to about 5, from about 4 to about 5.5, from about 4 to about 6, from about 4 to about 6.5, from about 4 to about 7, from about 4 to about 7.5, from about 4 to about 8, from about 4 to about 8.5, from about 4 to about 9, from about 4 to about 10, from about 5 to about 5.5, from about 5 to about 6, from about 5 to about 6.5, from about 5 to about 7, from about 5 to about 7.5, from about 5 to about 8, from about 5 to about 8.5, 7.5, about 7 to about 8, about 7 to about 8.5, about 7 to about 9, about 7 to about 10, about 7.5 to about 8, about 5.5 to about 8.5, about 5.5 to about 9, about 5.5 to about 10, about 6 to about 6.5, about 6 to about 7, about 6 to about 7.5, about 6 to about 8, about 6 to about 8.5, about 6 to about 9, about 6 to about 10, about 6.5 to about 7, about 6.5 to about 7.5, about 6.5 to about 8, about 6.5 to about 8.5, about 6.5 to about 9, about 6.5 to about 10, about 7 to about 7.5, about 7 to about 8, about 7 to about 8.5, about 7 to about 9, about 7 to about 10, about 7.5 to about 8, about 7.5 to about 8.5, about 7.5 to about 9, about 7.5 to about 10, about 8 to about 8.5, about 8 to about 9, about 8 to about 10, about 8.5 to about 9, about 8.5 to about 10, or about 9 to about 10.

[0217] In some embodiments, the pH of the solution can be about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the temperature of the solution can be at least about 5°C, at least about 10°C, at least about 15°C, at least about 20°C, at least about 25°C, at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, at least about 65°C, at least about 70°C, at least about 75°C, or greater than about 75°C. In some embodiments, the temperature of the solution can be at most about 75°C, at most about 70°C, at most about 65°C, at most about 60°C, at most about 55°C, at most about 50°C, at most about 45°C, at most about 40°C, at most about 35°C, at most about 30°C, at most about 25°C, at most about 20°C, at most about 15°C, at most about 10°C, at most about 5°C, or less than about 5°C.

[0218] In some embodiments, the temperature of the solution can be from about 5°C to about 70°C. In some embodiments, the temperature of the solution can be from about 5°C to about 10°C, from about 5°C to about 15°C, from about 5°C to about 20°C, from about 5°C to about 25°C, from about 5°C to about 30°C, from about 5°C to about 35°C, from about 5°C to about 40°C, from about 5°C to about 45°C, from about 5°C to about 50°C, from about 5°C to about 60°C, from about 5°C to about 70°C, from about 10°C to about 15°C, from about 10°C to about 20°C, from about 10°C to about 25°C, from about 10°C to about 30°C, from about 10 ... 10°C to about 40°C, about 10°C to about 45°C, about 10°C to about 50°C, about 10°C to about 60°C, about 10°C to about 70°C, about 15°C to about 20°C, about 15°C to about 25°C, about 15°C to about 30°C, about 15°C to about 35°C, about 15°C to about 40°C, about 15°C to about 45°C, about 15°C to about 50°C, about 15°C to about 60°C, about 15°C to about 70°C, about 20°C to about 25°C, about 20°C to about 30°C, about 20°C to about 35 ℃, about 20℃ to about 40℃, about 20℃ to about 45℃, about 20℃ to about 50℃, about 20℃ to about 60℃, about 20℃ to about 70℃, about 25℃ to about 30℃, about 25℃ to about 35℃, about 25℃ to about 40℃, about 25℃ to about 45℃, about 25℃ to about 50℃, about 25℃ to about 60℃, about 25℃ to about 70℃, about 30℃ to about 35℃, about 30℃ to about 40℃, about 30℃ to about 45℃, about 30℃ to about 50℃, about 30℃ to about About 60°C, about 30°C to about 70°C, about 35°C to about 40°C, about 35°C to about 45°C, about 35°C to about 50°C, about 35°C to about 60°C, about 35°C to about 70°C, about 40°C to about 45°C, about 40°C to about 50°C, about 40°C to about 60°C, about 40°C to about 70°C, about 45°C to about 50°C, about 45°C to about 60°C, about 45°C to about 70°C, about 50°C to about 60°C, about 50°C to about 70°C, or about 60°C to about 70°C.

[0219] In some embodiments, the temperature of the solution can be about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or greater than about 75°C.

[0220] In some embodiments, the first side or cis chamber comprises a crowding agent or blocking agent that reduces unwanted non-specific protein adsorption. In one embodiment, the blocking agent is BSA.

[0221] In some embodiments, the system can include circuits that can both apply voltage and measure current. Alternatively, it can include one circuit to apply a voltage difference and another circuit to measure current. It can also generate a voltage difference by placing an asymmetric salt across the membrane. For example, one of the chambers may contain a solution of high ionic strength.

[0222] In some embodiments, mechanisms for detecting current flow between a first side (e.g., cis side) and a second side (e.g., trans side) are described in WO 00 / 79257, patent application numbers 6,46,594, 6,673, 6,673,615, 6,627,067, 6,464,842, 6,362,002, 6,267,872, 6,015,714, 6,428,959, 6,617,113, and 5,795,782, and U.S. publication numbers 2004 / 0121525, 2003 / 0104428, and 2003 / 0104428. These can include electrodes directly associated with channels or pores at or near the porous opening, electrodes placed within the first and second sides (or cis chambers and trans chambers), and insulated glass microelectrodes. The electrodes can detect the difference in ionic current around the two chambers or the tunneling current around the porous opening. In another configuration, the transport characteristic is the electron flow around the diameter of the pore, which can be monitored by electrodes placed adjacent to or in contact with the circumference of the nanopore. The electrodes can be connected to an Axopatch 200B amplifier to amplify the signal.

[0223] It should be understood that the acquisition system described herein is not limited and other systems for acquiring or measuring nanopore signals can be used. Alternative electrical schemes can also be used, for example, on an array chip platform, to achieve an equivalent voltage drop across the nanopore and / or membrane.

[0224] In some embodiments, the sensor system is advantageously integrated into a portable device that includes multiple sensor systems. For example, it is included in a point-of-care diagnostic medical device, which is an in vitro diagnostic used by healthcare professionals to quickly obtain results near or at the patient's site. These products can be used, for example, to quickly determine the markers responsible for a disease in a doctor's office or clinic.

[0225] Arrays and Packages

[0226] The present disclosure provides an array comprising a plurality of sensor systems according to the present invention. Preferably, the array comprises a plurality of discrete reservoirs, each reservoir comprising a nanopore modified with one or more different R-elements to allow detection of a different analyte.

[0227] In some embodiments, the array comprises nanopores pre-modified with L moieties, thereby allowing for end-user-defined functionalization with one or more selected recognition elements (e.g., protein recognition elements R). For example, the L moieties of the pre-modified pores are selected to allow for the formation of double-stranded nucleic acids between a selected oligonucleotide-conjugated nanobody or affimer or affinity and an oligonucleotide-conjugated porin. In one embodiment, the system of the invention comprises an array of pre-modified pores, all of which have the same linker L oligonucleotide sequence, to which R binding partners (comprising a single species of R or a mixture of different R species) can be coupled via appropriate complementary sequences to form duplexes. In alternative embodiments, the pre-modified nanopore array comprises different L moieties specific for a group of complementary R sequences.

[0228] In one aspect, L consists of a native strand and a complementary protective strand, allowing R to attach to the pre-modified nanopore via toehold-mediated strand displacement (TMSD).

[0229] Methods and kits for preparing such arrays are also provided.

[0230] Also provided herein are uses of systems or analytical devices according to the present invention for single molecule sensing assays, preferably for sensing the presence or concentration of one or more analytes (e.g., target analytes) in complex (clinically relevant) samples. In some embodiments, the present invention provides uses of methods, nanopore or sensor systems, arrays or kits for single protein detection, preferably in combination with high throughput analysis.

[0231] In one aspect, the present disclosure provides an array comprising a plurality of nanopore systems according to any of the preceding embodiments. In some embodiments, the array comprises a plurality of discrete reservoirs. In some cases, one or more of the plurality of nanopore systems comprises nanopores modified with different recognition elements to allow detection of different analytes.

[0232] In one aspect, the present disclosure provides a kit for preparing a system according to any of the foregoing embodiments. In some embodiments, the kit comprises a nanopore pre-modified with a linker. In some cases, the linker is part of a double-stranded DNA complex consisting of an original strand and a complementary protection strand.

[0233] In one aspect, the present disclosure provides for use of a method, nanopore, nanopore sensor system, array, or kit according to any of the preceding embodiments for single protein detection. In some embodiments, single protein detection can be combined with high-throughput analysis. In some embodiments, the sensor system is integrated into a portable device comprising multiple sensor systems.

[0234] Computer system

[0235] The present disclosure provides a computer system programmed to implement a method of determining one or more characteristics of an analyte. Figure 16 A computer system 1601 is shown that is programmed or otherwise configured to determine the presence or absence of an analyte. The computer system 1601 can regulate various aspects of detecting the presence or absence of an analyte. The computer system 1601 can be a user's electronic device or a computer system remotely located relative to the electronic device. The electronic device can be a mobile electronic device.

[0236] Computer system 1601 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1605, which can be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 3001 also includes memory or storage location 1610 (e.g., random access memory, read-only memory, flash memory), electronic storage unit 1615 (e.g., a hard disk), a communication interface 1620 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 1625 (e.g., cache memory), other memory, data storage, and / or electronic display adapters. Memory 1610, storage unit 1615, interface 1620, and peripherals 1625 communicate with CPU 1605 via a communication bus (solid lines) (e.g., a motherboard). Storage unit 1615 can be a data storage unit (or data repository) for storing data. Computer system 1601 can be operatively connected to a computer network ("network") 1630 via communication interface 1620. Network 1630 may be the Internet, an interconnected network and / or an extranet, or an intranet and / or extranet in communication with the Internet. In some cases, network 1630 is a telecommunications and / or data network. Network 1630 may include one or more computer servers that enable distributed computing, such as cloud computing. In some cases, network 1630, with the assistance of computer system 1601, may implement a peer-to-peer network that enables devices connected to computer system 1601 to behave as clients or servers.

[0237] The CPU 1605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a storage location, such as memory 1610. The instructions can be directed to the CPU 1605, which can then be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 1605 can include fetching, decoding, executing, and writing back.

[0238] CPU 1605 may be part of a circuit (e.g., an integrated circuit). One or more other components of system 1601 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0239] Storage unit 1615 can store files, such as drivers, libraries, and saved programs. Storage unit 1615 can also store user data, such as user preferences and user programs. In some cases, computer system 1601 may include one or more additional data storage units external to computer system 1601, such as on a remote server that communicates with computer system 1601 via an intranet or the Internet.

[0240] Computer system 1601 can communicate with one or more remote computer systems via network 1630. For example, computer system 1601 can communicate with a user's remote computer system (e.g., a personal computer). Examples of remote computer systems include a personal computer (e.g., a laptop PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a phone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access computer system 1601 via network 1630.

[0241] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored on an electronic storage location (e.g., memory 1610 or electronic storage unit 1615) of computer system 1601. Machine executable code or machine readable code may be provided in the form of software. During use, the code may be executed by processor 1605. In some cases, the code may be retrieved from storage unit 1615 and stored on memory 1610 for immediate access by processor 1605. In some cases, electronic storage unit 1615 may be eliminated and machine executable instructions may be stored on memory 1610.

[0242] The code may be precompiled and configured for use with a machine having a processor suitable for executing the code, or may be compiled during runtime. The code may be provided in a programming language that may be selected to enable the code to be executed in a precompiled or as-compiled manner.

[0243] The aspects of the system and method provided herein (e.g., computer system 1601) can be implemented with programming. Various aspects of the technology can be considered to be "products" or "articles" in the form of machine (or processor) executable code and / or related data, which are carried or embodied on the type of machine-readable media. Machine executable code can be stored on an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media can include any or all tangible memories of a computer, processor, etc., or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or part of the software can sometimes be communicated via the Internet or various other telecommunications networks. For example, such communication can be implemented by loading software from one computer or processor into another computer, for example, from a management server or a host computer into the computer platform of an application server. Thus, another type of media that can carry software elements includes optical, radio, and electromagnetic waves, such as those used over physical interfaces between local devices via wired and optical landline networks and over various air-links. Physical elements that carry such waves, such as wired or wireless connections, optical connections, etc., can also be considered media that carry software. As used herein, unless limited to non-transitory properties, the term "tangible" "storage" media, such as computer or machine "readable media," refers to any medium that participates in providing instructions to a processor for execution.

[0244] Thus, a machine-readable medium (e.g., computer executable code) can take a variety of forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer or the like, such as can be used to implement a database, etc. as shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example, a floppy disk, a flexible disk, a hard disk, magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punched card tape, any other physical storage medium with a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave that transports data or instructions, a cable or connection that transports such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0245] The computer system 1601 may include or communicate with an electronic display 1635 that includes a user interface (UI) 1640 for providing, for example, identification of an analyte. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0246] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 1605 .

[0247] Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, implements any of the methods above or elsewhere herein.

[0248] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory connected thereto. The computer memory comprises machine executable code that, when executed by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0249] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not intended to be interpreted in a limiting sense. Without departing from the present invention, many variations, changes, and substitutions can now be imagined by those skilled in the art. In addition, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions described herein that depend on various conditions and variables. It should be understood that various optional schemes of the embodiments of the present invention described herein can be adopted when implementing the present invention. Therefore, it is intended that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The appended claims are intended to define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are thereby encompassed. BRIEF DESCRIPTION OF THE DRAWINGS

[0250] Figure 1. Attachment of single-stranded DNA to the ClyA nanopore. (A) Side view (left) and top view (right) of the ClyA structure (PDB: 6mrt). The serine at position 110 (colored purple) was genetically mutated to cysteine to enable site-specific chemical modification. (B) Schematic model showing the conjugation strategy for attaching ssDNA to the ClyA nanopore. A 16-mer oligonucleotide, designated f, was conjugated to a ClyA monomer via a maleimide-PEG4-DBCO linker, where the maleimide reacts with the -SH group on the protein and the DBCO is clicked onto the azide group on the oligonucleotide. The ClyA-f monomers then oligomerize into ClyA-f oligomers in the presence of 0.2% DDM at 37°C. (C) SDS-PAGE analysis of conjugation efficiency. Lane 1: Protein ladder, Lane 2: ClyA-S110C monomer, Lane 3: ClyA-S110C after reaction with maleimide-PEG4-DBCO (ClyA-DBCO), Lane 4: Purified ClyA-DBCO after reaction with f-azide (ClyA-f). (D) Native polyacrylamide gel analysis of ClyA-f oligomerization. Lane 5: ClyA-f after oligomerization, Lane 6: ClyA with the S110C mutant after oligomerization.

[0251] Figure 2. Functionalization of ClyA nanopores with spike protein nanobody Ty1 and electrical characterization of the nanopores. (A) Schematic model showing the strategy for functionalizing ClyA nanopores with Ty1 nanobody, where Ty1-f' is immobilized on the ClyA-f nanopore via DNA strand hybridization. (B) IV curves of ClyA-S110C (blue triangles), ClyA-f (black squares), and ClyA-f-Ty1 (red circles) at applied potentials ranging from -90 mV to 90 mV (three independent experiments). (C) Histogram showing the conductance distribution of ClyA-f nanopores with (red) and without (black) Ty1 nanobody. (D) Representative current traces of ClyA-f-Ty1 at an applied potential of -20 mV. "In" and "out" indicate nanobodies located inside (blocked pore) and outside (open pore), respectively, of the nanopore. I o is the open pore current and I b is the blocked pore current. (E) Full-point histogram of the current trace shown in D, showing a well-defined distribution of the blockade signal. (F) Schematic model explaining the reversible conformational changes between the blocked (left) and open (right) states of ClyA-f-Ty1 at an applied potential of -20 mV, corresponding to movement of one of the Ty1 Nanobodies into and out of the pore vestibule. All experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5.

[0252] Figure 3. Single-channel recording traces of ClyA-f-Ty1 and the residual current I at different applied potentials. b / I o , t in and t out Analysis of the pore structure. (A) Representative current traces of ClyA-f-Ty1 under applied potentials ranging from -10 mV to -40 mV. (B) Full-point histogram of the current trace in A, showing that the Ty1 nanobody tends to reside in the ClyA nanopore as the applied potential increases. (C, D) Histograms of the logarithmic time Ty1 is located inside and outside ClyA, respectively. (E, F) Effect of applied potential on the average logarithmic time Ty1 is located inside and outside ClyA. These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5.

[0253] Figure 4. Attachment of Nanobodies to ClyA via DNA oligonucleotide hybridization, validated using DNase I. (A) Current traces of ClyA-f-Ty1 in the presence of 2.5 mM MgCl2 at an applied potential of -20 mV, before and after the addition of 5 U DNase I. (B) Zoomed-in representative current trace from A, showing the removal of Nanobodies attached to the ClyA nanopore approximately 30 minutes after the addition of DNase I. Full-point histograms are displayed on top of the insets showing the current distribution before and after the addition of DNase I. The schematic model shown above depicts how Nanobodies are removed from the ClyA nanopore. Experiments were performed in 150 mM NaCl, 2.5 mM MgCl2, 50 mM Tris-HCl, pH 7.5.

[0254] Figure 5. Detection of the spike protein using a nanopore functionalized with a nanobody. (A) Current traces of ClyA-f-Ty1 before and after the sequential addition of 6 μM BSA and 2.3 nM spike protein. (B) Zoomed-in representative current trace from A (top) and full-site histogram of the current distribution (bottom). Shown from left to right are before and after the addition of BSA and spike protein. Experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5.

[0255] Figure 6. Effect of BSA on nanobody internalization. (A-C) Before and after addition of 3 μM BSA or 6 μM BSA to the first side (i.e., cis side) of the ClyA-f-Ty1 nanopore system. out The histogram distribution is shown in Figure 2. The histogram is fitted with a single exponential function. (DG) are the percentage of blockage, percentage of openness, and average time (t) of the coupled Ty1 in the ClyA nanopore as the BSA concentration increases. in ), the average time that Ty1 stays outside the ClyA nanopore (t out ). (n = 4, each experiment was performed with an independent nanopore. Error bars represent standard deviation.) These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5.

[0256] Figure 7. Effect of adding spike protein to the ClyA-f-Ty1 pore. (A) Current trace showing the transition of the pore from a dynamic state (Ty1 alternating between being inside and outside the pore) to a fully open state (Ty1 trapped outside the pore by binding to spike protein) in the time period immediately following the addition of 2.3 nM spike protein. (B) Current trace of ClyA-f-Ty1 in the time period approximately 25 minutes after the addition of 2.3 nM spike protein. (C) Full-point histogram of the current trace presented in B. (D, E) Current trace of the pore after the addition of 2.3 nM spike protein in the time period approximately 25 minutes after the addition of spike protein. in and t out The histograms were plotted using the logarithm of the values of . The histograms were fitted with a Gaussian distribution function. These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, in the presence of 6 μM BSA.

[0257] Figure 8. The open probability of ClyA-f-Ty1 is positively correlated with the concentration of spike trimer protein. (A) Representative current traces of ClyA-f-Ty1 before and after the addition of increasing concentrations of spike trimer protein. (B) Full-point histogram showing the current distribution before and after the addition of increasing concentrations of spike protein. (C) Curve regression of the open probability as a function of spike concentration. The curves were fitted using the Hill-Langmuir equation (n = 1.31, K d = 760.6 pM). (D) Schematic model showing the kinetics of the interaction between ClyA-f-Ty1 and the spike protein. The Ty1 nanobody dynamically moves in and out of the ClyA nanopore under an applied potential. The spike protein presumably interacts reversibly with the nanopore-attached Ty1 nanobody in a multivalent manner at high concentrations of spike trimers. Experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, in the presence of 6 μM BSA.

[0258] Figure 9. Effect of spike protein concentration on the binding kinetics of ClyA-f-Ty1 pores. (AD) are logarithmic values at spike concentrations of 115 pM, 230 pM, 345 pM, and 460 pM, respectively. 10 (t out ). The data were fitted with a Gaussian distribution. (EH) are the logarithms of spike concentrations at 115 pM, 230 pM, 345 pM, and 460 pM, respectively. 10 (t in ). Fit the data with a Gaussian distribution. (I, J) t out and t inThese experiments were performed in the presence of 6 μM BSA in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5.

[0259] Figure 10. Behavior of ClyA-f-Ty1 in the presence of blood. (A) Schematic model showing electrical measurements of ClyA-f-Ty1 in the presence of blood. (B) Current traces showing the current changes before and after adding 1 μL of blood to a ClyA-f-Ty1 nanopore in 500 μL of electrolyte buffer. (C, E) Representative current traces in the presence of 6 μM BSA (C) and after adding 1 μL of blood (E). (D, F) Whole-point histograms of the current traces before (D) and after (F) adding 1 μL of blood. (G) Histogram of the logarithm of the residence time at level 0 before and after adding 1 μL of blood. (H) Histogram of the logarithm of the residence time at level 1 before and after adding 1 μL of blood. These experiments were performed in the presence of 6 μM BSA in an electrolyte buffer of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5.

[0260] Figure 11. Detection of Spike trimers in the presence of blood. (A, B) Representative current traces before (A) and after (B) the addition of 2.3 nM Spike protein in the presence of 1 μL of blood at a -20 mV bias. Experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, in the presence of 6 μM BSA.

[0261] Figure 12. Detection of Her2 using functionalized nanopores. (A) Representative current traces of ClyA attached by the 2Rs15d nanobody (ClyA-f-15d) at an applied potential of -20 mV before and after addition of 32.8 nM Her2 protein. (B) Representative current traces of ClyA attached by the 2Rb17c nanobody (ClyA-f-17c) at the same applied potential before and after addition of 20.8 nM Her2 protein. Reported binding affinity of 2Rs15d to Her2: k on = 2.14 x10 5 M -1 s -1 , k off = 5.71 x 10 -4 s -1 , K D = 2.7 nM. Reported binding affinity of 2Rb17c to Her2: k on= 7.6 x 10 6 M -1 s -1 , k off = 4.58 x 10 -2 s -1 , K D = 6 nM. These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, in the presence of 6 μM BSA.

[0262] Figure 13. Functionalized ClyA nanopore for the detection of muPA. (A) Crystal structure of muPA (purple) in complex with the nb22 nanobody (green) (PDB: 5LHR). 56 Binding affinity: k on = (4.6 ±0.8) x 10 5 M -1 s -1 , k off = (7.8 ± 2.2) x 10 -5 s -1 , K D = 0.2 ± 0.03 nM. (B) Representative current traces of ClyA-f-nb22 before and after adding 3 nM muPA at an applied potential of -15 mV. (C) Amplified representative current traces after adding 3 nM muPA at -15 mV. Except for the open pore level (level 0), the signal consists of three blockage levels, wherein the current blockage percentage is 13.7% ± 0.1% (level 1), 34.1% ± 0.5% (level 2) and 63.6% ± 0.1% (level 3). (D) Heat map of the blockage events observed after adding 3 nM muPA, wherein the logarithm of the residence time is for the current blockage percentage. (E) Schematic model showing the conformational changes of ClyA-f-nb22 in response to muPA protein. The experiment was carried out in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 in the presence of 6 μM BSA.

[0263] Figure 14. Schematic diagram of some options for coupling the targeting moiety R to the nanopore N via hybridization of a double-stranded oligonucleotide (e.g., dsDNA) linker L (where one oligonucleotide strand of the double-stranded linker L is coupled to the nanopore N, while the other, complementary strand is coupled to the binding moiety R). The figure shows three possible options for coupling the components. A) The N and R components are located at opposite ends of the duplex linker L. This can be readily achieved, for example, by coupling the components to both 5' ends of each strand, or to both 3' ends. The distance "d" between the N and R coupling positions is primarily controlled by the lengths of the oligonucleotide strands forming the duplex, and the flexibility of the system (which determines the ability of R to enter the nanopore) depends in part on the flexibility of the double-stranded oligonucleotide (e.g., dsDNA), which is less flexible than single-stranded oligonucleotides. B) The N and R components are located at the same end of the hybridized duplex linker L, e.g., one component is coupled to the 5' end of strand 1 and the other to the 3' end of strand 2, or vice versa. This coupling method facilitates positioning the N and R coupling points closer together to reduce the distance "d" while still allowing for much longer oligonucleotide chains as desired. C) One or both of the N and R components are coupled to the oligonucleotide chain of the hybridized duplex linker L at an interior position along the chain (for simplicity, only R is shown coupled at a midpoint), for example via coupling to the backbone or bases of the polynucleotide.

[0264] In all of the above cases, the oligonucleotides may have single-stranded segments that are not duplexed (eg, ssDNA overhangs) to further control the distance and optimize the flexibility of the coupled components.

[0265] Figure 15. Schematic diagram of a nanopore N with a linker L initially in a protected state (A) comprising hybridized protection polynucleotide chains (i) that can be removed (B) by applying a voltage to the nanopore in the membrane system to capture and strip the protection chains from the linker L. The deprotected nanopore (C) can then be combined with a selected binding moiety R that hybridizes to the linker L to produce a functional NLR nanopore system (D).

[0266] Figure 16 . Describes a computer system that is programmed or otherwise configured to implement the methods provided herein.

[0267] Experimental part

[0268] Material

[0269] Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich. The unnatural amino acid (UAA) 4-azido-L-phenylalanine (pAzF) used in this study was prepared according to the reported protocol. 57All DNA oligonucleotides were purchased from IDT.

[0270] Expression and purification of pAzF-modified nanobodies

[0271] Ty1 will be encoded 52 、nb22 55 , 2Rs15d, and 2Rb17c 54 Nanobody DNA was cloned into the pET22b (+) plasmid (Addgene) with a pelB leader sequence at the N-terminus and a hexahistidine tag (6xHis) at the C-terminus. An amber stop codon (TAG) was added before 6xHis to incorporate UAA into the nanobody. 38 Preparation of pAzF modified nanobody. First, the constructed plasmid was transformed into BL21 Escherichia coli ( E. coli ) cells. Cells were cultured in 1 L of TB medium supplemented with 100 mL of saline buffer (0.17 M KH2PO4, 0.7 M K2HPO4), 1 mL of 2 M MgCl2, 1 mL of 100 mg / mL ampicillin, 1 mL of 50 mg / mL spectinomycin, 10 mL of 10% glucose, and 250 mg of 4-azido-L-phenylalanine at 37°C and 200 rpm. When the OD600 reached 0.6-0.9, IPTG was added to a final concentration of 1 mM. Protein induction was achieved by shaking overnight at 25°C. Cells were harvested by centrifugation at 4500 rpm for 15 minutes at 4°C and resuspended in 24 mL of cold TES buffer (0.2 M Tris, pH 8, 0.5 mM EDTA, 0.5 M sucrose). The suspension was incubated at 4°C and 200 rpm (swinging rotor) for 6 h, followed by the addition of 48 mL of 1 / 4 TES buffer and incubation at 4°C and 200 rpm overnight. Subsequently, the cell suspension was centrifuged at 4°C and 12,000 g for 30 minutes. The supernatant was collected and supplemented with 5 mM MgCl2 and then further purified by FPLC (GE Healthcare) using a 5 mL HisTrap column (GE Healthcare). The binding buffer and elution buffer used here were 20 mM and 500 mM imidazole, respectively, both supplemented with 20 mM sodium phosphate, pH 7.4, 500 mM NaCl. The purity of the protein was analyzed on a 4%-12% SDS-PAGE gel.

[0272] Conjugation of nanobodies to f'-oligonucleotides

[0273] First, the oligonucleotide f' (NH2-C6-5'-ATCCGCGGGTGTCGGG-3') with an amine group at the 5' end was reacted with a 20-fold excess of NHS-DBCO in 60% DMSO at pH 8.0 and 25°C overnight. After ethanol precipitation and subsequent reverse phase HPLC purification, the DBCO-oligonucleotide was incubated with the azide-modified nanobody in PBS at 25°C overnight. The reaction was optimized by adding different ratios of nanobody and f'-DBCO oligonucleotide. When the molar ratio was 5:1, the conjugation yield was above 70%. Therefore, this ratio was used for the conjugation of all four nanobodies. Subsequently, the nanobody-f' conjugate was purified by ion exchange chromatography and verified by 16% denaturing urea polyacrylamide gel electrophoresis or SDS-PAGE.

[0274] Expression and purification of ClyA-S110C nanopore

[0275] The ClyA-S110C construct was prepared by mutating the serine at position 110 to cysteine in the cysteine-free variant ClyA-CS as previously reported. 41 . The constructed plasmid was transformed into Escherichia coli BL21 (DE3) electrocompetent cells by electroporation. The cells were cultured in 2x YT medium containing 100 μg / mL ampicillin at 37°C and 200 rpm until OD600 reached 0.8-1. Protein expression was induced by adding 0.5 mM IPTG and incubating overnight at 20°C and 200 rpm. The cells were harvested by centrifugation at 6500 rpm and 4°C for 15 minutes. The pellet was stored in a -80°C refrigerator for at least 1 hour, then thawed at 37°C and resuspended in 20 mL lysis buffer (10 mM imidazole pH 8.0, 150 mM NaCl, 50 mM Tris.HCl pH 7.5, 1 mM MgCl2, 5 mM TCEP) supplemented with 0.2 mg / mL lysozyme. After incubation at 4°C for 25 minutes on a rotor, the cells were further lysed by sonication. The lysate was then centrifuged at 6500 rpm and 4°C for 30 minutes, and the supernatant was collected and incubated with Ni-NTA beads (Qiagen) on a rotor at room temperature for 1 hour. Non-specific binding proteins were removed by at least 20 column volumes of wash buffer (10 mM imidazole pH 8.0, 150 mM NaCl, 50 mM Tris.HCl, pH 7.5), and proteins were eluted from the beads in elution buffer (200 mM EDTA pH 7.5, 150 mM NaCl, 50 mM Tris.HCl, pH 7.5). Protein purity was analyzed on a 4%-12% SDS-PAGE gel.

[0276] Preparation of ClyA-f-nb nanopores

[0277] Freshly purified ClyA-S110C was first incubated with a 20x molar excess of DBCO-PEG4-maleimide at pH 7.5 and 4°C overnight with gentle agitation. Unreacted DBCO-PEG4-maleimide was removed using a 3 kDa cutoff Amicon filter (Millipore) in standard buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). Purified ClyA-PEG4-DBCO was then incubated with a 1.5-fold excess of f-azide oligonucleotide at 4°C overnight with gentle agitation to produce ssDNA-modified "ClyA-f" monomers. The f-azide oligonucleotide linker was prepared by reacting an oligonucleotide with an amino modification at the 5' end (NH2-C6-5'-CCCGACACCCGCGGAT-3') with azidobutyric acid NHS ester. The efficiency of the click reaction was examined using SDS-PAGE gels. ClyA-f monomers were oligomerized by incubation at 37°C for 30 minutes in the presence of 0.2% n-dodecyl-β-D-maltoside (DDM). Subsequently, oligomerized ClyA-S110C and ClyA-f were analyzed and purified by blue native polyacrylamide gel electrophoresis (BN-PAGE, Bio-Rad). Due to the negative charge of DNA oligonucleotides 29 , ClyA-f oligomers migrate slightly faster than unmodified ClyA-S110C oligomers. 41 , the lowest oligomeric band of ClyA-S110C and ClyA-f is type I nanopore (12-mer). Therefore, ClyA-S110C and ClyA-f dodecamers are obtained by cutting these bands from gel. After eluting from the gel slice using 30 μL of standard buffer (in the presence of 0.02% DDM), the ClyA-f oligomer solution is divided into 5 μL / tubes. The concentration of the ClyA-f dodecamers eluted from the gel is too low to be measured by Nanodrop or Bradford assay. Therefore, before the single-channel recording experiment, an excess of nanobody-f' (about 40 pmol) and 5 μL of ClyA-f oligomers are incubated at room temperature for at least 30 minutes to ensure that each ClyA nanopore is modified to the greatest extent with the nanobody forming a double chain.

[0278] Single-channel recording experiment

[0279] Use as previously described 58The vertical plane lipid membrane device of the recording chamber is electrically recorded. In brief, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, purchased from Avanti Polar Lipids) lipid bilayer is formed on the hole of Teflon membrane, and the first side (for example, cis side) and the second side (for example, trans side) of the fluid chamber of the recording chamber are separated by the Teflon membrane. After being connected with a patch clamp amplifier (Axopatch 200B, Axon Instruments) using Ag / AgCl electrode, the trans side and the first side of the chamber are filled with electrolyte buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). ClyA nanopore is added to the first side (for example, cis side) of the chamber connected to the ground electrode. After the hole is inserted, excess ClyA is removed by buffer exchange. Unless otherwise specified, DNAse I (Sigma-Aldrich), BSA, muPA (kindly provided by Emil Oldenburg), Her2 (obtained from SinoBiological) and various concentrations of spike protein (SARS-CoV-2 S protein, purchased from ACROBiosystems) were all added to the first side (e.g., cis side). All recordings were performed using a 2 kHz Bessel low-pass filter and a sampling rate of 10 kHz. All electrical recording current traces were filtered by a Gaussian low-pass filter with a cutoff value of 1 kHz before analysis. The data analysis software we used in this study was Clampfit.

[0280] Example 1: Functionalization of ClyA nanopores with nanobodies

[0281] To specifically detect proteins of various sizes, we designed ClyA nanopores that were functionalized with multiple nanobodies at the wide end of the pore via a 16-base-pair DNA duplex linker. We hypothesized that protein binding to the nanobody would alter the ion flux through the nanopore, leading to a distinguishable current signal indicative of protein detection. To achieve site-specific attachment of the DNA linker to ClyA, we mutated ClyA-CS by replacing serine with cysteine at position 110. 41 Variant (ClyA-S110C, Figure 1A Then, a 16 nt DNA oligonucleotide with an azide group at the 3' end (f-azide) was attached to ClyA-S110C by using a maleimide-PEG4-DBCO linker ( Figure 1B With the addition of a 20-fold excess of linker to ClyA-S110C, the bands of the products all shifted upward compared to ClyA-S110C in the SDS-PAGE gel, indicating a high yield of the ClyA-DBCO product ( Figure 1C Subsequently, the purified ClyA-DBCO was reacted with a 1.5-fold excess of f-azide to give the ClyA-f construct in full yield ( Figure 1C ). In addition, after self-assembly to form oligomeric pores in the presence of detergent, ClyA-S110C and ClyA-f dodecamers were extracted from blue native polyacrylamide gels. 41 ( Figure 1D , strip I) 42 Based on the high conjugation efficiency of ClyA-f monomers and the uniformity of oligomerization, it can be assumed that on each ClyA-f dodecamer, there are roughly 12 oligonucleotides available for Nanobody attachment.

[0282] To allow the nanobody to anchor on the ClyA nanopore, the amber codon was used to suppress 38 Unnatural amino acids were incorporated to produce nanobodies with an azide group at the N-terminus and conjugated to the complementary chain (f'-DBCO) of the oligonucleotide f containing a DBCO group at the 5' end via click chemistry. As a proof of concept, the Ty1 nanobody, which can reversibly bind to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, was conjugated to f'. The binding activity of the oligonucleotide-attached nanobody was examined using biolayer interferometry, which showed that the attachment of the oligonucleotide did not affect the binding affinity of the Ty1 nanobody to the RBD (data not shown). In addition, in order to test the feasibility of nanobody attachment to ClyA, the ClyA-f monomer was incubated with a 5-fold excess of the Ty1-f' conjugate and analyzed by SDS-polyacrylamide gel. It shows that ClyA-f has a significant mobility shift due to the attachment of the nanobody and indicates that the attachment efficiency is up to 100% (data not shown). Finally, nanobody-functionalized ClyA nanopores (ClyA-f-nb) were prepared by incubating ClyA-f dodecamers with the corresponding nanobody-f′ modules.

[0283] Example 2: Characterization of Nanobody-functionalized ClyA nanopores.

[0284] First, we performed electrical characterization of ClyA-S110C, ClyA-f, and Ty1-modified ClyA (ClyA-f-Ty1) at different applied potentials using a single-channel recording system to investigate the effects of ssDNA and nanobody attachment. At an applied potential of ±35 mV, the current trace of ClyA-f was similar to that of ClyA-S110C, and no specific signal due to the entry of the attached oligonucleotide into the nanopore was observed (data not shown). However, the IV curve showed that within the positive bias range of 10 mV to 90 mV, the open pore current of ClyA-f was slightly smaller than that of ClyA-S110C ( Figure 2B ), indicating that the attached ssDNA entering ClyA partially blocks the pore under the applied positive potential. However, under negative bias, the conductive behavior of ClyA-f is not affected by the ssDNA attachment ( Figure 2B In contrast, at a positive potential (+35 mV), attachment of the Ty1 nanobody had no effect on the ClyA-f-Ty1 pore, whereas when a negative potential (-35 mV) was applied, the pore was partially blocked compared to ClyA-f. When the applied potential was lowered to -20 mV, we observed a transient and reversible blocking signal ( Figure 2D These signals consist of two current levels (in and out), one of which (in) is similar to the current level expected for an open pore current, while the other current level is consistent with a Nanobody entering the nanopore. At the same applied potential, the current blockade percentage (I o -I b ) / I o x 100 (or ΔI / I o x 100, I o is the open pore current, and I b is the blocked pore current) was 14.2 ± 0.3% (n = 3), and the dwell time of the blocked signal (t in ) was 21.09 ± 1.06 ms (n = 3). By fitting the full-point histogram of the current trace with a Gaussian function and calculating the ratio of the area under the curve, we found that the open probability of ClyA-f-Ty1 at -20 mV was 51% ( Figure 2E ). In addition, by measuring the blocked pore current at different applied potentials, the IV curve of ClyA-f-Ty1 was obtained, which showed that the current of ClyA-f-Ty1 was smaller than that of non-nanobody-attached ClyA-f at negative bias ranging from -10 mV to -90 mV ( Figure 2B Therefore, the conductance of ClyA-f-Ty1 (1.71 ± 0.01 nS, n = 22) at -35 mV was smaller than that of ClyA-f (1.92 ± 0.01 nS, n = 22) ( Figure 2C These results indicate that attachment of Ty1 leads to voltage-dependent gating of the ClyA nanopore.

[0285] To further confirm that these blocking signals are caused by the movement of the attached nanobodies, we studied the dependence of these blocking signals on the applied potential. As the applied potential increased from -10 mV to -40 mV, the blocking probability of the pore and the residence time of the blocking signal (t in ) increased significantly, while the interval time that the pore remained open (t out) was significantly reduced (Figure 3). For example, at potentials of -50 mV and above, the ClyA pore was almost permanently blocked for this particular nanobody. However, by reversing the applied potential, the ClyA-f-Ty1 nanopore could be restored to an unblocked state. These results suggest that the blocking signal is not caused by molecular translocation, as higher voltages generally drive molecules to translocate faster through the nanopore (evidenced by shorter blocking residence times at higher voltages). ClyA-AS is known to generate strong electroosmotic flow 43 , which induces the capture of various proteins under negative applied potentials 19 Considering the small size of the nanobody (2.5 nm in diameter and 4 nm in height), 44 ) and a flexible connection of a 16 bp DNA duplex (about 5.5 nm in length), and the current blockade is the result of the coupled Ty1 nanobody entering the interior of the nanopore very close to the constriction region of the pore ( Figure 2F ).

[0286] Furthermore, we observed that in the presence of Mg 2+ In the case of -20 mV, 5 U of DNAse I was added to the first side of the chamber (e.g., the cis side) for about 30 minutes before the irreversible opening of the pore (Figure 4) was the result of cleavage of the dsDNA linker. This result confirmed that the nanobody was successfully attached to the ClyA nanopore through DNA duplex formation and provided additional evidence for the interpretation of the blocking signal. In this paper, we use "in" and "out" to define the position of the nanobody inside or outside the nanopore vestibule, respectively, and use t in and t out to represent the time the Nanobody spends inside and outside the nanopore, respectively. These results indicate that the Nanobody is attached to the ClyA nanopore via a flexible oligonucleotide linker, which enables the coupled Nanobody to dynamically move in and out of the nanopore, partially blocking the ionic current when inside the nanopore. Furthermore, the results demonstrate the ability to control the dynamics between the in and out states by applying a voltage.

[0287] Example 3: Real-time detection of SARS-CoV-2 spike protein

[0288] Bovine serum albumin (BSA) has been widely used in sensing techniques such as ELISA 45 Blocking agents in the solution to eliminate nonspecific interactions such as protein-protein or protein-surface 46 In our case, when BSA was added to the first side (e.g., cis side) of the ClyA-f-Ty1 nanopore, no additional blocking signal caused by BSA translocation was observed. Surprisingly, we found that the t outBoth the open probability and the open probability (the probability of being in the out state) decreased with increasing BSA concentration (Figures 5 and 6). For example, in the presence of 6 μM BSA and at a bias of -20 mV, the t out The time taken for the ClyA nanopore to be unoccupied by the conjugated Ty1 nanobody decreased from 42.9 ± 38.9 ms to 4.64 ± 0.38 ms, and the probability of ClyA-f-Ty1 being in the open state decreased from 14.2 ± 7.5% to 2.1 ± 0.8%. These results indicate that the presence of BSA significantly reduces the time that the ClyA nanopore is not occupied by the conjugated Ty1 nanobody. Considering that BSA has a size of approximately 14 × 4 × 4 nm and a pI of 4.7 in aqueous solution, 47 , BSA is likely to create a crowded environment outside the nanopore, thereby increasing the probability of nanobodies entering the pore. In previous studies, similar crowding effects have been used to enhance the capture of large molecules. 9 48 49 Furthermore, the addition of BSA greatly minimized the well-to-well variation of ClyA-f-Ty1 ( Figure 6E 、 Figure 6G ). Therefore, for further sensing applications, 6 μM BSA was added to the first side of the fluid chamber to minimize the background signal.

[0289] Multivalent interactions have been widely used to improve binding affinity and enhance sensing sensitivity. 50 51 It was reported that the binding affinity between SARS-CoV-2 spike protein and Ty1 was significantly increased by multimerization of nanobodies 39 . Given the structure of the dodecamer and the well-defined distance, the ClyA nanopore is predicted to be the optimal scaffold for multimerizing nanobodies that bring them into close proximity, allowing multiple nanobodies to bind to a single protein simultaneously to increase the sensitivity of spike protein recognition. To test the feasibility of this sensing system, SARS-CoV-2 spike protein was added to the first side (e.g., cis side) of the fluid chamber of the ClyA-f-Ty1 nanopore system at a final concentration of 2.3 nM in the presence of 6 μM BSA. Significantly, after about 1 minute, we observed that the frequency of the blocking signal began to decrease and t out Increase (Figure 5, Figure 7A). Shortly thereafter, the current trace became almost completely locked to the "out" state, which is consistent with the ClyA-f-Ty1 nanopore returning to the open current state (about -38pA) (Figure 5). During the recording time of about 25 minutes after the addition of spike protein, the probability that Ty1 is located outside the ClyA cavity (open probability) increased from 3.9 ± 0.4% to 98.9 ± 0.6% (n = 3, Figure 7), indicating that after being captured by the spike protein, the Ty1 nanobody remains outside the ClyA cavity. In addition, in the presence of 2.3 nM spike protein, t out The histogram of the logarithm of shows two peaks ( Figure 7E ), with average interevent times of 5.03 ± 1.34 ms and 20230.19 ± 1.95 ms (n = 3), respectively. The interevent duration of the first peak was very close to that before the addition of the spike protein (4.48 ± 1.32 ms, n = 3), indicating that these events were attributed to unbound Ty1 nanobodies nonspecifically located inside and outside the nanopore, while the events of the second peak were likely caused by the binding of the spike protein to the nanobody. Compared with the first peak, the time of the second peak increased by 3 orders of magnitude, indicating that the binding interaction between the spike protein trimer and the multimerized Ty1 nanobody is very strong.

[0290] To create a calibration curve for spike detection and further investigate the binding kinetics of trimeric spike to multimeric Ty1 nanobody, we tested the response of the ClyA-f-Ty1 nanopore to different concentrations of spike protein. At lower concentrations (0-460 pM), the open probability of ClyA-f-Ty1 increased with increasing spike protein concentration over the entire range ( Figure 8A 、 Figure 8B We also found that the time Ty1 is outside the pore (t out ) increases approximately linearly with the increase of spike protein concentration, while the time Ty1 is located in the nanopore (t in ) is independent of concentration (100 μM to 500 pM, Figure 9). This confirms that the increased open probability is indeed caused by the spike protein binding to the Ty1 nanobody. Since the spike is a trimeric protein that can interact with three Ty1 nanobodies 52 , the occupation of any of the 12 Ty1s on the ClyA nanopore by a spike modulates ion flux. Therefore, this enables our platform to sensitively detect spikes at picomolar concentrations. When we further increase the spike concentration, we find that the open probability is positively correlated with concentration and reaches a plateau at approximately 2 nM ( Figure 8CThe data could be fitted by the Hill-Langmuir equation with a Hill coefficient greater than 1 (n = 1.31), indicating positive cooperativity between the binding of trimeric spike and multimeric Ty1. In addition, the long inter-event duration and small dissociation constant (K) previously observed for spike binding were consistent with the observed long inter-event duration and small dissociation constant (K) for spike binding. d = 760.6 pM) is consistent with the fact that cooperative binding between multiple ligands and the same receptor can produce much stronger binding affinity. 50 53 Therefore, we conclude that nanopores with multiple binding ligands for the same protein have great potential for high-sensitivity detection.

[0291] Example 4: Detection of SARS-CoV-2 Spike Protein in Blood

[0292] For clinical sensing applications, it is crucial that the sensing efficiency and specificity of the sensor are not affected by blood components such as proteins, red and white blood cells, and platelets. To test the effect of blood components using our ClyA-f-Ty1 pore sensor, 1 μL (final concentration: 0.2% v / v) of defibrinated sheep blood was added to the first side (e.g., cis side) of the fluid chamber in the presence of BSA ( Figure 10A Interestingly, the conductive behavior of the ClyA-f-Ty1 nanopore was only slightly affected by blood, and the membrane remained stable ( Figure 10B 、 Figure 10C 、 Figure 10E No obvious blood-induced blockage was observed, except for very few transient blockage signals and a current blockage of approximately 31.5% ± 0.1% ( Figure 10E , level 2). However, the residence time of those events was very short (about 0.6 ms), suggesting that they were likely due to transient collisions with proteins or platelets in the blood. In addition, the changes in the open probability, residence time, and inter-event time of the ClyA-f-Ty1 nanopore before and after the addition of blood were negligible ( Figures 10D-10H ).

[0293] After adding 2.3 nM spike, the nanopore shifted significantly to the open state due to the binding of the Ty1 nanobody. Some large and seconds-long blockage events were also observed in this state ( Figure 11A 、 Figure 11B ). Possibly, in the absence of the spike protein, steric hindrance of the nanobody on ClyA prevents blood components from entering the pore, whereas when the spike protein is bound to the nanobody, the pore remains open, allowing some large proteins in the blood to occasionally enter the nanopore.

[0294] Notably, unlike other methods, in this system, the protein does not need to enter the nanopore to be detected. This is important because the applied potential required to detect proteins in this assay is only -20 mV, which is much lower than the potential required to trap the protein in the nanopore. 22 The lower voltage reduces the possibility of capturing unwanted background contaminants in the nanopore. In addition, the coupled nanobodies at the nanopore entrance further prevent the capture and interference of unwanted proteins and contaminants (such as background proteins in blood) in the nanopore, which significantly increases the selectivity of the nanopore for proteins.

[0295] Example 5: General applicability of nanobody-functionalized nanopores as protein sensors.

[0296] This example demonstrates that the concepts exemplified for spike protein detection can be broadly applied to a variety of other proteins when appropriate nanobodies are used. Nanobodies have similar properties in size and shape. 34 Therefore, it is expected that various nanobodies can induce similar transient blockade signals when immobilized on ClyA nanopores, thereby allowing the detection of proteins of variable sizes. Taking advantage of the modularity of our approach, we functionalized ClyA nanopores with nanobodies 2Rs15d (ClyA-f-15d), 2Rb17c (ClyA-f-17c), and nb22 (ClyA-f-nb22), respectively. Among these nanobodies, 2Rs15d and 2Rb17c 54 Nanobody nb22 recognizes the N-terminal and C-terminal halves of the human epidermal growth factor receptor 2 (HER2) protein, which is highly expressed in breast cancer. 55 Identification of murine urokinase-type plasminogen activator (muPA), a biomarker associated with cancer progression.

[0297] All three Nanobodies were successfully conjugated to oligonucleotide f', and these Nanobodies could be functionalized on ClyA with high attachment efficiency. Due to their similarity in size, shape, and surface charge, we hypothesized that these Nanobodies have similar effects on the electrical behavior of ClyA as Ty1. Indeed, all Nanobody-conjugated ClyA nanopores induced similar blocking signals at an applied potential of -20 mV (Figure 12).

[0298] In the presence of 6 μM BSA, the percentages of blockage caused by 2Rs15d, 2Rb17c, and nb22 were 11.7% ± 0.1%, 14.2% ± 0.4%, and 13.7% ± 0.1%, respectively. To verify protein sensing capabilities, recombinant soluble protein Her2-hFc (96 kDa) was added to the ClyA-f-15d and ClyA-f-17c pores, respectively. Similar to the phenomenon observed for the interaction between the spike and ClyA-f-Ty1, both nanobody-functionalized nanopores showed a significant increase in open probability after addition of Her2-hFc due to protein binding to the conjugated nanobody (Figure 12).

[0299] In addition, we also tested the feasibility of ClyA-f-nb22 for protein detection. Interestingly, when the protein muPA (48 kDa, pI 8.53, Figure 13A ) is added to the first side (e.g., cis side) of the ClyA-f-nb22 pore ( Figure 13B ), at a potential of -15 mV, in addition to the pore-opening level (level 0) and the events caused by the nanobody (level 1), we also observed new types of blockade events (levels 2 and 3). At -15 mV, the new level 3 blockade showed a current blockade of 63.6 ± 0.1% and a relatively long duration of 45.45 ± 1.50 ms, while the level 2 blockade was 34.1 ± 0.5% and a very short duration of 1.60 ± 0.43 ms. Before the addition of muPA ( Figure 13B Level 3 blockades were not observed (left panel) or when muPA was added to the ClyA-f or ClyA-f-Ty1 nanopores (data not shown), indicating that Level 3 events are neither caused by free muPA protein itself nor by nonspecific interactions between the nanobody and the protein. More likely, Level 3 blockades reflect the entry of the nb22:muPA complex into the nanopore. Furthermore, the residence time of Level 3 events increased by approximately 1.5 orders of magnitude when the applied potential was increased from -5 mV to -15 mV (data not shown), consistent with the fact that positively charged muPA:nb22 complexes tend to reside in the nanopore for longer periods of time at more negative potentials. These results further confirm that muPA complexed with nb22 enters the ClyA pore, causing Level 3 blockade events.

[0300] In contrast, Level 2 blockade does not change significantly with applied voltage. Given its short residence time and voltage-independence, Level 2 blockade may reflect transient collisions of the nb22:muPA complex with the ClyA nanopore, rather than full entry into the nanopore.

[0301] Based on the above analysis, we established a model ( Figure 13E ), which shows a conformational transition of ClyA-f-nb22 in response to muPA, which corresponds to the multiple current levels observed. Taken together, these results demonstrate the ability to detect smaller analytes within nanopores by binding to conjugated nanobodies.

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Claims

1. A method for detecting the presence of at least one target analyte in a sample using a nanopore system, the nanopore system comprising a cis chamber in fluid communication with a trans chamber via a modified nanopore, the cis chamber comprising a first conductive liquid medium, the trans chamber comprising a second conductive liquid medium, the method comprising: (a) adding a sample to be analyzed for the presence of a target analyte to the cis chamber; (b) optionally applying an electric potential across the modified nanopore; and (c) measuring an ionic current passing through the modified nanopore; wherein the modified nanopore is a biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, capable of specifically binding to the target analyte, and wherein R dynamically moves in and out of the nanopore to cause transient current blockade events, and wherein binding of R to the target analyte modulates its dynamic movement, thereby causing a change in the frequency and / or amplitude of the current blockade events, and wherein the change in the frequency and / or amplitude of the current blockade events indicates the presence of the target analyte in the sample.

2. The method of claim 1, wherein the modified nanopore is an oligomeric assembly comprising or consisting of monomers of the general formula NLR, wherein N is a monomer of a pore-forming toxin having a maximum lumen diameter of 5 nm to 20 nm, and L is a flexible linker attached to the cis-entrance of the pore.

3. The method of claim 1 or 2, wherein binding of R to the target analyte increases the time that R resides outside the pore, thereby reducing the frequency and / or amplitude of the current blockade events.

4. The method according to any of the preceding claims, wherein the biological nanopore is functionalized with at least two different protein recognition elements R' and R", preferably wherein R' and R" bind to different sites on the target analyte.

5. The method according to any one of the preceding claims, wherein the target analyte is a protein, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer or any combination thereof, preferably wherein the target analyte is a protein, more preferably selected from the group consisting of folded / native proteins, clinically relevant proteins, biomarkers, pathogenic proteins, cell surface proteins.

6. The method according to any one of the preceding claims, wherein the sample is a complex sample comprising a mixture of proteins, preferably wherein the sample comprises a clinical sample, more preferably a body fluid, such as whole blood, plasma, urine, feces, saliva, cerebrospinal fluid, breast milk and sputum.

7. A modified protein nanopore having a minimum pore diameter of 5 nm, functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, via a flexible linker, which specifically reacts with a target analyte, preferably a target protein, and wherein R is able to move into and out of the pore to cause a blockade current.

8. A sensor system for protein analysis comprising a fluid-filled compartment separated by a membrane into a first chamber and a second chamber, electrodes capable of applying a potential across the membrane, and at least one biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, capable of specifically binding to a target analyte, and wherein R is positioned at the top of the nanopore via a flexible connection to allow movement in and out of the nanopore, thereby causing a transient current blockade event.

9. A nanopore sensor system comprising a cis chamber in liquid communication with a trans chamber via a modified nanopore, the cis chamber comprising a first conductive liquid medium, the trans chamber comprising a second conductive liquid medium, wherein the modified nanopore is a biological nanopore functionalized with a protein recognition element R of 5 kDa to 50 kDa, preferably 10 kDa to 40 kDa, capable of specifically binding to a target analyte, wherein R is tethered to the top of the nanopore and capable of internalizing in the pore and dynamically moving in and out of the lumen of the nanopore to cause a transient current blockade event.

10. The method, nanopore or sensor system according to any of the preceding claims, wherein R is an IgG-based moiety or a non-IgG-based moiety, preferably a Nanobody, a scFv fragment, a Fab fragment, an affimer, a monoclonal antibody, an Affibody, an Adnectin, a DARPin or an anticalin, more preferably wherein R is a Nanobody.

11. The method, nanopore or sensor system according to any of the preceding claims, wherein the biological nanopore is a pore-forming toxin, preferably having a maximum lumen diameter of 5 nm to 20 nm, more preferably selected from cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2 (PFN2, PDB_ID 6SB3), tripartite α-pore-forming toxin (Ah1B, PDB_ID 6GRJ), C9 (PDB_ID 6DLW), GspD secretin (PDB_ID 5WQ7), Helicobacter pylori ( Helicobacter pylori ) OMC (PDB_ID 6X6S), SpoIIIAG (PDB_ID 5WC3), Gasdermin-A3 (PDB_ID 6CB8), or their mutants that enable site-specific functionalization with protein recognition elements.

12. The method, nanopore or sensor system according to claim 10, wherein the biological nanopore is ClyA, preferably a mutant ClyA, more preferably ClyA comprising the mutation S110C.

13. The method, nanopore or sensor system according to any one of the preceding claims, wherein the flexible linker is an oligonucleotide, preferably duplex DNA, or a chemically modified RNA.

14. The method, nanopore or sensor system according to any of the preceding claims, wherein the nanopore is (reversibly) functionalized with R via a flexible linker L, preferably by nucleic acid hybridization between a first oligonucleotide, which is conjugated to the nanopore, and a second oligonucleotide, which is conjugated to R, which is complementary to the first oligonucleotide.

15. An array comprising a plurality of sensor systems according to one of claims 8 to 14, preferably wherein the array comprises a plurality of discrete reservoirs, each of said reservoirs comprising a nanopore modified with a different R-element to allow detection of a different analyte.

16. A kit for preparing an array according to claim 14, comprising nanopores pre-modified with a linker moiety, preferably as part of a double-stranded DNA complex consisting of an original strand and a complementary protection strand.

17. Use of the method, nanopore or sensor system, array, kit according to any of the preceding claims for single protein detection, preferably in combination with high throughput analysis.

18. Use according to claim 17, wherein the sensor system is integrated into a portable device comprising a plurality of sensor systems.

19. A method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a first side and a second side, wherein the nanopore is coupled to a recognition element; and (b) contacting the recognition element with an analyte.

20. The method of claim 19, wherein the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

21. The method of claim 20, wherein the recognition element is coupled to the nanopore via a linker.

22. The method of claim 21, wherein the linker has a length of about 4 to 8 nanometers.

23. The method of claim 21 or 22, wherein the linker comprises an oligonucleotide, a duplex DNA molecule, a chemically modified RNA molecule, or any combination thereof.

24. The method of any one of claims 21-23, wherein the nanopore is coupled to at least a portion of the linker.

25. The method of claim 24, wherein the nanopore is coupled to a first oligonucleotide and the linker is coupled to a second oligonucleotide.

26. The method of claim 25, wherein the first oligonucleotide and the second oligonucleotide are coupled together via nucleic acid hybridization.

27. The method of any one of claims 19-26, wherein the nanopore system further comprises an electrode pair.

28. The method of claim 27, wherein the electrode pair is configured to generate an electric potential across the nanopore.

29. The method of claim 28, wherein movement of the recognition element between the interior region of the nanopore and the exterior region of the nanopore effects a change in the current of the nanopore system.

30. The method of any one of claims 27-29, further comprising (c) measuring an ionic current through an interior region of the nanopore.

31. The method of claim 30, further comprising (d) detecting the presence or absence of the analyte via a change in the ionic current.

32. The method of any one of claims 19-31, wherein the recognition element is about 5 kilodaltons to about 50 kilodaltons.

33. The method of any one of claims 19-32, wherein the recognition element is coupled to the analyte.

34. The method of claim 33, wherein the recognition element is coupled to the analyte to effect movement of the recognition element.

35. The method of claim 34, wherein effecting movement of the recognition element produces a change in (i) or (ii) the frequency of movement of the recognition element or (ii) the noise or amplitude of the current of the nanopore system.

36. The method of claim 34 or 35, wherein the recognition element is unable to move between the interior region of the nanopore and the exterior region of the nanopore when coupled to the analyte.

37. The method of any one of claims 34-36, wherein the recognition element, when coupled to the analyte, moves between an interior region of the nanopore and an exterior region of the nanopore.

38. The method of claim 37, wherein when the recognition element is coupled to the analyte, a change in (i) or (ii) the frequency of movement of the recognition element or (ii) the noise or amplitude of current blockade is reduced.

39. The method of any one of claims 19-38, wherein the nanopore is coupled to another recognition element.

40. The method of claim 39, wherein the recognition element and the further recognition element bind to different regions of the analyte.

41. The method of claim 39 or 40, wherein the recognition element and the further recognition element bind to different analytes.

42. The method of any one of claims 19-41, wherein the analyte is a protein, a peptide, a small molecule, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof.

43. The method of any one of claims 19-42, wherein the analyte is a protein.

44. The method of claim 43, wherein the protein is a folded protein, a native protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, or any combination thereof.

45. The method of any one of claims 19-44, wherein the analyte is from a sample.

46. The method of claim 45, wherein the sample is a complex sample.

47. The method of claim 46, wherein the complex sample comprises a mixture of proteins.

48. The method of any one of claims 45-47, wherein the sample is a clinical sample.

49. The method of claim 48, wherein the clinical sample comprises a body fluid.

50. The method of claim 49, wherein the body fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

51. The method of any one of claims 19-50, wherein the recognition element is a protein recognition element.

52. The method of claim 51, wherein the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monoclonal antibody, an affimer, an affimer, an Adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

53. The method of any one of claims 19-52, wherein the nanopore comprises an oligomeric assembly.

54. The method of claim 53, wherein at least one subunit of the oligomeric assembly comprises a subunit of the nanopore coupled to a recognition element.

55. The method of claim 54, wherein the recognition element is coupled to at least one subunit of the nanopore via a linker.

56. The method of claim 54 or 55, wherein at least one subunit of the nanopore comprises a monomer of a pore-forming toxin.

57. The method of claim 56, wherein the pore-forming toxin comprises cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2, a tripartite α-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof.

58. The method of claim 56 or 57, wherein the pore-forming toxin comprises one or more mutations.

59. The method of claim 58, wherein the pore-forming toxin is ClyA.

60. The method of claim 59, wherein the ClyA comprises an S110C mutation.

61. The method of any one of claims 19-60, wherein the interior region of the nanopore comprises an inner diameter of about 5 nanometers to about 20 nanometers.

62. A method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluid chamber and (2) a membrane, the membrane comprising a nanopore, wherein the membrane separates the fluid chamber into a first side and a second side, wherein the nanopore is coupled to a protein recognition element, wherein the protein recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore; and (b) contacting the protein recognition element with an analyte.

63. A system comprising: (a) Fluid chamber; and (b) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into (1) a first side and (2) a second side, wherein the nanopore is coupled to a recognition element.

64. The system of claim 63, wherein the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

65. The system of claim 63 or 64, wherein the recognition element is coupled to the nanopore via a linker.

66. The system of claim 65, wherein the linker has a length of about 4 nanometers to about 8 nanometers.

67. The system of claim 65 or 66, wherein the linker comprises an oligonucleotide, a duplex DNA molecule, a chemically modified RNA molecule, or any combination thereof.

68. The system of any one of claims 65-67, wherein the nanopore is coupled to at least a portion of the linker.

69. The system of claim 68, wherein the nanopore is coupled to a first oligonucleotide and the linker is coupled to a second oligonucleotide.

70. The system of claim 69, wherein the first oligonucleotide and the second oligonucleotide are coupled together via nucleic acid hybridization.

71. The system of any one of claims 63-70, wherein the system further comprises an electrode pair.

72. The system of claim 71, wherein the electrode pair is configured to generate an electric potential across the nanopore.

73. The system of claim 72, wherein movement of the recognition element between an interior region of the nanopore and an exterior region of the nanopore effects a change in the current of the system.

74. The system of any one of claims 63-73, wherein the recognition element is about 5 kilodaltons to about 50 kilodaltons.

75. The system of any one of claims 63-74, wherein the recognition element is configured to couple to an analyte.

76. The system of claim 75, wherein the recognition element coupled to the analyte is configured to effect movement of the recognition element.

77. The system of claim 76, wherein movement of the identification element produces a change in (i) or (ii) the frequency of movement of the identification element or (ii) the noise or amplitude of the current of the system.

78. The system of claim 76 or 77, wherein the recognition element is not configured to move between an interior region of the nanopore and an exterior region of the nanopore when coupled to the analyte.

79. The system of any one of claims 76-78, wherein the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore when coupled to the analyte.

80. The system of claim 79, wherein when the recognition element is coupled to the analyte, a change in (i) or (ii) the frequency of movement of the recognition element or (ii) the noise or amplitude of current blockade is reduced.

81. The system of any one of claims 75-80, wherein the analyte is a protein, a peptide, a small molecule, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof.

82. The system of any one of claims 75-81, wherein the analyte is a protein.

83. The system of claim 82, wherein the protein is a folded protein, a native protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, or any combination thereof.

84. The system of any one of claims 75-83, wherein the analyte is from a sample.

85. The system of claim 84, wherein the sample is a complex sample.

86. The system of claim 85, wherein the complex sample comprises a mixture of proteins.

87. The system of claim 84, wherein the sample is a clinical sample.

88. The system of claim 87, wherein the clinical sample comprises a bodily fluid.

89. The system of claim 88, wherein the bodily fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

90. The system of any one of claims 63-89, wherein the nanopore is configured to couple to another recognition element.

91. The system of claim 90, wherein the recognition element and the further recognition element are configured to bind to different regions of the analyte.

92. The system of claim 90 or 91, wherein the recognition element and the further recognition element are configured to bind to different analytes.

93. The system of any one of claims 63-92, wherein the recognition element is a protein recognition element.

94. The system of claim 93, wherein the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monoclonal antibody, an affimer, an affimer, an Adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

95. The system of any one of claims 63-94, wherein the nanopore comprises an oligomeric assembly.

96. The system of claim 95, wherein at least one subunit of the oligomeric assembly comprises a subunit of the nanopore coupled to a recognition element.

97. The system of claim 96, wherein the recognition element is configured to be coupled to at least one subunit of the nanopore via a linker.

98. The system of claim 96 or 97, wherein at least one subunit of the nanopore comprises a monomer of a pore-forming toxin.

99. The system of claim 98, wherein the pore-forming toxin comprises cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2, a triple alpha-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof.

100. The system of claim 98 or 99, wherein the pore-forming toxin comprises one or more mutations.

101. The system of claim 100, wherein the pore-forming toxin is ClyA.

102. The system of claim 101, wherein the ClyA comprises an S110C mutation.

103. The system of any one of claims 63-102, wherein the interior region of the nanopore comprises an inner diameter of about 5 nanometers to about 20 nanometers.

104. A system comprising: (a) Fluid chamber; and (b) a membrane comprising a nanopore, wherein the membrane separates the fluid chamber into (1) a first side and (2) a second side, wherein the nanopore is coupled to a protein recognition element, wherein the protein recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

105. A nanopore comprising a region configured to couple to a recognition element, wherein the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

106. The nanopore of claim 105, wherein the recognition element is coupled to the nanopore via a linker.

107. The nanopore of claim 106, wherein the linker has a length of about 4 nanometers to about 8 nanometers.

108. The nanopore of claim 106 or 107, wherein the linker comprises an oligonucleotide, a duplex DNA complex, a chemically modified RNA complex, or any combination thereof.

109. The nanopore of any one of claims 106-108, wherein the nanopore is coupled to at least a portion of the linker.

110. The nanopore of claim 109, wherein the nanopore is coupled to a first oligonucleotide and the linker is coupled to a second oligonucleotide.

111. The nanopore of claim 110, wherein the first oligonucleotide and the second oligonucleotide are coupled together via nucleic acid hybridization.

112. The nanopore of any one of claims 105-111, wherein the recognition element is about 5 kilodaltons to about 50 kilodaltons.

113. The nanopore of any one of claims 105-112, wherein the recognition element is configured to couple to an analyte.

114. The nanopore of claim 113, wherein the recognition element is coupled to the analyte to enable movement of the recognition element.

115. The nanopore of claim 114, wherein the recognition element is configured not to move between an interior region of the nanopore and an exterior region of the nanopore when coupled to the analyte.

116. The nanopore of claim 114 or 115, wherein the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore when coupled to the analyte.

117. The nanopore of any one of claims 113-116, wherein the analyte is a protein, a peptide, a small molecule, a protein assembly, a protein / DNA assembly, a protein / RNA assembly, a steroid, a lipid, a lipid membrane, a lipid particle, a bacterium, a viral capsid, a viral particle, a cell, a dendrimer, a polymer, or any combination thereof.

118. The nanopore of any one of claims 113-117, wherein the analyte is a protein.

119. The nanopore of claim 118, wherein the protein is a folded protein, a native protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, or any combination thereof.

120. The nanopore of any one of claims 113-119, wherein the analyte is from a sample.

121. The nanopore of claim 120, wherein the sample is a complex sample.

122. The nanopore of claim 121, wherein the complex sample comprises a mixture of proteins.

123. The nanopore of claim 120, wherein the sample is a clinical sample.

124. The nanopore of claim 123, wherein the clinical sample comprises a bodily fluid.

125. The nanopore of claim 124, wherein the bodily fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

126. The nanopore of any one of claims 105-125, wherein the nanopore is configured to be coupled to another recognition element.

127. The nanopore of claim 126, wherein the recognition element and the further recognition element are configured to bind to different regions of an analyte.

128. The nanopore of claim 126 or 127, wherein the recognition element and the further recognition element are configured to bind different analytes.

129. The nanopore of any one of claims 105-128, wherein the recognition element is a protein recognition element.

130. The nanopore of claim 129, wherein the protein recognition element comprises a nanobody, a Fab fragment, a single-chain variable fragment (scFv), an antibody, a monoclonal antibody, an affimer, an affimer, an adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

131. The nanopore of any one of claims 105-130, wherein the nanopore comprises an oligomeric assembly.

132. The nanopore of claim 131, wherein at least one subunit of the oligomeric assembly comprises a subunit of the nanopore coupled to a recognition element.

133. The nanopore of claim 132, wherein the recognition element is configured to be coupled to at least one subunit of the nanopore via a linker.

134. The nanopore of claim 132 or 133, wherein at least one subunit of the nanopore comprises a monomer of a pore-forming toxin.

135. The nanopore of claim 134, wherein the pore-forming toxin comprises cytolysin A (ClyA), pleurolysin (PlyAB), YaxAB, perforin-2, a tripartite α-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof.

136. The nanopore of claim 134 or 135, wherein the pore-forming toxin comprises one or more mutations.

137. The nanopore of claim 136, wherein the pore-forming toxin is ClyA.

138. The nanopore of claim 137, wherein the ClyA comprises an S110C mutation.

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