Biological nanopores functionalized with nanobodies and related means and methods - Patent Application 20070122997

Abstract

The present invention relates to means and methods for analyte analysis using nanopore-based sensors, e.g., methods, nanopore systems, and devices for the stochastic detection of (label-free) analytes in complex samples, e.g., the specific detection of protein biomarkers in body samples. [0003] [0004] [0005] [0006] [0007] [0008] [0009] [0010] [0011] [0012] [0013] [0014] [0015] [0016] [0017] [0018] [0019] [0020] [0021] [0022] [0023] [0024] [0025] [0026] [0027] [0028] [0029] [0030] [0031] [0032] [0033] [0034] [0035] [0036] [0037] [0038] [0039] [0040] [0041] [0042] [0043] [0044] [0045] [0046] [0047] [0048] [0049] [0050] [0051] [0052] [0053] [0054] [0055] [0056] [0057] [0058] [0059] [0060] [0061] [0062] [0063] [0064] [006

Description

[Technical Field]

[0001] Incorporation by Reference This application claims the benefit of European Patent Application No. EP22211193.2, filed December 2, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Determining an analyte (e.g., a target analyte) in a sample is an important aspect of scientific research. The presence or absence of an analyte in a sample can be important from a clinical perspective. Summary of the Invention [Means for solving the problem]

[0003] In one aspect, the present disclosure provides a nanotube-based liquid transport device comprising a cis chamber containing a first conductive liquid medium in liquid communication with a trans chamber containing a second conductive liquid medium through a modified nanopore. and (c) measuring ionic current passing through the modified nanopore, wherein the modified nanopore is a biological nanopore functionalized with a proteinaceous recognition element R of 5-50 kDa, e.g., 10-40 kDa, capable of specifically binding to the target analyte, and wherein R dynamically translocates in and out of the nanopore to cause transient current blockage events, and wherein binding of R to the target analyte modulates its dynamic translocation, thereby inducing a change in the frequency and / or magnitude of the current blockage events, and wherein the change in the frequency and / or magnitude of the current blockage events indicates the presence of the target analyte in the sample.

[0004] In some embodiments, the engineered nanopore is an oligomeric assembly comprising or consisting of monomers of the general formula NLR, where N is a monomer of a pore-forming toxin having a maximum internal diameter (e.g., internal lumen diameter) of 5 nm to 20 nm, and L is a flexible linker attached to the cis entrance of the pore.

[0005] In some embodiments, binding of R to the target analyte increases the time that R resides outside the pore, thereby decreasing the frequency and / or magnitude of the current blockage events.

[0006] In some embodiments of any one of the preceding embodiments, the biological nanopore is functionalized with at least two different proteinaceous recognition elements, R' and R'', e.g., where R' and R'' bind to distinct sites on the target analyte.

[0007] In some embodiments of any one 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 the target analyte is a protein, and the protein is selected from the group consisting of a folded / native protein, a clinically relevant protein, a biomarker, a pathogenic protein, and a cell surface protein.

[0008] In some embodiments, the target analyte is a protein, preferably selected from the group consisting of a folded / native protein, a clinically relevant protein, a biomarker, a pathogenic protein, or a cell surface protein.

[0009] In some embodiments of any one of the preceding embodiments, the sample is a complex sample comprising a mixture of proteins, wherein the sample includes a clinical sample, e.g., a bodily fluid, e.g., whole blood, plasma, urine, feces, saliva, cerebrospinal fluid, breast milk, and sputum.

[0010] In one aspect, the present disclosure provides an engineered proteinaceous nanopore having a minimum pore diameter of 5 nm, the engineered proteinaceous nanopore functionalized via a flexible linker with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, that specifically reacts with a target analyte, preferably a target protein. In a preferred embodiment, R is capable of moving in and out of the pore to induce a blocking current.

[0011] In one aspect, the present disclosure provides a sensor system for protein analysis, the sensor system comprising a fluid-filled compartment separated by a membrane into first and second chambers, electrodes capable of applying an electric potential across the membrane, and at least one biological nanopore functionalized with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, capable of specifically binding to a target analyte, wherein R is positioned at the top of the nanopore via a flexible linker, allowing it to enter and exit the nanopore and trigger a transient current blockage event.

[0012] In one aspect, the present disclosure provides a nanopore sensor system comprising a cis chamber containing a first conductive liquid medium in liquid communication with a trans chamber containing a second conductive liquid medium through a modified nanopore, wherein the modified nanopore is a biological nanopore functionalized with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, capable of specifically binding to a target analyte, wherein R is tethered to the top of the nanopore and is capable of being internalized within the pore and dynamically moving in and out of the nanopore lumen to trigger a transient current blockage event.

[0013] In some embodiments of any one of the preceding embodiments, R is an IgG-based or non-IgG-based moiety, a nanobody, an scFv fragment, a Fab fragment, an affimer, a monobody, an affibody, an adnectin, a DARPin, or an anticalin, more preferably a nanobody.

[0014] In some embodiments of any one of the preceding embodiments, the biological nanopore is a pore-forming toxin, preferably having a maximum internal diameter (e.g., maximum lumen diameter) of 5 nm to 20 nm, more preferably cytolysin A (ClyA), pleurotolysin (PlyAB), YaxAB, perforin-2 (PFN2, PDB_ID 6SB3), tripartite alpha-pore-forming toxin (AhlB, 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 a mutant thereof that allows site-specific functionalization with a proteinaceous recognition element. In some embodiments, the biological nanopore is ClyA, preferably a mutant ClyA, more preferably a ClyA comprising the mutation S110C.

[0015] In some embodiments of any one of the preceding embodiments, the flexible linker is an oligonucleotide, preferably double-stranded DNA, or chemically modified RNA.

[0016] In some embodiments of any one 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 conjugated to the nanopore and a second oligonucleotide conjugated to R, the second oligonucleotide being complementary to the first oligonucleotide.

[0017] In one aspect, the present disclosure provides an array comprising a plurality of sensor systems according to any one of the preceding embodiments, the array comprising a plurality of separated reservoirs, each of the plurality of reservoirs comprising a nanopore modified with a different R element to enable detection of a different analyte.

[0018] In one aspect, the present disclosure provides a kit for preparing an array according to the preceding 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 protector strand.

[0019] In some embodiments of any one of the preceding embodiments, the use of the above-described method, nanopore, or sensor system, array, or kit can be used in 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.

[0020] In one aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, the nanopore system comprising: (1) a fluid chamber; and (2) a membrane comprising a nanopore, the membrane separating the fluid chamber into a first side and a second side, the nanopore being coupled to a recognition element; and (b) contacting the recognition element with an analyte.

[0021] In some embodiments, the recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore. In some embodiments, the recognition element is attached to the nanopore via a linker. In some embodiments, the linker is about 4 nanometers to about 8 nanometers in length. In some embodiments, the linker comprises an oligonucleotide, a double-stranded DNA molecule, a chemically modified RNA molecule, or any combination thereof. In some embodiments, the nanopore is attached to at least a portion of the linker. In some embodiments, the nanopore is attached to a first oligonucleotide, and the linker is attached to a second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide are attached together via nucleic acid hybridization.

[0022] In some embodiments of any one of the preceding embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to generate an electric potential across the nanopore. In some embodiments, movement of the recognition element between the interior region of the nanopore and the exterior region of the nanopore results in a change in the current of the nanopore system. In some embodiments, the method further comprises (c) measuring an ionic current passing through the interior region of the nanopore. In some embodiments, the method further comprises (d) detecting the presence or absence of the analyte via the change in the ionic current.

[0023] In some embodiments of any one of the preceding embodiments, the recognition element is between about 5 kilodaltons and about 50 kilodaltons. In some embodiments of any one of the preceding embodiments, the recognition element binds to the analyte. In some embodiments, the recognition element bound to the analyte causes translocation of the recognition element. In some embodiments, causing the translocation of the recognition element produces a change (i) in the frequency of the translocation of the recognition element or (ii) in the noise or magnitude of the current of the nanopore system. In some embodiments, when bound to the analyte, the recognition element cannot translocate between the interior region of the nanopore and the exterior region of the nanopore. In some embodiments, when bound to the analyte, the recognition element translocates between the interior region of the nanopore and the exterior region of the nanopore. In some embodiments, when bound to the analyte, the change (i) in the frequency of the translocation of the recognition element or (ii) in the noise or magnitude of the current blockade is reduced.

[0024] In some embodiments of any one of the preceding 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.

[0025] In some embodiments of any one 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.

[0026] In some embodiments of any one of the preceding embodiments, the analyte binds to a different analyte. In some embodiments, the protein is a folded protein, a naturally occurring protein, a clinically relevant protein, a biomarker, a pathogenic protein, a cell surface protein, or any combination thereof.

[0027] In some embodiments of any one of the preceding embodiments, the analytes are from a sample. In some embodiments, the sample binds to different analytes. 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 is a bodily fluid. In some embodiments, the bodily fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

[0028] In some embodiments of any one of the preceding 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 monobody, an affimer, an affibody, an adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

[0029] In some embodiments of any one of the preceding embodiments, the nanopore comprises an oligomer assembly. In some embodiments, at least one subunit of the oligomer assembly comprises a subunit of the nanopore bound to a recognition element. In some embodiments, the recognition element is bound to the at least one subunit of the nanopore via a linker. In some embodiments, the 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), pleurotolysin (PlyAB), YaxAB, perforin-2, tripartite alpha-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, the ClyA comprises an S110C mutation.

[0030] In some embodiments of any one of the preceding embodiments, the interior region of the nanopore comprises an interior diameter of about 5 nanometers to about 20 nanometers.

[0031] In one aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, the nanopore system comprising: (1) a fluid chamber; and (2) a membrane comprising a nanopore, the membrane separating the fluid chamber into a first side and a second side, the nanopore coupled to a protein recognition element, the protein recognition element 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.

[0032] In one aspect, the present disclosure provides a system comprising: (a) a fluid chamber; and (b) a membrane containing a nanopore, wherein the membrane separates the fluid chamber into (1) a first side and (2) a second side, and the nanopore is coupled to a recognition element.

[0033] In some embodiments, the recognition element is configured to translocate between an interior region of the nanopore and an exterior region of the nanopore.

[0034] In some embodiments, the recognition element is attached to the nanopore via a linker. In some embodiments, the linker is about 4 nanometers to about 8 nanometers in length. In some embodiments, the linker comprises an oligonucleotide, a double-stranded DNA molecule, a chemically modified RNA molecule, or any combination thereof. In some embodiments, the nanopore is attached to at least a portion of the linker. In some embodiments, the nanopore is attached to a first oligonucleotide, and the linker is attached to a second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide are attached together via nucleic acid hybridization.

[0035] In some embodiments of any one of the preceding embodiments, the system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to generate an electrical potential across the nanopore. In some embodiments, movement of the recognition element between an interior region of the nanopore and an exterior region of the nanopore results in a change in electrical current in the system.

[0036] In some embodiments of any one of the preceding embodiments, the recognition element is between about 5 kilodaltons and about 50 kilodaltons.

[0037] In some embodiments of any one of the preceding embodiments, the recognition element is configured to bind to an analyte. In some embodiments, the recognition element bound to the analyte is configured to cause movement of the recognition element. In some embodiments, causing the movement of the recognition element generates a change (i) in the frequency of the movement of the recognition element or (ii) in the noise or magnitude of the current of the system. In some embodiments, the recognition element is not configured to move between the internal region of the nanopore and the external region of the nanopore when bound to the analyte. In some embodiments, the recognition element is configured to move between the internal region of the nanopore and the external region of the nanopore when bound to the analyte. In some embodiments, when the recognition element is bound to the analyte, (i) the change in the frequency of the movement of the recognition element or (ii) the noise or magnitude of the current blockade is reduced. 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 bodily fluid. In some embodiments, the bodily fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

[0038] In some embodiments of any one of the preceding embodiments, the nanopore is configured to be coupled to another recognition element. In some embodiments, the recognition element and the another recognition element are configured to be coupled to another recognition element. In some embodiments, the recognition element and the another recognition element are configured to bind different analytes.

[0039] In some embodiments of any one of the preceding 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 monobody, an affimer, an affibody, an adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

[0040] In some embodiments of any one of the preceding embodiments, the nanopore comprises an oligomeric assembly. In some embodiments, at least one subunit of the oligomeric assembly comprises a subunit of the nanopore bound to a recognition element. In some embodiments, the recognition element is configured to be bound to the at least one subunit of the nanopore via a linker. In some embodiments, the 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), pleurotolysin (PlyAB), YaxAB, perforin-2, tripartite alpha-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, the ClyA comprises an S110C mutation.

[0041] In some embodiments of any one of the preceding embodiments, the interior region of the nanopore comprises an interior diameter of about 5 nanometers to about 20 nanometers.

[0042] In one aspect, the present disclosure provides the above system comprising: (a) a fluid chamber; and (b) a membrane including a nanopore, wherein the membrane separates the fluid chamber into (1) a first side and (2) a second side, and the nanopore is coupled to a protein recognition element, and the protein recognition element is configured to move between an interior region of the nanopore and an exterior region of the nanopore.

[0043] In one aspect, the present disclosure provides a nanopore comprising a region configured to bind a recognition element, wherein the recognition element is configured to translocate between an interior region of the nanopore and an exterior region of the nanopore.

[0044] In some embodiments, the recognition element is attached to the nanopore via a linker. In some embodiments, the linker is about 4 nanometers to about 8 nanometers in length. In some embodiments, the linker comprises an oligonucleotide, a double-stranded DNA complex, a chemically modified RNA complex, or any combination thereof. In some embodiments, the nanopore is attached to at least a portion of the linker. In some embodiments, the nanopore is attached to a first oligonucleotide, and the linker is attached to a second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide are attached together via nucleic acid hybridization.

[0045] In some embodiments of any one of the preceding embodiments, the recognition element is between about 5 kilodaltons and about 50 kilodaltons.

[0046] In some embodiments of any one of the preceding embodiments, the recognition element is configured to bind to an analyte. In some embodiments, the recognition element bound to the analyte causes the recognition element to move. In some embodiments, the recognition element is configured not to move between the interior region of the nanopore and the exterior region of the nanopore when bound to 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 virus capsid, a virus 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 naturally occurring 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 bodily fluid, hi some embodiments, the bodily fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, sputum, or any combination thereof.

[0047] In some embodiments of any one of the preceding embodiments, the nanopore is configured to be coupled to another recognition element. In some embodiments, the recognition element and the another recognition element are configured to bind to different regions of an analyte. In some embodiments, the recognition element and the another recognition element are configured to bind to different analytes.

[0048] In some embodiments of any one of the preceding 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 monobody, an affimer, an affibody, an adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

[0049] In some embodiments of any one of the preceding embodiments, the nanopore comprises an oligomer assembly. In some embodiments, at least one subunit of the oligomer assembly comprises a subunit of the nanopore bound to a recognition element. In some embodiments, the recognition element is bound to the at least one subunit of the nanopore via a linker. In some embodiments, the 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), pleurotolysin (PlyAB), YaxAB, perforin-2, tripartite alpha-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, the ClyA comprises an S110C mutation.

[0050] In one aspect, the present disclosure provides an array comprising a plurality of nanopore systems according to any one of the preceding embodiments, the array comprising a plurality of separated reservoirs, one or more of the nanopore systems comprising nanopores modified with different recognition elements to enable detection of different analytes.

[0051] In one aspect, the present disclosure provides a kit for preparing a system according to any one 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 protector strand.

[0052] In one aspect, the present disclosure provides a use of the 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.

[0053] Another aspect of the present disclosure provides a non-transitory readable storage medium containing machine-executable code that, upon execution by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0054] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto, the computer memory containing machine-executable code that, upon execution by the one or more computer processors, implements any of the methods described above or elsewhere herein.

[0055] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not as restrictive.

[0056] Incorporation by Reference

[0057] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification supersedes and / or is intended to take precedence over such conflicting material. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention relates to means 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 the probabilistic detection of analytes in complex samples (e.g., the detection of (label-free) protein biomarkers in body samples).

[0059] Nanopores are capable of stochastically sensing single molecules in real time and various analytes, e.g., metal ions. 1,2 , biomolecules 3,4 , nucleic acid 5,6,7 , polypeptide 8,9 For example, protein sensing using this technique has been used to detect 10,11,12 Protein characterization 13 and quantification 14 and the protein sensing can also be used to study protein unfolding kinetics. 15 , conformation changes 16,17,16 and ligand binding affinity 18,19Nanopores have additional advantages over existing techniques, such as enzyme-linked immunosorbent assays (ELISAs) and mass spectrometry, because they can provide insight into the mechanisms underlying the cellular processes involved in cellular processes. Furthermore, nanopores can be easily integrated into small, portable devices. 20 , making it highly suitable for applications in point-of-care diagnostics.

[0060] So far, various nanopore-based strategies have been explored for protein sensing. Directly, protein detection can be achieved by monitoring the current modulation induced by the direct binding / translocation of the protein inside or through the lumen of the pore. The key to this strategy is to select a pore with an appropriate shape that can accommodate the analyte. In the past decade, nanopores with large lumen areas, such as FraC 21 , Cytolysin (ClyA) 22 , PlyAB 23,24 , have been utilized to study folded proteins. For example, ClyA, which has a relatively large (approximately 6x6x10 nm) cylindrical lumen, has shown the ability to capture and characterize various folded proteins. 25 , as well as the interaction of peptide or DNA ligands with proteins. 22 Although these biological nanopores have proven effective, the fixed size and limited variety of protein pores available in nature limit their general application for sensing a wider variety of folded proteins of various sizes. In comparison, binder-assisted indirect detection of proteins outside the nanopore is emerging as a more versatile strategy for sensing folded proteins. 14,26,27,28,29,30These approaches allow for the detection of large proteins that do not fit within the nanopore, and by utilizing specific binding interactions to proteins, can enhance the specificity of protein sensing compared to naked nanopores. The strategies involve capturing the protein near the entrance of the nanopore, causing a change in current from the presence of an analyte, or transmitting binding interactions that occur outside the nanopore to the inside of the pore, thereby resulting in an altered ion flow through the pore. To date, various binding agents, such as biotin, have been used. 10 , aptamers 29 ,peptide 26 , protein domain 30 , have been chemically or genetically functionalized onto nanopores and have therefore been widely used for protein detection or protein-ligand binding studies.

[0061] In one example, Thakur and Movileanu established a platform for investigating protein-protein interactions. 30 In this study, a protein domain (RNase barnase, Bn) containing a flexible 12-amino acid peptide adaptor at its N-terminus was fused to the monomeric pore t-FhuA. Once the cognate ligand protein (Barstar, Bs) bound to the protein binder, the adaptor was pulled away from the pore opening, triggering distinct unblocking current events. 30This nanopore sensor demonstrated the ability to detect and quantify protein analytes in the presence of small amounts of serum; however, it had several drawbacks that limited its application in protein sensing. First, constructing nanopores with different protein ligands genetically linked (i.e., fused) to the nanopore was laborious, and this approach was not optimal for detecting a variety of different proteins. In addition, the preparation of the nanopore required protein refolding in urea and detergent, which carries the risk of causing many protein ligands to lose their function.

[0062] In another example, Bayley et al. (which is incorporated herein by reference in its entirety) demonstrated that an aptamer-modified α-hemolysin (α-HL) nanopore in which a 15-mer DNA aptamer (TBA) was hybridized to an oligonucleotide covalently linked to a cysteine ​​near the mouth of the pore allowed thrombin to be detected. 29 It is worth noting that the immobilization of DNA adapters on the pore confers modularity, allowing various analytes to be detected using the same nanopore structure by changing the aptamer. Nevertheless, given the diversity of aptamer structures and lengths (ranging from 15 to 80 bases) for different analytes, it is not possible to expect different aptamers to behave identically every time without extensive experimentation (e.g., the linker on each aptamer must be carefully tailored to enable detection possibilities), and therefore it is not possible to create a universal system using different aptamer binders for different desired analyses.

[0063] Soskine et al. 18(which is incorporated herein by reference in its entirety) attached an aptamer to the top of a ClyA nanopore and detected folded proteins by selective exoassociation and entry into the pore. In this approach, proteins that bind to the aptamer on the nanopore are allowed to enter the nanopore, while non-proteins that do not bind to the aptamer are prevented from entering the nanopore.

[0064] Importantly, problems can arise when using this platform with biological samples, such as blood, as aptamers can be rapidly degraded by nucleases.

[0065] In some embodiments, the present disclosure provides novel modular nanopore sensors that enable stochastic sensing of (label-free) protein targets without suffering from the drawbacks of known nanopore-based sensors. In some embodiments, the present disclosure provides a general, versatile system that enables specific and sensitive detection of proteins and protein-containing pathogens. The present disclosure provides novel modular nanopore sensors that enable stochastic sensing of analytes (e.g., proteins). In some embodiments, the nanopore sensors are general, versatile systems that enable specific and sensitive detection of proteins and protein-containing analytes (e.g., viruses, bacteria) in complex samples, such as blood or serum, including analytes larger than the pore diameter.

[0066] In some embodiments, the nanopore can be functionalized at the top (or mouth) of a large vestibule nanopore with a small (up to about 50 kDa) recognition element (e.g., a proteinaceous recognition element), such as a nanobody, that can translocate in and out of the pore to induce a blocking current. Adding an analyte (e.g., a target analyte) to the solution outside the pore (at the first or cis end) and forming a target-recognition element complex results in a change in the capture of the recognition element within the nanopore, thereby resulting in a change in the ionic current through the open pore. Nanobodies (or other small binding molecules) occlude the pore in the resting state, restricting the entry of other proteins. Importantly, in the resting state, the recognition element remains mostly within the nanopore, and proteins and other unwanted nonspecific background molecules from solution cannot enter the nanopore. Therefore, this approach does not suffer from background noise from non-cognate proteins in solution that could block the signal or block the nanopore.

[0067] In some embodiments, the recognition element can be a small recognition element (e.g., a proteinaceous recognition element), and this approach is highly specific for detecting a wide range of entities, such as proteins. Second, the recognition element is conveniently tethered to the nanopore as an exchangeable module, for example, by complementary strand hybridization. Therefore, nanopores functionalized with different recognition elements (e.g., nanobodies) can be easily obtained, and the preparation process is less laborious than existing methods. Third, this nuclease-tolerant nanopore design enables the sensing of proteins in biological fluids regardless of their size, where large proteins are detected outside the nanopore, while small proteins are detected within the lumen of the pore.

[0068] In some embodiments, pore design using nanobodies as exchangeable modules immobilized on a ClyA dodecamer via DNA duplex formation is demonstrated. By simply exchanging modules, nanopores functionalized with four different nanobodies were constructed, and all nanopore constructs demonstrated the ability to detect analytes. For example, by benefiting from the multivalent interaction between the SARS-CoV-2 spike protein and the multimerized Ty1 nanobody, this approach enabled the detection of proteins in the picomolar to low nanomolar range, even in the presence of blood. Thus, the present invention provides a novel and versatile strategy for highly specific and sensitive detection of various proteins in biofluids, regardless of their size, shape, and charge.

[0069] Accordingly, in one embodiment, the present invention provides a nanopore sensor system comprising a cis chamber containing a first conductive liquid medium in liquid communication with a trans chamber containing a second conductive liquid medium through a modified nanopore, wherein the modified nanopore is a biological nanopore functionalized with a proteinaceous recognition element R of 5 to 50 kDa, preferably 10 to 40 kDa, capable of specifically binding to the target analyte. In one embodiment, the invention provides a nanopore sensor system comprising a first side of a fluid chamber (e.g., a cis chamber) containing a first conductive liquid medium in fluid communication with a second side of a fluid chamber (e.g., a trans chamber) containing a second conductive liquid medium through a modified nanopore (e.g., a biological nanopore) functionalized with a 5-50 kDa, e.g., 10-40 kDa, recognition element (e.g., a proteinaceous recognition element) R capable of specifically binding to an analyte (e.g., a target analyte). The recognition element R is preferably tethered to the top of the nanopore and, based on its small size relative to the pore's large lumen dimension, is capable of being internalized within the pore. The recognition element R dynamically enters and exits the nanopore lumen (vestibule) to trigger a transient current blockage event. Binding of R to the analyte modulates its dynamic movement, thereby inducing a change in the frequency and / or magnitude of the current-blocking events, which change in the frequency and / or magnitude of the current-blocking events indicates the presence of the analyte in the sample.

[0070] In some embodiments, the nanopore of the nanopore sensor system of the present disclosure can be a biological nanopore. Alternatively, in some embodiments, the nanopore of the nanopore sensor system of the present disclosure can be a solid-state nanopore.

[0071] In some embodiments, the recognition element can be smaller than the internal diameter (e.g., lumen diameter) of the nanopore. In some cases, the recognition element can be about 0.1% to about 500% smaller than the internal diameter (e.g., lumen diameter). In some examples, the recognition element can be about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, or 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%, Approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% to approximately 360%, approximately 3 It can be 60% 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% or less.In some examples, the recognition element is about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 550%, at least about 600%, at least about 650 %, 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% smaller.In some examples, the recognition element is about 500% or less, about 490% or less, about 480% or less, about 470% or less, about 460% or less, about 450% or less, about 440% or less, about 430% or less, about 420% or less, about 410% or less, about 400% or less, about 390% or less, about 380% or less, about 370% or less, about 360% or less, about 350% or less, about 340% or less, about 330% or less, about 320% or less, about 310% or less, about 300% or less, about 290% or less, about 280% or less, about 270% or less, about 260% or less, about 250% or less, about 240% or less, about 230% or less, or about 220% or less than the internal diameter (e.g., lumen diameter) of the nanopore. , about 210% or less, about 200% or less, about 190% or less, about 180% or less, about 170% or less, about 160% or less, about 150% or less, about 140% or less, about 130% or less, about 120% or less, about 110% or less, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.5% or less, about 0.1%, or less than 0.1%. In some examples, the recognition element may be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about The increase can be 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% smaller.

[0072] In some embodiments, the recognition element can move through the interior region of the nanopore and the exterior region of the nanopore. In some cases, the interior region of the nanopore can be the channel of the nanopore. In some cases, the interior region of the nanopore can be the lumen of the nanopore on the first side of the fluid chamber. In some instances, the exterior region can be any region of the nanopore or of the lumen outside the channel of the nanopore. In some cases, the recognition element can move freely between the interior region and the exterior region of the nanopore.

[0073] In some embodiments, movement of the recognition element between the interior region of the nanopore and the exterior region of the nanopore can result in a change in the ionic current of the nanopore. In some embodiments, the movement of the recognition element into the interior region of the nanopore can reduce the ionic current (e.g., the magnitude or noise of the ionic current) traveling through the channel of the nanopore. In some instances, the reduction in the ionic current traveling through the channel of the nanopore can be measured.

[0074] In some embodiments, the 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, the movement of the recognition element into the interior region of the nanopore can increase the ionic current traveling through the channel of the nanopore.

[0075] In some embodiments, the movement of the recognition element into the exterior region of the nanopore can open the channel of the nanopore. In some cases, the movement of the recognition element into the exterior region of the nanopore can increase the ionic current traveling through the channel of the nanopore. In some instances, the increase in the ionic current traveling through the channel of the nanopore can be measured.

[0076] In some embodiments, the nanopore system can exist in two states: (i) the recognition element is in an interior region of the nanopore, and (ii) the recognition element is in an exterior region of the nanopore. In some cases, the change of the nanopore system from (i) to (ii) can be measured. In some cases, the frequency of the nanopore system can be measured between (i) and (ii). In some cases, the nanopore system can measure a change in the frequency of the movement of the recognition element. In some instances, the change in frequency can indicate the presence of the analyte. In some instances, the change in frequency can indicate the absence of the analyte. In some cases, the change in frequency can be used to determine the concentration of the analyte in solution. In some cases, the frequency of the movement of the recognition element in the nanopore system can be from 0.1 kilohertz (kHz) to about 1 megahertz (MHz). In some cases, the frequency of the nanopore system can be between 0.1 kHz and about 1 kHz, between about 1 kHz and about 100 kHz, or between about 100 kHz and about 1 MHz. In some cases, the frequency of the 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 the movement of the recognition element in the nanopore system can be about 1 MHz or less, about 1,000 kHz or less, about 900 kHz or less, about 800 kHz or less, about 700 kHz or less, about 600 kHz or less, about 500 kHz or less, about 400 kHz or less, about 300 kHz or less, about 200 kHz or less, about 100 kHz or less, about 90 kHz or less, about 80 kHz or less, about 70 kHz or less, about 60 kHz or less, about 50 kHz or less, about 40 kHz or less, about 30 kHz or less, about 20 kHz or less, about 10 kHz or less, about 5 kHz or less, about 1 kHz or less, about 0.1 kHz, or less than 0.1 kHz. In some cases, the frequency of the 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.

[0077] In some embodiments, the nanopore system can measure a change in the magnitude of the ionic current traveling through the nanopore. In some cases, the ionic current traveling through the nanopore can be increased when the recognition element is within an interior region of the nanopore. In some cases, the ionic current traveling through the nanopore can be decreased when the recognition element is within an interior region of the nanopore. In some cases, the ionic current traveling through the nanopore can be increased when the recognition element is within an exterior region of the nanopore. In some instances, a change in the magnitude of the ionic current can indicate the presence of the analyte. In some instances, a change in the magnitude of the ionic current can indicate the absence of the analyte. In some instances, the magnitude of the ionic current can be from about 1 picoampere (pA) to about 1,000 pA. In some cases, the magnitude 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 magnitude 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 magnitude of the ionic current can be about 1,000 pA or less, about 900 pA or less, about 800 pA or less, about 700 pA or less, about 600 pA or less, about 500 pA or less, about 400 pA or less, about 300 pA or less, about 200 pA or less, about 100 pA or less, about 90 pA or less, about 80 pA or less, about 70 pA or less, about 60 pA or less, about 50 pA or less, about 40 pA or less, about 30 pA or less, about 20 pA or less, about 10 pA or less, about 5 pA or less, about 1 pA, or less than 1 pA.In some cases, the magnitude of the ion 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.

[0078] In some embodiments, the nanopore system can measure changes in the noise of the ionic current traveling through the nanopore. In some cases, the change in the noise of the ionic current can refer to fluctuations (e.g., statistical fluctuations) of the ionic current. In some cases, the noise of the ionic current can increase when the recognition element is within the interior region of the nanopore. In some cases, the noise of the ionic current can decrease when the recognition element is within the interior region of the nanopore. In some cases, the noise of the ionic current can decrease when the recognition element is within the exterior region of the nanopore. In some cases, the noise of the ionic current can decrease when the recognition element is within the exterior region of the nanopore. 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 from 0.1 kilohertz (kHz) to about 1 megahertz (MHz). In some cases, the frequency of the nanopore system can be between 0.1 kHz and about 1 kHz, between about 1 kHz and about 100 kHz, or between about 100 kHz and 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 about 1 MHz or less, about 1,000 kHz or less, about 900 kHz or less, about 800 kHz or less, about 700 kHz or less, about 600 kHz or less, about 500 kHz or less, about 400 kHz or less, about 300 kHz or less, about 200 kHz or less, about 100 kHz or less, about 90 kHz or less, about 80 kHz or less, about 70 kHz or less, about 60 kHz or less, about 50 kHz or less, about 40 kHz or less, about 30 kHz or less, about 20 kHz or less, about 10 kHz or less, about 5 kHz or less, about 1 kHz or less, 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 change in the magnitude (e.g., standard deviation) of the noise. In some cases, the magnitude of the noise can be about 1 picoampere (pA) to about 1,000 pA. In some cases, the magnitude 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 magnitude 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 magnitude of the noise can be about 1,000 pA or less, about 900 pA or less, about 800 pA or less, about 700 pA or less, about 600 pA or less, about 500 pA or less, about 400 pA or less, about 300 pA or less, about 200 pA or less, about 100 pA or less, about 90 pA or less, about 80 pA or less, about 70 pA or less, about 60 pA or less, about 50 pA or less, about 40 pA or less, about 30 pA or less, about 20 pA or less, about 10 pA or less, about 5 pA or less, about 1 pA, or less than 1 pA. In some cases, the magnitude 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.

[0079] In some embodiments, the recognition element can specifically bind to an analyte. In some cases, the recognition element can bind to a small analyte (e.g., 0.1 nm to 10 nm). In some cases, the recognition element bound to the small analyte can move between the interior region of the nanopore and the exterior region of the nanopore. In some cases, the recognition element bound to the small analyte can move into the interior region of the nanopore. In some cases, the recognition element can bind to a large analyte (e.g., 10 nm or larger). In some cases, the recognition element bound to the large analyte cannot move between the interior region of the nanopore and the exterior region of the nanopore. In some cases, the recognition element bound to the large analyte cannot move into the interior region of the nanopore.

[0080] In some embodiments, the recognition element can specifically bind to an analyte. In some cases, the recognition element can specifically bind to the analyte. In some embodiments, binding of the recognition element to the analyte can result in the movement of the recognition element. In some cases, the recognition element, when bound to the analyte, may not be able to move into the interior region of the nanopore.

[0081] In some embodiments, the recognition element bound to the analyte can be larger than the internal diameter (e.g., lumen diameter) of the nanopore. In some cases, the recognition element bound to the analyte can be between about 0.1% and about 500% larger than the internal diameter (e.g., lumen diameter) of the nanopore. In some examples, the recognition element bound to the analyte can be between about 0.1% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, between about 60% and about 65%, between about 65% and about 70%, between about 70% and about 80%, or between about 80% and about 90%. % to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% to approximately 360%, It can be 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% or greater.In some examples, the recognition element bound to the analyte is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180% smaller than the internal diameter (e.g., lumen diameter) of the nanopore. The increase in the pore size can be at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or more than 500% greater.In some examples, the recognition element bound to the analyte is about 500% or less, about 490% or less, about 480% or less, about 470% or less, about 460% or less, about 450% or less, about 440% or less, about 430% or less, about 420% or less, about 410% or less, about 400% or less, about 390% or less, about 380% or less, about 370% or less, about 360% or less, about 350% or less, about 340% or less, about 330% or less, about 320% or less, about 310% or less, about 300% or less, about 290% or less, about 280% or less, about 270% or less, about 260% or less, about 250% or less, about 240% or less, about 230% or less, about 250% or less, about 260% or less, about 270% or less, about 280% or less, about 290% or less, about 280% or less, about 290% or less, about 260% or less, about 250% or less, about 240% or less, about 230% or less, about 250% or less, about 260% or less, about 270% or less, about 260% or less, about 270% or less, about 280% or less, about 280% or less, about 280% or less, about 280% or less, about 290% or less, about 290% or less, about 290% or less, about 260% or less, about 250% or less, about 240% or less, about 230% or less, about It can be 20% or less, about 210% or less, about 200% or less, about 190% or less, about 180% or less, about 170% or less, about 160% or less, about 150% or less, about 140% or less, about 130% or less, about 120% or less, about 110% or less, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.5% or less, about 0.1%, or less than 0.1% or greater. In some examples, the recognition element bound to the analyte is about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 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 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 610%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about The increase can be 80%, 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% greater.

[0082] In some embodiments, the recognition element bound to the analyte can be between about 0.1% and about 500% smaller than the internal diameter (e.g., lumen diameter) of the nanopore. In some examples, the recognition element bound to the analyte can be between about 0.1% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, between about 60% and about 65%, between about 65% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 100%, between about 100% and about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 45%, between about 45% and about 50%, between about 50% and about 55%, between about 55% and about 60%, between about 60% and about 65%, between about 65% and about 70%, between about 70% and about 100%, or between about 100% and about 200%. % to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, approximately 100% to approximately 110%, approximately 110% to approximately 120%, approximately 120% to approximately 130%, approximately 130% to approximately 140%, approximately 140% to approximately 150%, approximately 150% to approximately 160%, approximately 160% to approximately 170%, approximately 170% to approximately 180%, approximately 180% to approximately 190%, approximately 190% to approximately 200%, approximately 200% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 350% to approximately 360%, It can be 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% or less.In some examples, the recognition element bound to the analyte is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about or 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% smaller.In some examples, the recognition element bound to the analyte is about 500% or less, about 490% or less, about 480% or less, about 470% or less, about 460% or less, about 450% or less, about 440% or less, about 430% or less, about 420% or less, about 410% or less, about 400% or less, about 390% or less, about 380% or less, about 370% or less, about 360% or less, about 350% or less, about 340% or less, about 330% or less, about 320% or less, about 310% or less, about 300% or less, about 290% or less, about 280% or less, about 270% or less, about 260% or less, about 250% or less, about 240% or less, about 230% or less, about 250% or less, about 260% or less, about 270% or less, about 280% or less, about 290% or less, about 280% or less, about 290% or less, about 260% or less, about 250% or less, about 240% or less, about 230% or less, about 250% or less, about 260% or less, about 270% or less, about 260% or less, about 270% or less, about 280% or less, about 280% or less, about 280% or less, about 280% or less, about 290% or less, about 290% or less, about 290% or less, about 260% or less, about 250% or less, about 240% or less, about 230% or less, about It can be 20% or less, about 210% or less, about 200% or less, about 190% or less, about 180% or less, about 170% or less, about 160% or less, about 150% or less, about 140% or less, about 130% or less, about 120% or less, about 110% or less, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.5% or less, about 0.1%, or less than 0.1%. In some examples, the recognition element bound to the analyte is about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 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 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 610%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about The increase can be 80%, 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% smaller.

[0083] In some cases, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore can be reduced. In some examples, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore can be reduced by about 0.1% to about 500% compared to when the recognition element is not bound to the analyte. In some cases, when the recognition element is bound to the analyte, the 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% 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% 0% to approximately 200%, approximately 200% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 3 The decrease may be from 50% to about 360%, from about 360% to about 370%, from about 370% to about 380%, from about 380% to about 390%, from about 390% to about 400%, from about 400% to about 410%, from about 410% to about 420%, from about 420% to about 430%, from about 430% to about 440%, from about 440% to about 450%, 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, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 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%, at least about 5 The decrease can be 0%, 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, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore is about 500% or less, about 490% or less, about 480% or less, about 470% or less, about 460% or less, about 450% or less, about 440% or less, about 430% or less, about 420% or less, about 410% or less, about 400% or less, about 390% or less, about 380% or less, about 370% or less, about 360% or less, about 350% or less, about 340% or less, about 330% or less, about 320% or less, about 310% or less, about 300% or less, about 290% or less, about 280% or less, about 270% or less, about 260% or less, about 250% or less, about 260% or less, about 270% or less, about 280% or less, about 290% or less, about 280% or less, about 290% or less, about 260% or less, about 25 ...90% or less, about 260% or less, about 250% or less, about The decrease can be as low as 40% or less, about 230% or less, about 220% or less, about 210% or less, about 200% or less, about 190% or less, about 180% or less, about 170% or less, about 160% or less, about 150% or less, about 140% or less, about 130% or less, about 120% or less, about 110% or less, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.5% or less, about 0.1%, or less than 0.1%.In some cases, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore is about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 610%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 7 %, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0084] In some cases, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore can be reduced. In some examples, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore can be reduced by about 0.1% to about 500% compared to when the recognition element is not bound to the analyte. In some cases, when the recognition element is bound to the analyte, the 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% 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% 0% to approximately 200%, approximately 200% to approximately 210%, approximately 210% to approximately 220%, approximately 220% to approximately 230%, approximately 230% to approximately 240%, approximately 240% to approximately 250%, approximately 250% to approximately 260%, approximately 260% to approximately 270%, approximately 270% to approximately 280%, approximately 280% to approximately 290%, approximately 290% to approximately 300%, approximately 300% to approximately 310%, approximately 310% to approximately 320%, approximately 320% to approximately 330%, approximately 330% to approximately 340%, approximately 340% to approximately 350%, approximately 3 The decrease may be from 50% to about 360%, from about 360% to about 370%, from about 370% to about 380%, from about 380% to about 390%, from about 390% to about 400%, from about 400% to about 410%, from about 410% to about 420%, from about 420% to about 430%, from about 430% to about 440%, from about 440% to about 450%, 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, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore is at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 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%, at least about 5 The decrease can be 0%, 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, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore is about 500% or less, about 490% or less, about 480% or less, about 470% or less, about 460% or less, about 450% or less, about 440% or less, about 430% or less, about 420% or less, about 410% or less, about 400% or less, about 390% or less, about 380% or less, about 370% or less, about 360% or less, about 350% or less, about 340% or less, about 330% or less, about 320% or less, about 310% or less, about 300% or less, about 290% or less, about 280% or less, about 270% or less, about 260% or less, about 250% or less, about 260% or less, about 270% or less, about 280% or less, about 290% or less, about 280% or less, about 290% or less, about 260% or less, about 25 ...90% or less, about 260% or less, about 250% or less, about The decrease can be as low as 40% or less, about 230% or less, about 220% or less, about 210% or less, about 200% or less, about 190% or less, about 180% or less, about 170% or less, about 160% or less, about 150% or less, about 140% or less, about 130% or less, about 120% or less, about 110% or less, about 100% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, about 0.5% or less, about 0.1%, or less than 0.1%.In some cases, when the recognition element is bound to the analyte, the movement of the recognition element into the interior region of the nanopore is about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 610%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 7 %, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.

[0085] In some embodiments, when the recognition element is not bound to the analyte, the recognition element can move between the interior region of the nanopore and the exterior region of the nanopore. In some cases, when the recognition element is within the interior region of the nanopore, the recognition element can reduce the ionic current (e.g., the magnitude or noise of the ionic current) passing through the nanopore. In some cases, when the recognition element is within the interior region of the nanopore, the recognition element can increase the ionic current passing through the nanopore. In some instances, an increase and / or decrease in the ionic current can be measured. In some cases, an increase and / or decrease in the ionic current can indicate the absence of the analyte.

[0086] In some embodiments, when the recognition element is bound to the analyte, an interior region of the nanopore can open. In some cases, when the interior region of the nanopore is open, an ionic current can pass through the nanopore. In some cases, the passage of the ionic current through the nanopore can be measured. In some instances, measuring the ionic current through the nanopore can indicate the presence of the analyte.

[0087] In some embodiments, when the recognition element is bound to the analyte, there can be a decrease in the movement of the recognition element into the interior region of the nanopore. In some cases, when there is a decrease in the movement of the recognition element into the interior region of the nanopore, there can be an increase in the ionic current passing through the nanopore. In some cases, the increase in the ionic current passing through the nanopore can be measured. In some instances, measuring the increase in the ionic current passing through the nanopore can indicate the presence of the analyte. In some instances, measuring the increase in the ionic current passing through the nanopore can indicate the absence of the analyte. In some instances, measuring the decrease in the ionic current passing through the nanopore can indicate the presence of the analyte. In some instances, measuring the decrease in the ionic current passing through the nanopore can indicate the absence of the analyte.

[0088] The present disclosure provides a method for detecting the presence of at least one analyte in a sample using a nanopore system comprising a first side of a fluid chamber comprising a first conductive liquid medium in liquid communication with a second side of the fluid chamber comprising a second conductive liquid medium through a modified nanopore, the method comprising: (a) adding to the first side the sample to be analyzed for the presence of the analyte; (b) optionally applying a potential across the modified nanopore; and (c) measuring the ionic current passing through the modified nanopore, wherein the modified nanopore is a nanopore (e.g., a biological nanopore) functionalized with a 5-50 kDa, e.g., 10-40 kDa, recognition element (e.g., a proteinaceous recognition element) R capable of specifically binding to the analyte, as described herein above.

[0089] The present disclosure provides a method for detecting the presence of at least one target analyte in a sample using a nanopore system comprising a cis chamber containing a first conductive liquid medium in liquid communication with a trans chamber containing a second conductive liquid medium through a modified nanopore, the method comprising: (a) adding to the cis chamber a sample to be analyzed for the presence of a target analyte; (b) optionally applying a potential across the modified nanopore; and (c) measuring the ionic current passing through the modified nanopore. Including, Further provided is the above method, wherein the modified nanopore is a biological nanopore, as defined herein above, functionalized with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, capable of specifically binding to the target analyte, wherein R preferably dynamically translocates in and out of the nanopore to cause transient current blockage events, and wherein binding of R to the target analyte modulates its dynamic translocation, thereby inducing a change in the frequency and / or magnitude of the current blockage events, and wherein the change in the frequency and / or magnitude of current blockage events indicates the presence of the target analyte in the sample.

[0090] International Publication No. WO 2016 / 166232 relates to a nanopore-based sensor system comprising a nanopore and a protein adaptor internalized in the lumen of the nanopore. The present disclosure provides a nanopore in which a recognition element (e.g., a nanobody) is attached to the top of the nanopore and freely moves in and out of the lumen.

[0091] 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 instances, 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, an scFv, an Fab fragment, or any combination thereof. In some embodiments, the protein recognition element can be a single domain antibody, also known as a nanobody. For example, nanobodies (V) derived from heavy chain antibodies found in camelids are also known. H H fragments), or nanobodies derived from heavy chain antibodies of cartilaginous fish (variable neoantigen receptor V NARAlternatively, R can be a Fab fragment, such as an IgG-based moiety, such as a single-chain variable fragment (scFv).

[0092] Alternatively, in some embodiments, the protein recognition element can be a non-antibody-based protein molecule. In some instances, the non-antibody-based protein molecule can be an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, fynomer, gastrobody, Kunitz domain peptide, monobody, nanoCLAMP, optimer, repebody, pronectin, centyrin, obody, or any combination thereof. In some cases, the non-antibody-based protein molecule (e.g., R) can be a non-IgG based moiety, such as an affimer, an affibody (based on the Z domain of protein A from Staphylococcus aureus), a monobody and adnectin (based on the fibronectin type III domain), a DARPin (designed ankyrin repeat protein), or an anticalin (based on lipocalin). In some embodiments, the protein recognition element can be an antibody, nanobody, scFv, Fab fragment, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, gastrobody, Kunitz domain peptide, monobody, nanoCLAMP, optimer, repebody, pronectin, centilin, obody, or any combination thereof.

[0093] In some embodiments, the nanopore is capable of specifically binding to an analyte of 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. Da, 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, 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 or less, about 47 kDa, at least about 48 kDa, at least about 49 kDa, at least about 50 kDa, or greater than about 50 kDa.In some embodiments, the nanopore is capable of specifically binding to an analyte of about 50 kDa or less, about 49 kDa or less, about 48 kDa or less, about 47 kDa or less, about 46 kDa or less, about 45 kDa or less, about 44 kDa or less, about 43 kDa or less, about 42 kDa or less, about 41 kDa or less, about 40 kDa or less, about 39 kDa or less, about 38 kDa or less, about 37 kDa or less, about 36 kDa or less, about 35 kDa or less, about 34 kDa or less, about 33 kDa or less, about 32 kDa or less, about 31 kDa or less, about 30 kDa or less, about 29 kDa or less, about 28 kDa or less, about 27 kDa or less. Da or less, about 26 kDa or less, about 25 kDa or less, about 24 kDa or less, about 23 kDa or less, about 22 kDa or less, about 21 kDa or less, about 20 kDa or less, about 19 kDa or less, about 18 kDa or less, about 17 kDa or less, about 16 kDa or less, about 15 kDa or less, about 14 kDa or less, about 13 kDa or less, about 12 kDa or less, about 11 kDa or less, about 10 kDa or less, about 9 kDa or less, about 8 kDa or less, about 7 kDa or less, about 6 kDa or less, about 5 kDa or less, about 4 kDa or less, about 3 kDa or less, about 2 kDa or less, about 1 kDa, or less than about 1 kDa.

[0094] 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 capable of specifically binding to an analyte, 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 15 ... Da ~ about 35kDa, about 10kDa - about 40kDa, about 10kDa - about 45kDa, about 10kDa - about 50kDa, about 10kDa - about 55kDa, about 10kDa - about 60kDa, about 15kDa - about 20kDa, about 15kDa - about 25kDa, about 15kDa - about 30kD a, about 15kDa - about 35kDa, about 15kDa - about 40kDa, about 15kDa - about 45kDa, about 15kDa - about 50kDa, about 15kDa - about 55kDa, about 15kDa - about 60kDa, about 20kDa - about 25kDa, about 20kDa - about 30kDa, about 20kDa - Approximately 35kDa, approximately 20kDa to approximately 40kDa, approximately 20kDa to approximately 45kDa, approximately 20kDa to approximately 50kDa, approximately 20kDa to approximately 55kDa, approximately 20kDa to approximately 60kDa, approximately 25kDa to approximately 30kDa, approximately 25kDa to approximately 35kDa, approx. 25kDa to about 45kDa, about 25kDa to about 50kDa, about 25kDa to about 55kDa, about 25kDa to about 60kDa, about 30kDa to about 35kDa, about 30kDa to about 40kDa, about 30kDa to about 45kDa, about 30kDa to about 50kDa, about 30kDa 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 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.

[0095] In some embodiments, the nanopore is capable of specifically binding to an analyte of 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.

[0096] 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 (XNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), or any combination thereof. In some examples, the nucleic acid recognition element can be a naturally occurring nucleic acid molecule. In some examples, the nucleic acid recognition element can be a synthetic (e.g., laboratory-generated) nucleic acid molecule. In some examples, the nucleic acid recognition element can be a recombinant nucleic acid molecule.

[0097] In some embodiments, the nucleic acid recognition element can be about 1 kDa to about 50 kDa in size. In some cases, the nucleic acid recognition element can be about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 10 kDa to about 15 kDa, about 15 kDa to about 20 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 40 kDa to about 45 kDa, or about 45 kDa to about 50 kDa in size. In some cases, the nucleic acid recognition element is 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, 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, at least about 51 kDa, 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, 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 size.In some cases, the nucleic acid recognition element is about 50 kDa or less, about 49 kDa or less, about 48 kDa or less, about 47 kDa or less, about 46 kDa or less, about 45 kDa or less, about 44 kDa or less, about 43 kDa or less, about 42 kDa or less, about 41 kDa or less, about 40 kDa or less, about 39 kDa or less, about 38 kDa or less, about 37 kDa or less, about 36 kDa or less, about 35 kDa or less, about 34 kDa or less, about 33 kDa or less, about 32 kDa or less, about 31 kDa or less, about 30 kDa or less, about 29 kDa or less, about 28 kDa or less, about 27 kDa or less, about 26 kDa or less. The size can be less than about 25 kDa, less than about 24 kDa, less than about 23 kDa, less than about 22 kDa, less than about 21 kDa, less than about 20 kDa, less than about 19 kDa, less than about 18 kDa, less than about 17 kDa, less than about 16 kDa, less than about 15 kDa, less than about 14 kDa, less than about 13 kDa, less than about 12 kDa, less than about 11 kDa, less than about 10 kDa, less than about 9 kDa, less than about 8 kDa, less than about 7 kDa, less than about 6 kDa, less than about 5 kDa, less than about 4 kDa, less than about 3 kDa, less than about 2 kDa, less than about 1 kDa, or even smaller. In some cases, the nucleic acid recognition element 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 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, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kD 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 in size.

[0098] In some embodiments, the nanopore (e.g., a biological nanopore) may be functionalized with at least two different recognition elements (e.g., proteinaceous recognition elements), R' and R'', e.g., where R' and R'' bind to distinct sites (epitopes) on a given analyte (e.g., a target analyte), or where R' and R'' bind to different analytes (e.g., target analytes).

[0099] 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 selected from the group consisting of a folded / native protein, a peptide, a digested folded protein, a clinically relevant protein, a biomarker, a pathogen protein, a bacterial protein, a viral protein, a prokaryotic protein, a eukaryotic protein, a parasite protein, an antibody, a contractile protein, an enzyme, a hormone protein, a structural protein, a storage protein, a transport protein, a cell surface protein, or any combination thereof.

[0100] The sample can be of any type. The sample can be an aqueous solution containing one or more (biological) components. In one aspect, the sample is a complex sample containing a mixture of proteins, preferably wherein the sample is a clinical sample, more preferably a body fluid, such as whole blood, plasma, serum, semen, amniotic fluid, mucus, ascitic fluid, peritoneum, peritoneal fluid, extracellular fluid, intercellular fluid, lymphatic fluid, joint fluid, synovial fluid, tears, breast milk, bile, pericardial fluid, gastric acid, pleural effusion, sputum, urine, feces, saliva, cerebrospinal fluid, aqueous dilutions thereof, or combinations thereof.

[0101] The nanopore (eg, a biological nanopore) can be a pore-forming toxin, preferably a pore-forming toxin having a maximum internal diameter (eg, lumen diameter) of between 5 nm and 20 nm. In some cases, the pore is suitably selected from the group consisting of cytolysin A (ClyA), pleurotolysin (PlyAB), YaxAB, perforin-2 (PFN2, PDB_ID 6SB3), tripartite alpha-pore-forming toxin (AhlB, 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 variants thereof that allow site-specific functionalization with a recognition element (e.g., a proteinaceous recognition element).

[0102] In some embodiments, the internal 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 internal diameter (e.g., lumen diameter) can be about 20 nm or less, about 19 nm or less, about 18 nm or less, about 17 nm or less, about 16 nm or less, about 15 nm or less, about 14 nm or less, about 13 nm or less, about 12 nm or less, about 11 nm or less, about 10 nm or less, about 9 nm or less, about 8 nm or less, about 7 nm or less, about 6 nm or less, about 5 nm, or less than about 5 nm.

[0103] In some embodiments, the internal diameter (e.g., lumen diameter) can be about 5 nm to about 25 nm. In some embodiments, the internal diameter (e.g., lumen diameter) can be about 5 nm to about 6 nm, about 5 nm to about 7 nm, about 5 nm to about 8 nm, about 5 nm to about 9 nm, about 5 nm to about 10 nm, about 5 nm to about 12 nm, about 5 nm to about 14 nm, about 5 nm to about 16 nm, about 5 nm to about 18 nm, about 5 nm to about 20 nm, about 5 nm to about 25 nm, about 6 nm to about 7 nm, about 6 nm to about 8 nm, about 6 nm to about 9 nm, about 6 nm to about 10 nm, or about 6 nm to about 1 2nm, about 6nm to about 14nm, about 6nm to about 16nm, about 6nm to about 18nm, about 6nm to about 20nm, about 6nm to about 25nm, about 7nm to about 8nm, about 7nm to about 9nm, about 7nm to about 10nm, about 7nm to about 1 2nm, about 7nm to about 14nm, about 7nm to about 16nm, about 7nm to about 18nm, about 7nm to about 20nm, about 7nm to about 25nm, about 8nm to about 9nm, about 8nm to about 10nm, about 8nm to about 12nm, about 8nm to about 14nm, about 8nm to about 16nm, about 8nm to about 18nm, about 8nm to about 20nm, about 8nm to about 25nm, about 9nm to about 10nm, about 9nm to about 12nm, about 9nm to about 14nm, about 9nm to about 16nm, about 9nm ~about 18nm, about 9nm to about 20nm, about 9nm to about 25nm, about 10nm to about 12nm, about 10nm to about 14nm, about 10nm to about 16nm, about 10nm to about 18nm, about 10nm to about 20nm, about 10nm to about 2 5 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, about 14 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.

[0104] In some embodiments, the internal 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.

[0105] In some embodiments, the engineered nanopore may be an oligomeric assembly comprising or consisting of monomers of the general formula NLR, where N is a monomer of a pore-forming toxin having a maximum internal 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 proteinaceous recognition element) capable of specifically binding to the analyte (e.g., a target analyte). The flexible linker may have any size as long as it allows functional positioning of R relative to the pore entry / opening. In one embodiment, L has a length of about 4 to 8 nm, preferably 5 to 6 nm. L may be an oligonucleotide, preferably DNA, or chemically modified RNA (e.g., locked nucleic acid or RNA chemically modified at the 2' position with -F, -OMe for increased stability). In one embodiment, L comprises 8 to 20 nucleotides, for example, 10 to 18, 12 to 20, 8 to 14, or 16 to 20 nucleotides.

[0106] In some embodiments, the recognition element can be coupled to the nanopore on the first side of the nanopore system. In some embodiments, the recognition element can be coupled to the nanopore on the second side of the nanopore system. In some embodiments, the recognition element can be coupled to the nanopore on the first side of the nanopore system and the second side of the nanopore system.

[0107] In some embodiments, the analyte can be added to the first side of the nanopore system. In some embodiments, the analyte can be added to the second side of the nanopore system. In some embodiments, the analyte can be added to the first side of the nanopore system and the second side of the nanopore system.

[0108] In some embodiments, the recognition element can be bound to the nanopore on the first side of the nanopore system, and the analyte can be added to the first side of the nanopore system. In some embodiments, the recognition element can be bound to the nanopore on the second side of the nanopore system, and the analyte can be added to the second side of the nanopore system. In some embodiments, the recognition element can be bound to the nanopore on the first side of the nanopore and the second side of the nanopore, and the analyte can be added to the first side of the nanopore system and the second side of the nanopore system.

[0109] 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.

[0110] 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 instances, the covalent bond can be a non-polar covalent bond. In some instances, the covalent bond can be a polar covalent bond. In some instances, the recognition element can be directly coupled to the nanopore via a non-covalent bond. In some instances, the non-covalent bond can be a hydrophobic interaction, a van der Waals interaction, an electrostatic interaction, a hydrogen bond, or any combination thereof.

[0111] 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 linker. In some instances, the linker can be a flexible linker. In some cases, the nanopore can be coupled to the linker via a conjugation reaction. In some instances, the conjugation reaction can be a sulfide-based conjugation reaction, an ester reaction, a thioester reaction, an amide reaction, a native chemical ligation reaction, or any combination thereof. In some cases, the nanopore can be coupled to the linker via a bioconjugation reaction.In some examples, the bioconjugation reaction is a reaction of lysine with an N-hydroxysuccinimidyl (NHS) ester, an acylation reaction of lysine, a reaction of lysine with an isocyanate, a reaction of lysine with an isothiocyanate, a reaction of lysine with benzoyl fluoride, a reaction of cysteine ​​with maleimide, a reaction of cysteine ​​with iodoacetamide, a reaction of cysteine ​​with 2-thiopyridine, a reaction of cysteine ​​with 3-arylpropiolonitrile, an electrophilic aromatic substitution reaction, a reaction of tyrosine with a diazonium salt, a reaction of tyrosine with 4-phenyl-1,2,4-triazole-3,5-dione (PTAD), a Mannich reaction, a reaction of N-terminal serine or threonine with NaIO, a reaction of N-terminal cysteine ​​with iodoacetamide, a reaction of the N-terminus of an analyte with pyridoxal phosphate, a Staudinger ligation with an azide, a Huisgen cyclization of an azide, or the like. The alkylation may include rhodium cyclization, 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.

[0112] In some cases, the linker can be an amino acid linker. In some examples, the amino acid linker can include any combination of amino acids. In some cases, standard 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, the amino acid can be an unnatural amino acid. In some cases, the unnatural amino acid is selected from the group consisting of hydroproline, β-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, pyroglutamic acid, naphthylalanine, Abu, DAB, methionine sulfoxide, methionine sulfone, α-amino-n-butyric acid, norvaline, alloisoleucine, t-leucine, α-amino-n-heptanoic acid, pipecolic acid, allothreonine, homocysteine, homoserine, α,β-diamin-3-amino-4-methyl-2-propanol, α-amino-n-butyric acid, α-amino-n-butanoic ... The amino acid linker may comprise any combination of standard amino acids and unnatural amino acids, such as α-hydroxy-γ-aminobutyric acid, β-aminopropionic acid, α,γ-diaminobutyric acid, β-alanine, β-amino-n-butyric 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 linker may comprise any combination of standard amino acids and unnatural amino acids. In some cases, the amino acid linker may be a combination of glycine and serine amino acids. In some cases, the amino acid linker may be a combination of aspartic acid and serine amino acids. In some examples, the amino acid linker may comprise from about 1 amino acid to about 10 amino acids.In some examples, the amino acid linker can comprise at least 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 10 amino acids, at least about 10 amino acids, or more than 10 amino acids. In some examples, the amino acid linker can comprise no more than about 10 amino acids, no more than about 9 amino acids, no more than about 8 amino acids, no more than about 7 amino acids, no more than about 6 amino acids, no more than about 5 amino acids, no more than about 4 amino acids, no more than about 3 amino acids, no more than about 2 amino acids, no more than about 1 amino acid, or less than 1 amino acid. In some examples, the amino acid linker can comprise about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, or about 10 amino acids.

[0113] In some cases, the linker can be a polymer linker. In some instances, the polymer linker can be ethylene glycol, polyethylene glycol, or a combination thereof.

[0114] 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.

[0115] 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-interacting linker. In some cases, the chemical linker can be a click chemistry linker. In some cases, the click chemistry linker can include one or more click reagents. In some cases, the one or more click reagents can include 1,3-dipolar families, epoxides, aziridines, cyclic sulfates, epoxides, aziridines, cyclic sulfates, oxine ethers, hydrazones, aromatic heterocycles, or any combination thereof.

[0116] In some cases, the linker can be a nucleic acid linker. In some cases, the nucleic acid linker can be a polynucleic acid linker.

[0117] In some embodiments, the linker comprises 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 comprises about 20 nucleotides or less, about 19 nucleotides or less, about 18 nucleotides or less, about 17 nucleotides or less, about 16 nucleotides or less, about 15 nucleotides or less, about 14 nucleotides or less, about 13 nucleotides or less, about 12 nucleotides or less, about 11 nucleotides or less, about 10 nucleotides or less, about 9 nucleotides or less, about 8 nucleotides or less, about 7 nucleotides or less, about 6 nucleotides or less, about 5 nucleotides or less, about 4 nucleotides or less, about 3 nucleotides or less, about 2 nucleotides or less, about 1 nucleotide, or less than about 1 nucleotide.

[0118] In some embodiments, the linker comprises from about 1 nucleotide to about 20 nucleotides. In some embodiments, the linker is from about 1 nucleotide to about 2 nucleotides, from about 2 nucleotides to about 3 nucleotides, from about 3 nucleotides to about 4 nucleotides, from about 4 nucleotides to about 5 nucleotides, from about 5 nucleotides to about 6 nucleotides, from about 6 nucleotides to about 7 nucleotides, from about 7 nucleotides to about 8 nucleotides, from about 8 nucleotides to about 9 nucleotides, from about 8 nucleotides to about 10 nucleotides, from about 8 nucleotides to about 12 nucleotides, from about 8 nucleotides to about 13 nucleotides, from about 8 nucleotides to about 14 nucleotides, from about 8 nucleotides to about 15 nucleotides, from about 8 nucleotides to about 16 nucleotides, from about 8 nucleotides to about 17 nucleotides, from about 8 nucleotides to about 18 nucleotides, from about 8 nucleotides to about 19 nucleotides, from about 8 nucleotides to about 20 nucleotides, from about 9 nucleotides to about 10 nucleotides, from about 9 nucleotides to about 12 nucleotides, from about 9 nucleotides to about 13 nucleotides, from about 9 nucleotides to about 14 nucleotides, from 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, 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 1 6 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.

[0119] In some embodiments, the linker comprises about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 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.

[0120] 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 conjugated to the nanopore and a second oligonucleotide conjugated to R that is complementary to the first oligonucleotide.

[0121] In one embodiment, the invention provides a nanopore (e.g., an engineered nanopore, e.g., a proteinaceous nanopore) having a minimum pore diameter of 5 nm that is functionalized via a flexible linker with a recognition element (e.g., a proteinaceous recognition element) R of 5 to 50 kDa, e.g., 10 to 40 kDa, 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 translocate in and out of the pore, causing a blocking current. In some cases, the recognition element R is tethered to the top of the nanopore.

[0122] In one aspect, the present invention provides an engineered proteinaceous nanopore with a minimum pore diameter of 5 nm functionalized via a flexible linker with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, that specifically reacts with a target analyte, preferably a target protein. In a preferred embodiment, R is capable of translocating in and out of the pore to induce a blocking current. In a preferred embodiment, the recognition element R is tethered to the top of the nanopore.

[0123] 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) functionalized with a recognition element (e.g., a proteinaceous recognition element) R of 5-50 kDa, preferably 10-40 kDa, capable of specifically binding to an analyte, wherein R is positioned, e.g., via a flexible linker, at the top of the nanopore to allow it to enter and exit the nanopore and trigger a transient current blockade event.

[0124] 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 an electric potential across the membrane, and at least one biological nanopore functionalized with a recognition element (e.g., a proteinaceous recognition element) R of 5-50 kDa, preferably 10-40 kDa, capable of specifically binding to a target analyte, wherein R is positioned, preferably via a flexible linker at the top of the nanopore, to allow it to enter and exit the nanopore and trigger a transient current blockage event.

[0125] In one embodiment, the invention provides an array comprising a plurality of sensor systems according to the invention, as well as methods and kits for preparing such an array. Preferably, the array comprises a plurality of separate reservoirs, each of which contains a nanopore modified with a different R element to allow for the detection of a different analyte.

[0126] In one aspect, the present disclosure provides a kit for preparing an array according to the present invention, the kit 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 protector strand complementary to the original strand.

[0127] Also provided herein are methods of use of the methods, nanopore or sensor systems, arrays or kits in single protein detection, preferably in combination with high-throughput analysis.

[0128] definition

[0129] Recognition element R

[0130] In some embodiments, the recognition element (e.g., a proteinaceous recognition element) R is tethered to the top of the nanopore and can dynamically move in and out of the nanopore lumen (vestibule) to trigger transient current blockage events. Binding of R to the analyte (e.g., target analyte) modulates this dynamic movement, thereby inducing a change in the frequency and / or magnitude of the current blockage events, where the change in the frequency and / or magnitude of the current blockage events indicates the presence of the analyte (e.g., target analyte) in the sample. Typically, binding of R to the analyte (e.g., target analyte) increases the time that R resides outside the bore, thereby decreasing the frequency of the current blockage events.

[0131] In some embodiments, to enable dynamic movement in and out of the nanopore, the R moiety for use in the present invention is much smaller than a conventional IgG antibody, having a molecular weight of approximately 150 kDa and composed of two distinct polypeptide chains. Typical dimensions of IgG are approximately 14.5 nm x 8.5 nm x 4.0 nm, with a 13.7 nm spacing between antigen-binding sites. The molecular weight of R ranges from 5 to 50 kDa, preferably 10 to 40 kDa, 10 to 35 kDa, 10 to 30 kDa, and more preferably 12 to 15 kDa. Preferred R moieties have dimensions in the single-digit nanometer range, e.g., 1 to 5 x 1 to 5 nm.

[0132] 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 about 50 kDa or less, about 45 kDa or less, about 40 kDa or less, about 35 kDa or less, about 30 kDa or less, about 25 kDa or less, about 20 kDa or less, about 15 kDa or less, about 10 kDa or less, about 5 kDa or less, or less than about 5 kDa.

[0133] In some embodiments, the molecular weight of the recognition element can be from about 5 kDa to about 60 kDa. In some embodiments, the molecular weight of the recognition element 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, Approximately 10kDa to approximately 40kDa, approximately 10kDa to approximately 45kDa, approximately 10kDa to approximately 50kDa, approximately 10kDa to approximately 55kDa, approximately 10kDa to approximately 60kDa, approximately 15kDa to approximately 20kDa, approximately 15kDa to approximately 25kDa, approximately 15kDa to approximately 30kDa, approximately 15kDa ~about 35kDa, about 15kDa to about 40kDa, about 15kDa to about 45kDa, about 15kDa to about 50kDa, about 15kDa to about 55kDa, about 15kDa to about 60kDa, about 20kDa to about 25kDa, about 20kDa to about 30kDa, about 20kDa to about 35kDa , about 20kDa to about 40kDa, about 20kDa to about 45kDa, about 20kDa to about 50kDa, about 20kDa to about 55kDa, about 20kDa to about 60kDa, about 25kDa to about 30kDa, about 25kDa to about 35kDa, about 25kDa to about 40kDa, about 25kD a ~ about 45kDa, about 25kDa - about 50kDa, about 25kDa - about 55kDa, about 25kDa - about 60kDa, about 30kDa - about 35kDa, about 30kDa - about 40kDa, about 30kDa - about 45kDa, about 30kDa - about 50kDa, about 30kDa - about 55kD a, 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 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.

[0134] 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.

[0135] In some embodiments, the recognition element (e.g., R) can be a single domain antibody, also known as a nanobody. For example, nanobodies derived from heavy chain antibodies found in camelids (also known as VHH fragments) or nanobodies derived from heavy chain antibodies of cartilaginous fish (also known as novel variable antigen receptor (VNAR) fragments). Alternatively, R can be a Fab fragment, e.g., an IgG-based moiety, e.g., a single-chain variable fragment (scFv). Alternatively, R can be a non-IgG-based moiety, e.g., an affimer, an affibody (based on the Z domain of protein A from Staphylococcus aureus), a monobody, and an adnectin (based on the fibronectin type III domain), a DARPin (designed ankyrin repeat protein), or an anticalin (based on lipocalin).

[0136] In one aspect, R is a Fab fragment. In some embodiments, the fragment antigen-binding region (Fab region) is the region on an antibody that binds to an antigen. It is composed of one constant domain and one variable domain from each of the heavy and light chains. Fab fragment antibodies can be generated by papain digestion of a whole IgG antibody to remove the entire Fc fragment, including the hinge region. These antibodies are monovalent and contain only one antigen-binding site. The molecular weight of a Fab fragment is approximately 50 kDa. The variable domain contains a paratope (antigen-binding site) that contains a pair of complementarity-determining regions at the amino terminus of the monomer. Thus, each arm of the Y binds to an epitope on an antigen.

[0137] In another embodiment, R is based on a single-chain variable fragment (scFv), a fusion protein of approximately 30-35 kDa and 2 x 3 nm, linking the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. See Asaadi et al., Biomarker Research volume 9, Article number: 87 (2021), which is incorporated herein by reference in its entirety.

[0138] In some embodiments, R is a nanobody, or so-called VHH antibody, originally referred to as a heavy chain antibody (HCAb), also known as a single domain antibody. Nanobodies, consisting of only the variable domains of camelid-derived heavy chain antibodies, are a rapidly growing family of potent protein binders. 31,32,33 Ablynx, Belgium 登録商標The nomenclature "nanobody", originally adopted by [the authors], comes from their nanometer size, i.e., 4 nm in length, 2.5 nm in width, and molecular weight of only 12-14 kD. Nanobodies are nuclease-resistant, which is more advantageous for indirect protein sensing compared to aptamers. Apart from that, nanobodies can be easily produced as recombinant proteins in bacterial expression systems and can be easily equipped with customized tags without affecting their function. 35,36,37,38 Furthermore, multimerization of nanobodies can improve their binding affinity. 39 , and the detection sensitivity can be improved. 40 It has been reported that:

[0139] In some embodiments, R is a non-IgG-based moiety, such as an Affimer. 登録商標 Affimer molecules are small proteins that bind to analytes (e.g., target analytes) with affinities in the nanomolar range. Affimer reagents, 12–14 kDa, are small non-antibody binding proteins, approximately 10 times smaller than IgG antibodies, and they are less than 4 nm in length. These engineered non-antibody binding proteins have been designed to mimic the molecular recognition properties of monoclonal antibodies in a variety of applications.

[0140] In a preferred aspect, R is a nanobody, or so-called VHH antibody, originally referred to as a heavy chain antibody (HCAb), also known as a single domain antibody. In some embodiments, R is a non-IgG-based moiety, such as an Affimer. Affimer 登録商標 The molecules are small proteins that bind to target proteins with affinities in the nanomolar range, as described herein above.

[0141] See also Bedford et al. (Biophysical Reviews volume 9, pp. 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 that are suitable for use in nanopore functionalization.

[0142] In some embodiments, a single nanopore may be conjugated to R moieties of the same type, e.g., all nanobodies, scFvs, or affimers, or mixed types of R moieties, a combination of two or more of the types described herein above, e.g., scFvs and affimers, or scFvs and nanobodies.

[0143] In some embodiments, the nanopore can be coupled to one or more recognition elements, hi 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 about 50 or fewer recognition elements, about 45 or fewer recognition elements, about 40 or fewer recognition elements, about 35 or fewer recognition elements, about 30 or fewer recognition elements, about 25 or fewer recognition elements, about 20 or fewer recognition elements, about 18 or fewer recognition elements, about 15 or fewer recognition elements, about 12 or fewer recognition elements, about 10 or fewer recognition elements, about 5 or fewer recognition elements, about 4 or fewer recognition elements, about 3 or fewer recognition elements, about 2 or fewer recognition elements, about 1 or fewer recognition elements, or less than 1 recognition element.

[0144] 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 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 elements, about 3 to about 30 recognition elements, about 3 to about 40 recognition elements, about 3 to about 50 recognition elements, about 4 to about 5 recognition elements, about 4 to about 10 recognition elements, about 4 to about 15 recognition elements, about 4 to about 20 recognition elements, about 4 to about 25 recognition elements, about 4 to about 30 recognition elements, about 4 to about 40 recognition elements, about 4 to about 50 recognition elements, about 5 to about 10 recognition elements, about 5 to about 15 recognition elements, about 5 to about 20 recognition elements recognition elements, about 5 recognition elements to about 25 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,It can be bound to about 15 to about 40 recognition elements, about 15 to about 50 recognition elements, about 20 to about 25 recognition elements, about 20 to about 30 recognition elements, about 20 to about 40 recognition elements, about 20 to about 50 recognition elements, about 25 to about 30 recognition elements, about 25 to about 40 recognition elements, about 25 to about 50 recognition elements, about 30 to about 40 recognition elements, about 30 to about 50 recognition elements, or about 40 to about 50 recognition elements.

[0145] 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.

[0146] In some embodiments, the one or more recognition elements can be bound to the same region of the analyte. In some embodiments, the one or more recognition elements can be bound to different regions of the analyte. In some embodiments, the one or more recognition elements can be bound to different analytes.

[0147] In some embodiments, R is attached or positioned at the top of the nanopore (e.g., the cis side of the top of the nanopore) via a flexible tether, allowing contact with an analyte added to the first side (e.g., the cis chamber). The linker coupling site to R is selected on the surface, loop, or end of the protein so as to leave the binding domain motif of R free and sterically unhindered. Common conjugation sites (e.g., sites for attaching R to beads or surfaces) are well known for many suitable R binders. The site of nanopore modification by R is selected to allow R to dynamically enter and exit the interior of the nanopore, or at least to cause transient current blockade events in the absence of an analyte (e.g., a target analyte), where binding of the analyte (e.g., a target molecule) to R modulates its dynamic movement, thereby inducing a change in the frequency and / or magnitude of current blockade events.

[0148] In some embodiments, binding of R to the analyte (e.g., target analyte) increases the time that R resides 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 the current blockage events. Alternatively, binding of R to the analyte (e.g., target analyte) decreases the time that R resides outside the pore, thereby increasing the frequency of current blockage events. For example, binding to a highly charged analyte can promote internalization through a change in the electrophoretic forces acting on the R-analyte complex.

[0149] In other embodiments, binding of R to the analyte (e.g., a target analyte) alters the ionic current flowing through the nanopore when the R-analyte complex is within the nanopore. For example, in embodiments where the R-analyte complex can enter the nanopore cavity, the presence of the analyte either increases or decreases the ionic current flowing through the nanopore relative to the unbound R current level as a result of changes in excluded volume or electrostatics. In some embodiments, the R-analyte complex exhibits multiple current levels as a result of the complex being located at different locations within the nanopore. The change in current level can be used to detect the presence of the analyte. The change and absolute value of the current level associated with the R-analyte complex within the nanopore can 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 may be designed to universally bind to a particular analyte (e.g., a particular target protein analyte) that exists in multiple post-translationally or otherwise modified forms in a mixture, e.g., by binding to an unmodified epitope region of the protein, altering the ionic current in a manner that is characteristic of the modified region of the protein analyte facing the interior of the nanopore.

[0150] In some embodiments, the nanopore (e.g., a biological nanopore) may be functionalized with a single type of R to enable sensing of a single analyte (e.g., a target analyte), or may 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", each of which specifically binds to a different analyte (e.g., a target analyte), thus 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", each of which specifically binds to a distinct site (epitope) on the same analyte (e.g., a target analyte). In this way, the binding strength and duration of the bound state of the analyte can be increased, as well as the specificity for binding to a given analyte (e.g., a target analyte) over other background analytes can be increased.

[0151] In some embodiments, R is preferably positioned on the first (or cis) side of the top of the nanopore via a flexible tether that allows R to move in and out of the pore as described herein.

[0152] In one aspect, the recognition element R is directly attached to the nanopore. In one aspect, flexibility, such as flexibility that allows R to move in and out of the pore as described herein to allow rotation or bending of R, can be achieved by the bond that attaches R to the nanopore. In one aspect, such flexibility can be achieved by flexibility within R or within the nanopore. In some examples, the nanopore can be conjugated to a flexible region of R, such as a flexible N- or C-terminus of R, or a flexible loop on the outer surface of R. Alternatively, or in combination, R can be attached to a flexible region, such as a flexible N- or C-terminus of the nanopore, or a flexible loop on the outer surface of the nanopore.

[0153] In one aspect, the engineered nanopore is an oligomeric assembly comprising or consisting of monomers of the general formula NLR, where N is a monomer of a pore-forming toxin having a maximum internal 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).

[0154] In some embodiments, the nanopore is a monomeric protein. In some cases, the nanopore can be formed from a single beta-barrel similar to an outer membrane porin structure. In some embodiments, the nanopore can be monomeric, formed by genetic fusion or conjugation of multiple monomeric protein units.

[0155] In some embodiments, the nanopore can comprise an oligomeric assembly. In some cases, at least one subunit of the oligomeric assembly comprises a subunit of the nanopore bound to a recognition element. In some cases, the at least one subunit can be directly bound to the recognition element. In some cases, the at least one subunit can be indirectly bound to the recognition element. In some cases, the at least one subunit can be bound to the at least one subunit of the nanopore via a linker. In some examples, the at least one subunit of the nanopore comprises a monomer of a pore-forming toxin. In some instances, the pore-forming toxin can be cytolysin A (ClyA), pleurotolysin (PlyAB), YaxAB, perforin-2, tripartite alpha-pore-forming toxin, secretin, Helicobacter pylori OMC, SpoIIIAG, Gasdermin-A3, or any combination thereof. In some instances, the pore-forming toxin can include one or more mutations. In some instances, the pore-forming toxin is ClyA. In some instances, the ClyA pore-forming toxin can have an S110C mutation.

[0156] In some embodiments, the nanopore has an internal diameter (e.g., lumen diameter) that 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 nanopore has an internal diameter (e.g., lumen diameter) that can be about 20 nm or less, about 19 nm or less, about 18 nm or less, about 17 nm or less, about 16 nm or less, about 15 nm or less, about 14 nm or less, about 13 nm or less, about 12 nm or less, about 11 nm or less, about 10 nm or less, about 9 nm or less, about 8 nm or less, about 7 nm or less, about 6 nm or less, about 5 nm or less, or less than about 5 nm.

[0157] In some embodiments, the nanopore can have an internal diameter (e.g., lumen diameter) that is from about 5 nm to about 25 nm. In some embodiments, the nanopore can have an internal diameter (e.g., lumen diameter) that is 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, from about 7 nm to about 16 nm, from about 7 nm to about 18 nm, from about 7 nm to about 20 nm, from about 7 nm to about 25 nm, from about 8 nm to about 9 nm, from about 8 nm to about 10 nm, from about 8 nm to about 12 nm, from about 8 nm to about 14 nm, from about 8 nm to about 16 nm, from about 8 nm to about 18 nm, from about 8 nm to about 20 nm, from about 8 nm to about 25 nm, from about 9 nm to about 10 nm, from about 9 nm to about 12 nm, from about 9 nm to about 14 nm, from about 9 nm to about 16 nm, from about 9 nm to about 18 nm, from about 9 nm to about 20 nm, from about 9 nm to about 25 nm, from about 10 nm to about 12 nm, from about 10 nm to about 14 nm, from about 10 nm to about 16 nm, from about 10 nm to about 18 nm, from about 10 nm to about 20 nm, from about 10 nm to about 25 nm, from about 12 nm to about 14 nm, from about 12 nm to about 16 nm, from about 12 nm to about 18 nm, from about 12 nm to about 20 nm, from about 12 nm to about 25 nm, from about 14 nm to about 16 nm, from about 14 nm to about 18 nm, from about 14 nm to about 20 nm, from about 14 nm to about 25 nm, from about 16 nm to about 18 nm, from about 16 nm to about 20 nm, from about 16 nm to about 25 nm, from about 18 nm to about 20 nm, from about 18 nm to about 25 nm, or from about 20 nm to about 25 nm.

[0158] In some embodiments, the nanopore has an internal diameter (e.g., lumen diameter) that 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.

[0159] In some embodiments, the linker size is variable and depends, among other things, on the binding site of R, the dimensions of the pore, and / or the shape of the pore. One of skill in the art can readily select a suitable linker length and shape. In some embodiments, a suitable linker length and shape, combined with the location of the linker attachment point on the nanopore, can be a linker that provides a suitable distance of R from the entrance of the pore.

[0160] In some embodiments, the flexible linker can be of any size, so long as it allows for functional positioning of R relative to the pore entry / opening. In one embodiment, L has a length of about 2-8 nm, preferably 4-6 nm. It should be understood that this length of L is the shortest distance between the nanopore attachment point and the R attachment point, and that the remainder of the linker portion can be of almost any length.

[0161] 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 more than about 8 nm. In some embodiments, the linker has a length of about 8 nm or less, about 7.5 nm or less, about 7 nm or less, about 6.5 nm or less, about 6 nm or less, about 5.5 nm or less, about 5 nm or less, about 4.5 nm or less, about 4 nm or less, about 3.5 nm or less, about 3 nm or less, about 2.5 nm or less, about 2 nm or less, about 1.5 nm or less, about 1.0 nm or less, about 0.5 nm or less, or less than about 0.5 nm.

[0162] 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 m ~ about 3 nm, about 2.5 nm - about 3.5 nm, about 2.5 nm - about 4 nm, about 2.5 nm - about 4.5 nm, about 2.5 nm - about 5 nm, about 2.5 nm - about 5.5 nm, about 2.5 nm - about 6 nm, about 2 .5nm to about 7nm, about 2.5nm to about 8nm, about 3nm to about 3.5nm, about 3nm to about 4nm, about 3nm to about 4.5nm, about 3nm to about 5nm, about 3nm to about 5.5nm, about 3nm to about 6nm, Approximately 3 nm to approximately 7 nm, approximately 3 nm to approximately 8 nm, approximately 3.5 nm to approximately 4 nm, approximately 3.5 nm to approximately 4.5 nm, approximately 3.5 nm to approximately 5 nm, approximately 3.5 nm to approximately 5.5 nm, approximately 3.5 nm to approximately 6 nm, approximately 3. 5nm to about 7nm, about 3.5nm to about 8nm, about 4nm to about 4.5nm, about 4nm to about 5nm, about 4nm to about 5.5nm, about 4nm to about 6nm, about 4nm to about 7nm, about 4nm to about 8nm, about 4. The length is 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, about 5.5 nm to about 6 nm, about 5.5 nm to about 7 nm, about 5.5 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.

[0163] 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.

[0164] In some embodiments, the linker can be comprised of many well-known types, including, for example, polymers such as PEG, DNA, RNA, LNA, PNA, or any combination thereof. In some cases, the one or more polymer molecules can be polyethylene glycol, ethylene, polystyrene, vinyl chloride, polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene, polyamide, silicone-based polymers, PMOXA polymers, polyglycans, polyacrylamide polymers, polyacrylic acid polymers, polyamines, polyethyleneimines, quaternary ammonium polymers, polyvinyl alcohol polymers, pluronics, etc. 登録商標 The conjugation can include polymers, ethylene oxide polymers, propylene oxide polymers, polyvinylpyrrolidone polymers, carboxypolymethylene polymers, or any combination thereof. Conjugation can be performed using any suitable, well-known chemical attachment method, such as vapor chemical attachment methods, including reactions involving cysteine ​​(e.g., maleimide coupling), lysine, or click chemistry. The conjugation chemistry is preferably located at the end of the linker, but can also be located midway through the molecule as needed to position R relative to N. The linker can be composed of a single unit (e.g., a single polymer strand directly linking N to R) or multiple units (e.g., hybridized oligonucleotides in which R and N are each attached to one of the two strands). R can be directly attached to N as long as the N attachment point is given sufficient length and flexibility to allow R to enter and exit the nanopore. This can be achieved, for example, by attaching R to a flexible loop present in or introduced into the N sequence at an appropriate position. Alternatively, additional sequences in R (e.g., internal loops, or N- or C-terminal extensions, if attached) can be designed to create flexible linkers, which can then be attached directly to surface residues on N.

[0165] In one embodiment, L is an oligonucleotide, preferably a duplex made from complementary strands of DNA, or chemically modified RNA (e.g., a locked nucleic acid or RNA chemically modified with -F, -OMe at the 2' position for increased stability). L may comprise a stretch of at least 8, at least 10, preferably at least 14, more preferably at least 18 nucleotides.

[0166] In some embodiments, the nanopore is (reversibly) functionalized with R via a linker L, preferably where L is an oligonucleotide duplex formed by nucleic acid hybridization between a first oligonucleotide conjugated to the nanopore and a second oligonucleotide conjugated to R that is complementary to the first oligonucleotide.

[0167] In some embodiments, various orientations are possible to advantageously position R relative to the attachment of L on the nanopore and the entrance to the nanopore (see Figure 14). For example, N and R may be attached to the same end of the oligonucleotide duplex linker L. Alternatively, N and R may be attached to the midpoint or distal end of the oligonucleotide duplex linker L.

[0168] In some embodiments, duplex formation and exchange of components attached to the nanopore is preferably achieved by a toehold-mediated strand displacement (TMSD) reaction, which involves a process in which an invading strand displaces an existing strand from a gate strand, starting from an exposed toehold domain. For example, TMSD can be used to exchange strands duplexed in the nanopore. For example, in the case of a nanopore comprising N-L1 (where L1 is one strand of a duplex linker), initially duplexed to L2-R1, TMSD can be used to exchange the binding entity to L3-R2, changing the bound binder R and thus the analyte (e.g., target) that the nanopore can detect.

[0169] In another embodiment, the linker L on the nanopore is initially protected and thus activated only when necessary. For example, the nanopore is initially duplexed with a blank protecting strand, which is then removed channel by channel based on a chip array containing multiple nanopores. For example, the blank protecting oligonucleotide strand can be removed from a desired nanopore channel by channel by applying a voltage to a selected channel containing the nanopore, capturing the protecting strand and electrophoretically peeling it from the nanopore (FIG. 15).

[0170] Nanopore

[0171] In some embodiments, the nanopore suitably has a maximum internal diameter (e.g., lumen diameter) of 5 nm to 20 nm, e.g., 5 nm to 10 nm. The pore may 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 of the channel through the nanopore that can determine or control the signal obtained when a substrate (e.g., a target substrate) translocates relative to the nanopore.

[0172] In some embodiments, the internal 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 internal diameter (e.g., lumen diameter) can be about 20 nm or less, about 19 nm or less, about 18 nm or less, about 17 nm or less, about 16 nm or less, about 15 nm or less, about 14 nm or less, about 13 nm or less, about 12 nm or less, about 11 nm or less, about 10 nm or less, about 9 nm or less, about 8 nm or less, about 7 nm or less, about 6 nm or less, about 5 nm, or less than about 5 nm.

[0173] In some embodiments, the internal diameter (e.g., lumen diameter) can be about 5 nm to about 25 nm. In some embodiments, the internal diameter (e.g., lumen diameter) can be about 5 nm to about 6 nm, about 5 nm to about 7 nm, about 5 nm to about 8 nm, about 5 nm to about 9 nm, about 5 nm to about 10 nm, about 5 nm to about 12 nm, about 5 nm to about 14 nm, about 5 nm to about 16 nm, about 5 nm to about 18 nm, about 5 nm to about 20 nm, about 5 nm to about 25 nm, about 6 nm to about 7 nm, about 6 nm to about 8 nm, about 6 nm to about 9 nm, about 6 nm to about 10 nm, or about 6 nm to about 1 2nm, about 6nm to about 14nm, about 6nm to about 16nm, about 6nm to about 18nm, about 6nm to about 20nm, about 6nm to about 25nm, about 7nm to about 8nm, about 7nm to about 9nm, about 7nm to about 10nm, about 7nm to about 1 2nm, about 7nm to about 14nm, about 7nm to about 16nm, about 7nm to about 18nm, about 7nm to about 20nm, about 7nm to about 25nm, about 8nm to about 9nm, about 8nm to about 10nm, about 8nm to about 12nm, about 8nm to about 14nm, about 8nm to about 16nm, about 8nm to about 18nm, about 8nm to about 20nm, about 8nm to about 25nm, about 9nm to about 10nm, about 9nm to about 12nm, about 9nm to about 14nm, about 9nm to about 16nm, about 9nm ~about 18nm, about 9nm to about 20nm, about 9nm to about 25nm, about 10nm to about 12nm, about 10nm to about 14nm, about 10nm to about 16nm, about 10nm to about 18nm, about 10nm to about 20nm, about 10nm to about 2 5 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, about 14 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.

[0174] In some embodiments, the internal 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.

[0175] In some embodiments, the nanopore (e.g., a biological nanopore) may be a pore-forming toxin. Pore-forming toxins (PFTs) of pathogenic bacteria are well-characterized virulence factors. They belong to an ancient and highly diverse protein family. PFTs are present across phyla of Gram-negative and Gram-positive bacteria, with members found among human, insect, and plant pathogens. Depending on the nature of the secondary structure of the membrane-perforating channel, PFTs are classified into two families: α-PFTs form α-helical pores, while β-PFTs form β-barrel pores.

[0176] In one aspect, the nanopore is a member of the cytolysin A (ClyA) family of toxins, or a variant thereof that allows for site-specific functionalization with a recognition element (e.g., a proteinaceous recognition element). ClyA-like toxins include PDB IDs: 1QOY (soluble ClyA), 2WCD (protomeric ClyA), 6EK7 (soluble YaxA), 6EL1 (protomeric YaxA, protomeric YaxB), 6EK4 (soluble PaxB), 4K1P (soluble NheA), 5KUC (Cry6AA), and 2NRJ (Hbl-B). See Brauning et al. (Toxins (Basel). 2018 Sep;10(9):343, which is incorporated herein by reference in its entirety) and the references cited therein, which are incorporated herein by reference in their entirety.

[0177] In some embodiments, the nanopore is ClyA, preferably a mutant ClyA, that is functionalized with R in the region encompassing amino acids F101-S110, as found in GenBank sequence AJ313032.1. ClyA is an α-helical PFT with a relatively large diameter (3-6 nm depending on the entrance).

[0178] In one aspect, the modified nanopore is based on the ClyA variant ClyA-AS and has the following sequence: MTGIFAEQTVEVVKSAIETADGALDLYNKYLDQVIPWKTFDETIKELSRFKQEYSQEASVLVGDIKVLLMDSQDKYFEATQTVYEWAGVVTQLLSAYIQLFDGYNEKKASAQKDILIRILDDGVKKLNEAQKSLLTSSQSFNNASGKLLALDSQLT NDFSEKSSYYQSQVDRIRKEAYAGAAAGIVAGPFGLIISYSIAAGVVEGKLIPELNNRLKTVQNFFTSLSATVKQANKDIDAAKLKLATEIAAIGEIKTETETTRFYVDYDDLMLSLLKGAAKKMINTSNEYQQRHGRKTLFEVPDVGSSYHHHHH. This mutant contains the following mutations compared to the wild-type ClyA protein: C87A, L99Q, E103G, F166Y, I203V, C285S, K294R, and an additional S110C mutation is introduced to allow R functionalization.

[0179] In some embodiments, suitable water-facing amino acids near the entrance (e.g., the cis entrance) of the nanopore can be modified from a structural model. In some instances, other useful regions of ClyA for functionalization include residues D267-S272, lumen-exposed residues in the helix comprising A111-Q139, and the helix comprising D71-D64. Non-limiting examples of preferred residues for modification include D114, E129, K132, S133, V136, Q139, E78, D71, and D64. ClyA residues outside the ClyA nanopore that should be modified include residues S272 through the terminus of the protein, F101 through K66, and D267 through K230.

[0180] In one aspect, ClyA is suitably functionalized at position 110 by using the mutant S110C.

[0181] In one aspect, the nanopore is an engineered member of the YaxAB family. The Yersinia YaxAB system represents a binary α-PFT family with orthologues in human, insect, and plant pathogens.

[0182] In some embodiments, the nanopore is pleurotolysin (PlyAB; PDB ID 4V2T) from the oyster mushroom (Pleurotus ostreatus), or a variant thereof that allows site-specific functionalization with a recognition element (e.g., a proteinaceous recognition element). PlyAB is composed of two distinct components: pleurotolysin A (PlyA, 16 kDa), which serves as a scaffold to recruit a second component, pleurotolysin B (PlyB, 54 kDa), which spans the lipid bilayer. Cryogenic electron microscopy revealed a nanopore with an entrance of approximately 10.5 nm (e.g., cis entrance), an entrance of approximately 7.2 nm (e.g., trans entrance), and a constriction with a diameter of approximately 5.5 nm. Suitable regions for attaching R include, based on residue numbering in AJ313032.1, S49 to D71, R100 to S89, G181 to G205, D298 to E316, and V329 to P336.

[0183] In some embodiments, the concept of analyte-dependent dynamics of the recognition element R entering and exiting the lumen of the nanopore is also advantageously applied to de novo fabricated nanopores, such as DNA-based membrane nanopores with pore shapes and tunable lumen widths of tens of nanometers or less (Xingh et al. 2022, Nature Nanotechnology vol. 17, pp. 708-713, which is incorporated herein by reference in its entirety) or DNA origami nanopores with internal diameters of 30 nm or less (Fragrasso et al. ACS Nano 2021, 15, 8, 12768-12779, which is incorporated herein by reference in its entirety). Furthermore, the nanopore is a novel nanopore based on a de novo alpha-helical transmembrane region or a beta-barrel transmembrane region (see, for example, Shimizu et al. 2022, Nature Nanotechnology volume 17, pg. 67-75 (incorporated herein by reference in its entirety), or Scott et al. 2021, Nature Chemistry volume, 13, pg. 643-650 (incorporated herein by reference in its entirety), or Vorobieva et al. 2021, Science, Vol 371, Issue 6531 (incorporated herein by reference in its entirety)).

[0184] Accordingly, the present invention also provides methods and sensor systems comprising an artificial, non-solid-state nanopore functionalized with a 5-50 kDa recognition element (e.g., a protein recognition element) R capable of specifically binding to an analyte (e.g., a target analyte), wherein R dynamically moves in and out of the interior of the nanopore, triggering transient current blockage events, and wherein binding of R to an analyte (e.g., a target analyte, e.g., a target protein) modulates its dynamic movement, thereby inducing a change in the frequency and / or magnitude of the current blockage events, which change in the frequency and / or magnitude of the current blockage events indicates the presence of the analyte (e.g., the target analyte) in a sample. In one embodiment, it comprises a DNA-based membrane nanopore or a DNA origami nanopore functionalized with a nanobody.

[0185] In some embodiments, the nanopore (e.g., an artificial or non-solid-state nanopore) is capable of specifically binding to an analyte (e.g., a target analyte) 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 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, 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 The antibody is linked to a recognition element of 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, 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 greater than about 50 kDa.In some embodiments, the nanopore (e.g., an artificial or non-solid-state nanopore) is about 50 kDa or less, about 49 kDa or less, about 48 kDa or less, about 47 kDa or less, about 46 kDa or less, about 45 kDa or less, about 44 kDa or less, about 43 kDa or less, about 42 kDa or less, about 41 kDa or less, about 40 kDa or less, about 39 kDa or less, about 38 kDa or less, about 37 kDa or less, about 36 kDa or less, about 35 kDa or less, about 34 kDa or less, about 33 kDa or less, about 32 kDa or less, about 31 kDa or less, about 30 kDa or less, about 29 kDa or less, capable of specifically binding to an analyte (e.g., a target analyte). or less, about 28 kDa or less, about 27 kDa or less, about 26 kDa or less, about 25 kDa or less, about 24 kDa or less, about 23 kDa or less, about 22 kDa or less, about 21 kDa or less, about 20 kDa or less, about 19 kDa or less, about 18 kDa or less, about 17 kDa or less, about 16 kDa or less, about 15 kDa or less, about 14 kDa or less, about 13 kDa or less, about 12 kDa or less, about 11 kDa or less, about 10 kDa or less, about 9 kDa or less, about 8 kDa or less, about 7 kDa or less, about 6 kDa or less, about 5 kDa or less, about 4 kDa or less, about 3 kDa or less, about 2 kDa or less, about 1 kDa, or less than about 1 kDa.

[0186] In some embodiments, the 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 capable of specifically binding to an analyte, and 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 ...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 kDa ~ about 30kDa, about 10kDa - about 35kDa, about 10kDa - about 40kDa, about 10kDa - about 45kDa, about 10kDa - about 50kDa, about 10kDa - about 55kDa, about 10kDa - about 60kDa, about 15kDa - about 20kDa, about 15kDa - about 25kDa, Approximately 15kDa to approximately 30kDa, approximately 15kDa to approximately 35kDa, approximately 15kDa to approximately 40kDa, approximately 15kDa to approximately 45kDa, approximately 15kDa to approximately 50kDa, approximately 15kDa to approximately 55kDa, approximately 15kDa to approximately 60kDa, approximately 20kDa to approximately 25kDa, approximately 20kDa to approximately 30k Da, approx. 20kDa ~ approx. 35kDa, approx. 20kDa ~ approx. 40kDa, approx. 20kDa ~ approx. 45kDa, approx. 20kDa ~ approx. 50kDa, approx. 20kDa ~ approx. 55kDa, approx. 40kDa, about 25kDa to about 45kDa, about 25kDa to about 50kDa, about 25kDa to about 55kDa, about 25kDa to about 60kDa, about 30kDa to about 35kDa, about 30kDa to about 40kDa, about 30kDa to about 45kDa, about 30kDa to about 50kDa, about 30kDa and is bound to a recognition element of about 1 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 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.

[0187] In some embodiments, the nanopore (e.g., an artificial or non-solid-state nanopore) is capable of specifically binding to an analyte, and 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, or about 25 kDa. 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 recognition elements.

[0188] Analyte

[0189] In some embodiments, the methods or sensor systems of the present invention can be readily designed to detect any analyte (e.g., target analyte) or multiple analytes (e.g., multiple target analytes). The present invention is advantageously used to detect label-free analytes (e.g., target analytes).

[0190] In one embodiment, the present invention provides a method for detecting an analyte / antigen (e.g., a target analyte / antigen) selected from the group consisting of 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.

[0191] In some cases, the analyte (e.g., target analyte) is a protein. In some instances, 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, or a cell surface protein.

[0192] The present invention is particularly suited for detecting protein targets spanning a very wide range of masses and sizes. In one aspect, the present invention detects proteins (e.g., protein targets) spanning a very wide range of masses and sizes, from very small proteins and peptides to very large proteins and complexes. The recognition element R is dynamic and moves in and out of the nanopore, and the present systems and methods are sensitive to R binding not only very small analytes, but also very large analytes that cannot fit within the nanopore.

[0193] In some embodiments, the present invention is particularly suitable for detecting multi-analyte or single-analyte (e.g., multi-protein analyte or single-protein analyte) complexes that are greater than 50 Da, preferably greater than 100 Da, and most preferably greater than 150 Da.

[0194] 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 about 1,000 kDa or less, about 750 kDa or less, about 500 kDa or less, about 400 kDa or less, about 300 kDa or less, about 250 kDa or less, about 200 kDa or less, about 175 kDa or less, about 150 kDa or less, about 140 kDa or less, about 130 kDa or less, about 120 kDa or less, about 110 kDa or less, about 100 kDa or less, about 90 kDa or less, about 80 kDa or less, about 70 kDa or less, about 60 kDa or less, about 50 kDa or less, about 25 kDa, or less than about 25 kD.

[0195] In some embodiments, the analyte or analyte complex is between about 50 kDa and about 500 kDa. In some embodiments, the analyte or analyte complex is from about 50 kDa to about 60 kDa, from about 50 kDa to about 70 kDa, from about 50 kDa to about 80 kDa, from about 50 kDa to about 90 kDa, from about 50 kDa to about 100 kDa, from about 50 kDa to about 125 kDa, from about 50 kDa to about 150 kDa, from about 50 kDa to about 175 kDa, from about 50 kDa to about 200 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 500 kDa, from about 60 kDa to about 70 kDa, from about 60 kDa to about 80 kDa, from about 60 kDa to about 90 kDa, from about 60 kDa to about 100 kDa, Approximately 100kDa, approximately 60kDa to approximately 125kDa, approximately 60kDa to approximately 150kDa, approximately 60kDa to approximately 175kDa, approximately 60kDa to approximately 200kDa, approximately 60kDa to approximately 250kDa, approximately 60kDa to approximately 500kDa, approximately 70kDa to approximately 80kDa, approximately 70kDa ~about 90kDa, about 70kDa to about 100kDa, about 70kDa to about 125kDa, about 70kDa to about 150kDa, about 70kDa to about 175kDa, about 70kDa to about 200kDa, about 70kDa to about 250kDa, about 70kDa to about 500kDa, about 80kDa ~ about 90kDa, about 80kDa to about 100kDa, about 80kDa to about 125kDa, about 80kDa to about 150kDa, about 80kDa to about 175kDa, about 80kDa to about 200kDa, about 80kDa to about 250kDa, about 80kDa to about 500kDa, about 90kDa to about 100kDa, about 90kDa to about 125kDa, about 90kDa to about 150kDa, about 90kDa to about 175kDa, about 90kDa to about 200kDa, about 90kDa to about 250kDa, about 90kDa to about 500kDa, about 100kDa to about 125kDa, about 10 0kDa to about 150kDa, about 100kDa to about 175kDa, about 100kDa to about 200kDa, about 100kDa to about 250kDa, about 100kDa to about 500kDa, about 125kDa to about 150kDa, about 125kDa to about 175kDa, about 125kDa to about 200kDa, about 125kDa to about 250kDa, about 125kDa to about 500kDa, about 150kDa to about 175kDa, about 150kDa to about 200kDa, about 150kDa to about 250kDa, about 150kDa to about 500kDa, about 175kDa to about 200kDa,It is 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.

[0196] 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.

[0197] The methods and systems are capable of detecting analytes much larger than can be accommodated within very large nanopores. In some embodiments, the present invention is capable of binding and detecting a broader range of biologically important biomarkers, such as large proteins, protein complexes (including those containing intact viruses, bacteria, and cells).

[0198] In some embodiments, the disclosed methods are for detecting or characterizing modifications in an analyte (e.g., an analyte, e.g., a target protein, e.g., a label-free 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).

[0199] In some embodiments, post-translational modifications include modifications with hydrophobic groups, modifications 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 laboratories for biotechnological or biomedical purposes. This allows the level of post-translational modification of laboratory-obtained peptides, polypeptides, or proteins to be monitored compared with their natural counterparts. Thus, the methods disclosed herein can be used to detect the presence or absence, degree, or number of positions of post-translational modifications in polypeptides.

[0200] In some embodiments, post-translational modifications with hydrophobic groups include myristoylation, palmitoylation, isoprenylation or prenylation (attachment of an isoprenoid group); farnesylation (attachment of a farnesol group); geranylgeranylation (attachment of a geranylgeraniol group); and glypiation (formation of a glycosylphosphatidylinositol (GPI) anchor via an amide bond). Examples of post-translational modifications by cofactors include lipoylation (attachment of a lipoate (Cs) functional group); flavination (attachment of a flavin moiety (e.g., flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD))); attachment of heme C (e.g., attachment to a cysteine ​​via a thioether bond); phosphopantetheinylation (attachment of a 4'-phosphopantetheinyl group); formation of a retinylidene Schiff base; or any combination thereof.

[0201] In some embodiments, the post-translational modification by the addition of a chemical group is by acylation (e.g., O-acylation (ester), N-acylation (amide), or S-acylation (thioester); acetylation (addition of an acetyl group, e.g., to the N-terminus or to lysine); formylation; alkylation (addition of an alkyl group, e.g., methyl or ethyl); methylation (addition of a methyl group, e.g., to lysine or arginine); amidation; butyrylation; gamma-carboxylation; glycosylation (enzymatic attachment of a glycosyl group, e.g., to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan); polysialylation (polysialic acid addition). acid attachment); malonylation; hydroxylation; iodination; bromination; citrullination; nucleotide addition (attachment of any nucleotide, such as any of the nucleotides described above); ADP-ribosylation (ADP phosphorylation (attachment of a phosphate group, e.g., to serine, threonine, or tyrosine (O-linked) or histidine (N-linked)), adenylylation (attachment of an adenylyl moiety, e.g., to tyrosine (O-linked) or histidine, or lysine (N-linked)); propionylation; pyroglutamate formation; S-glutathionylation; sumoylation; S-nitrosylation; succinylation (attachment of a succinyl group, e.g., to lysine); selenoylation (incorporation of selenium); ubiquitination (attachment of a ubiquitin subunit (N-linked)); or any combination thereof.

[0202] Nanopore Sensor System

[0203] A further embodiment relates to a nanopore system as described above, comprising a fluid-filled compartment separated by a membrane into a first chamber and a second chamber, an electrode capable of applying an electric potential across the membrane, and one or more functionalized nanopores according to the present invention inserted into the membrane.

[0204] In one aspect, the present invention provides a nanopore system comprising a membrane having an engineered nanopore therein, the membrane separating a fluid chamber into a first side and a second side, and means for providing a voltage difference between the first side and the second side of the membrane, wherein the engineered nanopore is a biological or newly formed non-solid-state nanopore functionalized with a recognition element of 5-50 kDa, preferably 10-40 kDa, more preferably 12-15 kDa, capable of specifically binding to an analyte (e.g., a target analyte).

[0205] In some embodiments, the biological or newly 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, capable of specifically binding to an analyte. In some embodiments, the biological or newly formed non-solid-state nanopore is coupled to a recognition element of about 50 kDa or less, about 45 kDa or less, about 40 kDa or less, about 35 kDa or less, about 30 kDa or less, about 25 kDa or less, about 20 kDa or less, about 15 kDa or less, about 10 kDa or less, about 5 kDa, or less than about 5 kDa.

[0206] In some embodiments, the biological or newly 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 newly formed non-solid-state nanopore is capable of specifically binding to an analyte, and is between about 5 kDa and about 10 kDa, between about 5 kDa and about 15 kDa, between about 5 kDa and about 20 kDa, between about 5 kDa and about 25 kDa, between about 5 kDa and about 30 kDa, between about 5 kDa and about 35 kDa, between about 5 kDa and about 40 kDa, between about 5 kDa and about 45 kDa, between about 5 kDa and about 50 kDa, between about 5 kDa and about 55 kDa, between about 5 kDa and about 60 kDa, between about 10 kDa and about 15 kDa, between about 10 kDa and about 20 kDa, between about 10 kDa and about 25 kDa, between about 10 ...10 kDa and about 35 kDa, between about 10 kDa and about 35 kDa, between about 10 kDa and about 35 kDa, between about 10 kDa and about 40 kDa, between about 10 kDa and about 45 kDa, between about 1 kDa ~ about 30kDa, about 10kDa - about 35kDa, about 10kDa - about 40kDa, about 10kDa - about 45kDa, about 10kDa - about 50kDa, about 10kDa - about 55kDa, about 10kDa - about 60kDa, about 15kDa - about 20kDa, about 15kDa - about 25kDa, Approximately 15kDa to approximately 30kDa, approximately 15kDa to approximately 35kDa, approximately 15kDa to approximately 40kDa, approximately 15kDa to approximately 45kDa, approximately 15kDa to approximately 50kDa, approximately 15kDa to approximately 55kDa, approximately 15kDa to approximately 60kDa, approximately 20kDa to approximately 25kDa, approximately 20kDa to approximately 30k Da, approx. 20kDa ~ approx. 35kDa, approx. 20kDa ~ approx. 40kDa, approx. 20kDa ~ approx. 45kDa, approx. 20kDa ~ approx. 50kDa, approx. 20kDa ~ approx. 55kDa, approx. 40kDa, about 25kDa to about 45kDa, about 25kDa to about 50kDa, about 25kDa to about 55kDa, about 25kDa to about 60kDa, about 30kDa to about 35kDa, about 30kDa to about 40kDa, about 30kDa to about 45kDa, about 30kDa to about 50kDa, about 30kDa and is bound to a recognition element of about 1 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 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.

[0207] In some embodiments, the biological or newly 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 can specifically bind to an analyte.

[0208] In some embodiments, the term "membrane" is used in its conventional sense to refer to a thin film-like structure that separates the chambers of the 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 the fluid chamber. The membrane separating the first side (or cis side) and the second side (or trans side) comprises a nanopore functionalized with at least one R. Membranes can generally be classified into synthetic membranes and biological membranes. Any membrane can be used in accordance with the present invention. Multiple nanopores may be present in a single membrane.

[0209] 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 from amphiphilic molecules, such as phospholipids, which have at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles that form monolayers 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).

[0210] In some embodiments, the nanopore system includes a first side of a fluid chamber or cis chamber containing a first conductive liquid medium in liquid communication with a second side of a fluid chamber or trans chamber containing a second conductive liquid medium. The conductive liquid medium in the chamber of the nanopore system has a wide range of ionic contents known to those skilled in the art, typically from 0.05 M to less than 3 M. In some embodiments, the conductive liquid medium can have an ionic 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 ionic content of about 5.0 M or less, about 4.5 M or less, about 4.0 M or less, about 3.5 M or less, about 3.0 M or less, about 2.5 M or less, about 2.0 M or less, about 1.5 M or less, about 1.0 M or less, about 0.5 M or less, about 0.1 M or less, about 0.05 M or less, about 0.01 M, or less than about 0.01 M.

[0211] 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 can have an ion content of about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 5 M, about 0.05 M to about 0.1 M, or about 0.05 M to about 0 .5M, about 0.05M to about 1M, about 0.05M to about 1.5M, about 0.05M to about 2M, about 0.05M to about 2.5M, about 0.05M to about 3M, about 0.05M to about 3.5M, about 0.05M to about 4M, about 0 .05M~about 5M, about 0.1M~about 0.5M, about 0.1M~about 1M, about 0.1M~about 1.5M, about 0.1M~about 2M, about 0.1M~about 2.5M, about 0.1M~about 3M, about 0.1M~about 3.5M, about 0. 1M~4M, 0.1M~5M, 0.5M~1M, 0.5M~1.5M, 0.5M~2M, 0.5M~2.5M, 0.5M~3M, 0.5M~3.5M, 0.5M~ Approximately 4M, approximately 0.5M to approximately 5M, approximately 1M to approximately 1.5M, approximately 1M to approximately 2M, approximately 1M to approximately 2.5M, approximately 1M to approximately 3M, approximately 1M to approximately 3.5M, approximately 1M to approximately 4M, approximately 1M to approximately 5M, approximately 1.5M to approximately 2M, approximately 1.5 M ~ about 2.5M, about 1.5M - about 3M, about 1.5M - about 3.5M, about 1.5M - about 4M, about 1.5M - about 5M, about 2M - about 2.5M, about 2M - about 3M, about 2M - about 3.5M, about 2M - about 4M, about 2M - Approximately 5M, approximately 2.5M to approximately 3M, approximately 2.5M to approximately 3.5M, approximately 2.5M to approximately 4M, approximately 2.5M to approximately 5M, approximately 3M to approximately 3.5M, approximately 3M to approximately 4M, approximately 3M to approximately 5M, approximately 3.5M to approximately 4M, approximately 3.5M to approximately 5 M, or from about 4M to about 5M.

[0212] In some embodiments, the conductive liquid medium can have an ionic content of about 0.01M, about 0.05M, about 0.1M, about 0.5M, about 1.0M, about 1.5M, about 2.0M, about 2.5M, about 3.0M, about 3.5M, about 4.0M, about 4.5M, or about 5.0M.

[0213] A wide variety of salts, such as NaCl and KCl, can be used. A suitable solution includes 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. The first and second sides of the fluid chamber can be symmetric or asymmetric. The cis and trans chambers can be symmetric or asymmetric. A wide range of pH and temperature conditions can be used, such as pH 5-9, 10-50°C, preferably 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 about 10 or less, about 9.5 or less, about 9 or less, about 8.5 or less, about 8 or less, about 7.5 or less, about 7 or less, about 6.5 or less, about 6 or less, about 5.5 or less, about 5 or less, about 4.5 or less, about 4 or less, about 3.5 or less, about 3, or less than about 3.

[0214] In some embodiments, the pH of the solution can be about 3 to about 10. In some embodiments, the pH of the solution can be about 3 to about 4, about 3 to about 5, about 3 to about 5.5, about 3 to about 6, about 3 to about 6.5, about 3 to about 7, about 3 to about 7.5, about 3 to about 8, about 3 to about 8.5, about 3 to about 9, about 3 to about 10, about 4 to about 5, about 4 to about 5.5, about 4 to about 6, about 4 to about 6.5, about 4 to about 7, about 4 to about 7.5, about 4 to about 8, about 4 to about 8.5, about 4 to about 9, about 4 to about 10, about 5 to about 5.5, about 5 to about 6, about 5 to about 6.5, about 5 to about 7, about 5 to about 7.5, about 5 to about 8, about 5 to about 8.5, about 5 to about 9, about 5 to about 10, about 5.5 to about 6, about 5.5 to about 6.5, about 5.5 to about 7, about 5.5 to about 7.5 , about 5.5 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. It can be 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.

[0215] 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 about 75°C or less, about 70°C or less, about 65°C or less, about 60°C or less, about 55°C or less, about 50°C or less, about 45°C or less, about 40°C or less, about 35°C or less, about 30°C or less, about 25°C or less, about 20°C or less, about 15°C or less, about 10°C or less, about 5°C or less, or less than about 5°C.

[0216] 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° C. to about 35° C., About 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°C, About 20°C to about 40°C, about 20°C to about 45°C, about 20°C to about 50°C, about 20°C to about 60°C, about 20°C to about 70°C, about 25°C to about 30°C, about 25°C to about 35°C, about 25°C to about 40°C, about 25°C to about 45°C, about 25°C to about 50°C, about 25°C to about 60°C, about 25°C to about 70°C, about 30°C to about 35°C, about 30°C to about 40°C, about 30°C to about 45°C, about 30°C to about 50°C, about 30°C to about 60°C, The temperature can be 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.

[0217] 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.

[0218] In some embodiments, the first side, i.e., the cis chamber, includes a crowding or blocking agent that reduces unwanted nonspecific protein adsorption. In one embodiment, the blocking agent is BSA.

[0219] In some embodiments, the system can be equipped with a circuit that can both apply voltage and measure current. Alternatively, the system can be equipped with one circuit for applying voltage difference and another circuit for measuring current. It can also generate voltage difference by using asymmetric salt across the membrane. For example, one of the chambers can contain a solution with high ionic strength.

[0220] In some embodiments, mechanisms for detecting current between the first side (e.g., the cis side) and the second side (e.g., the trans side) are described in International Publication No. WO 00 / 79257, U.S. Pat. Nos. 6,46,594, 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, U.S. Patent Application Publication Nos. US 2004 / 0121525, US 2003 / 0104428, and US 2003 / 0104428. They may include electrodes directly associated with the channel or pore at or near the porous opening, electrodes located within the first and second sides (or cis and trans chambers), and insulated glass microelectrodes. Electrodes may be capable of detecting differences in ionic currents around the two chambers or tunneling currents around the porous opening. In another configuration, the transport property is electron flow around the diameter of the opening, 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.

[0221] It is understood that the acquisition systems described herein are not limiting and that other systems for acquiring or measuring nanopore signals can be used. Alternative electrical schemes can also be employed to achieve an equivalent voltage drop across the nanopore and / or membrane, for example on an array chip platform.

[0222] In some embodiments, the sensor system is advantageously integrated into a portable device with multiple sensor systems, such as a point-of-care diagnostic medical device, which is an in vitro diagnostic used by medical professionals to obtain rapid results near or in the field of a patient. These products can be useful, for example, in a doctor's office or clinic, for rapidly determining causative markers for certain diseases.

[0223] Arrays and Kits

[0224] The present disclosure provides an array comprising a plurality of sensor systems according to the invention, preferably comprising a plurality of separate reservoirs, each of the plurality of reservoirs comprising a nanopore modified with one or more different R elements to enable detection of a different analyte.

[0225] In some embodiments, the array comprises nanopores pre-modified with an L moiety, thus allowing end-user-defined functionalization with one or more selected recognition elements (e.g., protein recognition elements R). For example, the L moiety of the pre-modified pore is selected to allow the formation of a duplex nucleic acid between a nanobody or affimer or affibody conjugated to a selected oligonucleotide and a pore protein conjugated to the oligonucleotide. In one embodiment, the system of the invention comprises an array of pre-modified pores all bearing the same linker L oligonucleotide sequence, to which an R binding partner (comprising a single species of R or a mixture of different R species) can bind with the appropriate complementary sequence to form a duplex. In an alternative embodiment, the array of pre-modified nanopores comprises different L moieties specific for a set of complementary R sequences.

[0226] In one aspect, L is composed of an original strand and a complementary protection strand, which allows attachment of R to a pre-modified nanopore by toehold mediated strand displacement (TMSD).

[0227] Methods and kits for preparing such arrays are also provided.

[0228] Also provided herein is the use of a system or analytical device according to the invention for single molecule sensing analysis, preferably for sensing the presence or concentration of one or more analytes (e.g., target analytes) in a complex (clinically relevant) sample. In some embodiments, the invention provides the use of a method, nanopore or sensor system, array, or kit in single protein detection, preferably in combination with high-throughput analysis.

[0229] In one aspect, the present disclosure provides an array comprising a plurality of nanopore systems according to any one of the preceding embodiments. In some embodiments, the array comprises a plurality of separate reservoirs. In some cases, one or more of the plurality of nanopore systems comprises nanopores modified with different recognition elements to enable detection of different analytes.

[0230] In one aspect, the present disclosure provides a kit for preparing the system of any one of the foregoing embodiments. In some embodiments, the kit includes 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 protected strand.

[0231] In one aspect, the present disclosure provides a method for using a method, nanopore, nanopore sensor system, array, or kit according to any one of the above-described embodiments in single protein detection. In some embodiments, the single protein detection can be combined with high-throughput analysis. In some embodiments, the sensor system is integrated into a portable device with multiple sensor systems.

[0232] Computer Systems

[0233] The present disclosure provides a computer system programmed to perform a method for determining one or more characteristics of an analyte. Figure 16 shows a computer system 1601 programmed or configured to determine the presence or absence of an analyte. The computer system 1601 can control various aspects of detecting the presence or absence of an analyte. The computer system 1601 can be a user's electronic device or can be a computer system located remotely relative to the electronic device. The electronic device can be a mobile electronic device.

[0234] The computer system 1601 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 1605, which can be a single-core processor or a multi-core processor, or multiple processors for parallel processing. The computer system 3001 also includes memory or memory locations 1610 (e.g., random access memory, read-only memory, flash memory), an 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, such as cache, other memory, data storage, and / or an electronic display adapter. The memory 1610, storage unit 1615, interface 1620, and peripherals 1625 communicate with the CPU 1605 via a communication bus (solid line), e.g., a motherboard. The storage unit 1615 can be a data storage unit (or data repository) for storing data. The computer system 1601 may be operatively connected to a computer network ("network") 1630 via a communication interface 1620. The network 1630 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. The network 1630 may, in some cases, be a telecommunications network and / or a data network. The network 1630 may include one or more computer servers that may enable distributed computing, e.g., cloud computing. The network 1630 may, in some cases, implement a peer-to-peer network via the computer system 1601, thereby enabling devices connected to the computer system 1601 to act as clients or servers.

[0235] The CPU 1605 can execute sequences of machine-readable instructions, which may be implemented in a program or software. The instructions may be stored in a memory location, such as memory 1610. The instructions may be sent to the CPU 1605, which may then program or otherwise configure the CPU 1605 to perform the methods of the present disclosure. Examples of operations performed by the CPU 1605 may include fetch, decode, execute, and writeback.

[0236] The CPU 1605 can be part of a circuit, such as an integrated circuit, within which one or more other components of the system 1601 can be included. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0237] The storage unit 1615 can store files, such as drivers, libraries, and saved programs. The storage unit 1615 can store user data, such as user settings and user programs. The computer system 1601 can optionally include one or more additional data storage units external to the computer system 1601, such as on a remote server that communicates with the computer system 1601 over an intranet or the Internet, for example, a storage unit located on a remote server that communicates with the computer system 1601 over an intranet or the Internet.

[0238] The computer system 1601 can communicate with one or more remote computer systems over a network 1630. For example, the 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 portable PC), a slate PC, or a tablet PC (e.g., an Apple登録商標 iPad, Samsung 登録商標 Galaxy Tab), phone, smartphone (e.g., Apple 登録商標 iPhone, Android-enabled device, Blackberry 登録商標 ), or a personal digital assistant. A user can access the computer system 1601 via a network 1630.

[0239] The methods described herein may be implemented by machine (e.g., a computer processor) executable code stored in an electronic storage location of the computer system 1601, such as in memory 1610 or electronic storage unit 1615. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by the processor 1605. In some cases, the code may be retrieved from storage unit 1615 and stored in memory 1610 so that it is easily accessible by the processor 1605. In some cases, the electronic storage unit 1615 may be eliminated, and machine-executable instructions are stored in memory 1610.

[0240] The code can be pre-compiled and configured for use on a machine having a suitable processor to execute the code, or it can be compiled at run time. The code can be provided in a programming language that can be selected to be able to execute in a pre-compiled or compiled state.

[0241] Aspects of the systems and methods provided herein, such as the computer system 1601, can be embodied in programming. Various aspects of the technology may be thought of as a "product" or "article of manufacture," typically taking the form of machine (or processor) executable code and / or associated data, held or embodied in some type of readable storage medium. The machine-executable code may be stored in an electronic storage unit, memory (e.g., read-only memory, random-access memory, flash memory), or hard disk. "Storage" type media may include some or all of the tangible memory of a computer, processor, etc., or their associated modules (various semiconductor memories, tape drives, disk drives, etc.), which may provide non-transitory storage for software programming at any time. All or part of the software may be communicated over the Internet or various other communication networks. Such communication may, for example, allow software to be loaded from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include the optical, electrical, and electromagnetic waves used over physical interfaces between local devices, wired and optical fixed line networks, and various wireless links. The physical elements that carry such waves, e.g., wired or wireless links, optical links, etc., may be considered to be media that carry software. As used herein, unless limited to non-transitory tangible "storage" media, a computer or machine "readable storage medium" refers to any medium that participates in providing instructions to a processor for execution.

[0242] Thus, a machine-readable storage medium, e.g., computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media are, for example, optical or magnetic disks, and include any storage device in any one or more computers, etc., that may be used to implement the databases, etc., 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 and fiber optics, including, for example, the wiring that comprises a bus within a computer system. Carrier-wave transmission media may take the form of electric 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, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards, paper tape, any other physical storage media with a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link carrying 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.

[0243] The computer system 1601 may include or be in communication with an electronic display 1635 that includes a user interface (UI) 1640 for providing, for example, analyte identification information. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0244] The methods and systems of the present disclosure may be implemented by one or more algorithms, which may be implemented by software in response to being executed by the central processing unit 1605.

[0245] Another aspect of the present disclosure provides a non-transitory computer-readable storage medium containing machine-executable code that, when executed by one or more computer processors, implements any of the methods described above or elsewhere herein.

[0246] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto, the computer memory including machine-executable code that, when executed by the one or more computer processors, implements any of the methods described above or elsewhere herein.

[0247] While 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 by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Various modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the present invention. It is therefore intended that the present invention also embrace such alternatives, modifications, variations, or equivalents. The claims appended hereto define the scope of the present invention, and methods and structures within the scope of these claims, and their equivalents, are intended to be embraced therein. [Brief explanation of the drawings]

[0248] [Figure 1] Figure 1: Binding of single-stranded DNA to the ClyA nanopore. (A) Side (left) and top (right) views of the ClyA structure (PDB: 6mrt). To enable site-specific chemical modification, the serine at position 110 (colored purple) was genetically mutated to a cysteine. (B) Schematic model showing the conjugation strategy for attaching ssDNA to the ClyA nanopore. A 16-mer oligonucleotide (designated f) was attached to a ClyA monomer via a maleimide-PEG4-DBCO linker, where the maleimide reacted with an -SH group on the protein and DBCO clicked onto an azide group on the oligo. The ClyA-f monomer was then oligomerized to a ClyA-f oligomer in the presence of 0.2% DDM at 37 °C. (C) SDS-PAGE analysis of the 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 the oligomerization of ClyA-f, Lane 5: ClyA-f after oligomerization, Lane 6: S110C-mutated ClyA after oligomerization. [Figure 2]Figure 2: Functionalization of ClyA nanopores with the spike nanobody Ty1 and electrical characterization of the nanopores. (A) Schematic model showing the strategy for functionalizing ClyA nanopores with the Ty1 nanobody, where Ty1-f' was immobilized on the ClyA-f nanopore by DNA strand hybridization. (B) I-V curves of ClyA-S110C (blue triangles), ClyA-f (black squares), and ClyA-f-Ty1 (red circles) at applied potentials ranging from -90 to 90 mV (three independent experiments). (C) Histogram showing the conductance distribution of the ClyA-f nanopore with (red) and without (black) the Ty1 nanobody. (D) Representative current traces of ClyA-f-Ty1 at an applied potential of -20 mV. "In" and "out" refer to nanobodies located inside (blocked pore) and outside (open pore) of the nanopore, respectively. Io is the open pore current, and Ib is the blocked pore current. (E) All-point histograms of the current traces shown in D demonstrate the clear distribution of the blockade signal. (F) A schematic model illustrating 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 the movement of a single Ty1 nanobody into and out of the pore vestibule. All experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. [Figure 3]Figure 3: Single channel recording traces of ClyA-f-Ty1 and analysis of the residual currents Ib / Io, tin, and tout under various applied potentials. (A) Representative current traces of ClyA-f-Ty1 under applied potentials ranging from -10 to -40 mV. (B) All-point histogram of the current traces in A, showing that the Ty1 nanobody tends to reside within the ClyA nanopore as the applied potential increases. (C, D) Log-time histograms of Ty1 located inside and outside ClyA, respectively. (E, F) Effect of applied potential on the mean log-time of Ty1 located inside and outside ClyA. These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, and pH 7.5. [Figure 4] Figure 4. Nanobody attachment to ClyA through DNA oligo hybridization verified using Dnase I. (A) Current traces of ClyA-f-Ty1 before and after the addition of 5 U of DNASE I in the presence of 2.5 mM MgCl2 at an applied potential of -20 mV. (B) A magnification of a representative current trace from A, showing that the nanobody attached to the ClyA nanopore was removed approximately 30 minutes after the addition of DNASE I. A full-point histogram showing the current distribution before and after the addition of Dnase I is displayed on the top of the panel. The schematic model described above illustrates how the nanobody was removed from the ClyA nanopore. The experiment was performed in 150 mM NaCl, 2.5 mM MgCl2, 50 mM Tris-HCl, pH 7.5. [Figure 5] Figure 5. Detection of spike proteins by nanobody-functionalized nanopores. (A) Current traces of ClyA-f-Ty1 before and after the sequential addition of 6 μM BSA and 2.3 nM spike protein. (B) A magnified view of a representative current trace from A (top) and a full-point histogram of the current distribution (bottom). From left to right, the traces are shown before and after the addition of BSA and spike protein. The experiment was performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. [Figure 6]Figure 6. Effect of BSA on nanobody internalization. (A-C) Histogram distribution of tout before and after adding 3 μM or 6 μM BSA to the first side (e.g., cis side) of the ClyA-f-Ty1 nanopore system. The histograms were fitted with a single exponential function. (D-G) Changes in the blocked percentage, open percentage, mean time that bound Ty1 resides inside the ClyA nanopore (tin), and mean time that Ty1 resides outside the ClyA nanopore (tout) with increasing BSA concentration (n = 4, each experiment was performed on an independent nanopore. Error bars represent standard deviation). These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. [Figure 7] Figure 7. Effect of spike protein addition on ClyA-f-Ty1 pores. (A) Current traces show the pore transition from a dynamic state (Ty1 moving alternately inside and outside the pore) to a fully open state (Ty1 trapped outside the pore through binding to spike e protein) immediately after the addition of 2.3 nM spike protein. (B) Current traces of ClyA-f-Ty1 approximately 25 min after the addition of 2.3 nM spike protein. (C) Full-point histograms of the current traces shown in B. (D, E) Histograms of the logarithms of tin and tout after the addition of 2.3 nM spike protein approximately 25 min after spike protein addition. The histograms were fitted with Gaussian distribution functions. These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 6 μM BSA. [Figure 8]Figure 8. The open probability of ClyA-f-Ty1 is positively correlated with spike trimer protein concentration. (A) Representative current traces of ClyA-f-Ty1 before and after increasing the spike trimer protein concentration. (B) All-point histograms are displayed to show the current distribution before and after increasing the spike protein concentration. (C) Curve regression of the open probability in function of spike concentration. The curve was fitted using the Hill-Langmuir equation (n = 1.31, Kd = 760.6 pM). (D) Schematic model showing the dynamics of the interaction between ClyA-f-Ty1 and spike proteins. Ty1 nanobodies dynamically enter and exit the ClyA nanopore under an applied potential. The spike proteins, at high concentrations of spike trimers, likely multivalently interact reversibly with Ty1 nanobodies attached on the nanopore. The experiments were carried out in the presence of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 6 μM BSA. [Figure 9A-D] Figure 9. Effect of spike protein concentration on binding kinetics to the ClyA-f-Ty1 pore. (A–D) Histograms of log(tout) at spike concentrations of 115 pM, 230 pM, 345 pM, and 460 pM, respectively. The data were fitted with a Gaussian distribution. [Figure 9E-H] Figure 9. Effect of spike protein concentration on binding kinetics to the ClyA-f-Ty1 pore. (E-H) Histograms of log(tin) at spike concentrations of 115 pM, 230 pM, 345 pM, and 460 pM, respectively. The data were fitted with a Gaussian distribution. [Figure 9I-J] Figure 9. Effect of spike protein concentration on binding kinetics to the ClyA-f-Ty1 pore. (I,J) Concentration dependence of the logarithm of tout and tin. These experiments were performed in the presence of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 6 μM BSA. [Figure 10]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 change in current before and after the addition of 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 the addition of 1 μL of blood (E). (D,F) Full-point histograms of the current traces before (D) and after (F) the addition of 1 μL of blood. (G) Histogram of the logarithm of the residence time at level 0 before and after the addition of 1 μL of blood. (H) Histogram of the logarithm of the residence time at level 1 before and after the addition of 1 μL of blood. These experiments were performed in the presence of an electrolyte buffer of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 6 μM BSA. [Figure 11] 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 bias of -20 mV. The experiment was performed in the presence of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 6 μM BSA. [Figure 12] Figure 12. Detection of nanopores functionalized with Her2. (A) Representative current traces of ClyA attached with the 2Rs15d nanobody (ClyA-f-15d) before and after the addition of 32.8 nM Her2 protein at an applied voltage of -20 mV. (B) Representative current traces of ClyA attached with the 2Rb17c nanobody (ClyA-f-17c) before and after the addition of 20.8 nM Her2 protein at the same applied potential. Reported binding affinity of 2Rs15d to Her2: k = 2.14 × 10 M s, k = 5.71 × 10 s, K = 2.7 nM. Reported binding affinity of 2Rb17c to Her2: k = 7.6 × 10 M s, k = 4.58 × 10 s, K = 6 nM. These experiments were carried out in the presence of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 6 μM BSA. [Figure 13] Figure 13. Functionalized ClyA nanopore for detection of muPA. (A) Crystal structure of muPA (purple) complexed with nb22 nanobody (green) (PDB: 5LHR). Reported binding affinity of nb22 for muPA: k = (4.6 ± 0.8) × 10 M s, k = (7.8 ± 2.2) × 10 s, K = 0.2 ± 0.03 nM. (B) Representative current traces of ClyA-f-nb22 before and after addition of 3 nM muPA at an applied potential of -15 mV. (C) Zoomed-in view of a representative current trace after addition of 3 nM muPA at an applied potential of -15 mV. The signal consisted of three blockade levels in addition to the open-pore level (level 0), with current blockade percentages of 13.7% ± 0.1% (level 1), 34.1% ± 0.5% (level 2), 6, and 3.6% ± 0.1% (level 3), respectively. (D) Heat map of blockade events observed after addition of 3 nM muPA, showing the logarithm of residence time versus current blockade percentage. (E) Schematic model showing the conformational changes of ClyA-f-nb22 in response to muPA protein. The experiment was performed in the presence of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 6 μM BSA. [Figure 14]Figure 14. Schematic diagram of several options for attaching a targeting moiety R to a nanopore N via hybridization of a duplex oligonucleotide (e.g., dsDNA) linker L (where one oligonucleotide strand of the duplex linker L binds to the nanopore N, and the other, complementary strand binds to the binding site R). The diagram illustrates three possible options for attaching the components: A) The N and R components are located at opposite ends of the duplex linker L. For example, this can be easily achieved by attaching components to both 5'-ends or both 3'-ends of each strand. The distance "d" between the binding sites between N and R is primarily controlled by the length of the duplex oligonucleotide strand, 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 a single-stranded oligonucleotide. B) The N and R components are located at the same end of the hybridized duplex linker L. For example, one component is attached to the 5'-end of strand 1 and the other to the 3'-end of strand 2, or vice versa. This coupling method is advantageous for positioning the coupling points of N and R closer together, shortening the distance "d," while still allowing for much longer oligonucleotide chains, if desired. c) One or both of the N and R components are attached to the oligonucleotide chain of the hybridized duplex linker L at an internal position along the chain, e.g., via a bond to the backbone of the polynucleotide or to a base of the polynucleotide (for simplicity, in the figures, R is attached only at the midpoint). In all cases described above, the oligonucleotide can have a non-duplexed single-stranded portion (e.g., an ssDNA overhang) to further control the distance and optimize the flexibility of the attached components. [Figure 15]Figure 15. Schematic of a nanopore N with a linker L initially in a protected state (A) containing a hybridized protecting polynucleotide strand (i). The protecting polynucleotide strand is removed by applying a voltage to the nanopore in a membrane system to capture and release it from the linker L (B). The deprotected nanopore (C) can then bind with a selected binding site R hybridized to the linker L to create a functional NLR nanopore system (D). [Figure 16] FIG. 16 illustrates a computer system programmed or otherwise configured to perform the methods provided herein.

[0249] Experimental section

[0250] material

[0251] Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich. The unnatural amino acid (UAA) used in this study, 4-azido-L-phenylalanine (pAzF), was synthesized according to published protocols. 57 All DNA oligos were purchased from IDT.

[0252] Expression and purification of nanobodies engineered with pAzF

[0253] Ty1 52 , nb22 55 , Rs15d and 2Rb17c 54DNA encoding each nanobody was cloned into the PET22b(+) plasmid (Addgene) containing 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 the 6xHis to incorporate a UAA into the nanobody. Production of nanobodies engineered with pAzF was performed using established protocols. 38 The procedure was carried out according to the method described above. First, the constructed plasmid was transformed into BL21 E. coli cells. The cells were cultured at 37°C and 200 rpm in 1 L of TB medium supplemented with 100 mL of salt buffer (0.17 M KH2PO4, 0.72 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. When the OD600 reached 0.6-0.9, IPTG was added to a final concentration of 1 mM. Protein induction was completed overnight at 25°C with shaking. The cells were harvested by centrifugation at 4500 rpm for 15 minutes at 4°C and then 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 200 rpm (horizontal rotator) at 4°C for 6 hours, after which 48 mL of 1 / 4 TES buffer was added and incubated overnight at 200 rpm at 4°C. The cell suspension was then centrifuged at 12000 g for 30 minutes at 4°C. The supernatant was collected and supplemented with 5 mM MgCl2, then further purified by FPLC (GE Healthcare) using a 5 mL HisTrap column (GE Healthcare). The binding and elution buffers used here were 20 mM and 500 mM imidazole, respectively, and both were supplemented with 20 mM sodium phosphate pH 7.4 and 500 mM NaCl. The purity of the protein was analyzed on a 4%-12% SDS-PAGE gel.

[0254] Conjugation of nanobodies with f'-oligos

[0255] First, oligo f' (NH2-C6-5'-ATCCGCGGGTGTCGGG-3') bearing an amine group at the 5' end was reacted with a 20-fold excess of NHS-DBCO in 60% DMSO at pH 8.0 at 25°C overnight. After purification by ethanol precipitation and subsequent reverse-phase HPLC, the DBCO-oligo was incubated with an azide-modified nanobody in PBS at 25°C overnight. The reaction was optimized by adding different ratios of nanobody to f'-DBCO oligo. When the molar ratio was 5:1, the conjugation yield was greater than 70%. Therefore, this ratio was applied to the conjugation of all four nanobodies. Subsequently, the nanobody-f' conjugate was purified by ion-exchange chromatography and verified by either 16% denaturing urea polyacrylamide gel electrophoresis or SDS-PAGE.

[0256] Expression and purification of ClyA-S110C nanopore

[0257] The ClyA-S110C construct was prepared by mutating the serine at position 110 to a cysteine ​​from the previously reported cysteine-free mutant ClyA-CS. 41The constructed plasmid was transformed into electrocompetent E. coli BL21(DE3) cells by electroporation. Cells were grown in 2xYT medium containing 100 μg / mL ampicillin at 37°C, 200 rpm, until the OD600 reached 0.8-1. Protein expression was induced by adding 0.5 mM IPTG and incubating overnight at 20°C, 200 rpm. Cells were harvested by centrifugation at 6500 rpm, 4°C, for 15 minutes. The pellet was stored in a -80°C freezer for at least 1 hour and then thawed at 37°C. It was then resuspended in 20 mL of 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 rotator, the cells were further lysed by sonication. The lysate was then centrifuged at 6500 rpm for 30 minutes at 4°C, and the supernatant was collected and incubated with Ni-NTA beads (Qiagen) at room temperature for 1 hour on a rotator. Nonspecifically bound proteins were removed with 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 the protein was eluted from the beads with 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.

[0258] Preparation of ClyA-f-nb nanopore

[0259] First, freshly purified ClyA-S110 was incubated with a 20-fold molar excess of DBCO-PEG4-maleimide at pH 7.5 overnight at 4°C with gentle shaking. 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). Next, purified ClyA-PEG4-DBCO was incubated with a 1.5-fold excess of f-azido oligonucleotides overnight at 4°C with gentle shaking to create the ssDNA-modified "ClyA-f" monomer. The f-azido oligo linker was prepared by reacting a 5'-amino-modified oligonucleotide (NH2-C6-5'-CCCGACACCCGCGGAT-3') with azidobutyric acid NHS ester. SDS-PAGE gel was used to examine the efficiency of the click reaction. ClyA-f monomer was oligomerized by incubation at 37°C for 30 minutes in the presence of 0.2% n-dodecyl-β-D-maltoside (DDM). Subsequently, the 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 the DNA oligos, 29 , the ClyA-f oligomer migrated slightly faster than the unmodified ClyA-S110C oligomer. 41According to the results, the lowest oligomer bands of ClyA-S110C and ClyA-f were type I nanopores (12-mer). Therefore, the dodecamers of ClyA-S110C and ClyA-f were obtained by slicing these bands from the gel. After elution from the gel slices with 30 μL of standard buffer containing 0.02% DDM, 5 μL of the ClyA-f oligomer solution was dispensed into tubes. The concentration of the ClyA-f dodecamer eluted from the gel was too low to be measured by either Nanodrop or Bradford assay. Therefore, prior to single-channel recording experiments, an excess of nanobody-f' (approximately 40 pmol) was incubated with 5 μL of ClyA-f oligomer for at least 30 min at room temperature to maximize the ClyA nanopore modification with the duplexed nanobodies.

[0260] Single-channel recording experiment

[0261] Electrical recordings were performed as previously described 58 This was performed using a vertical planar lipid membrane setup. Briefly, a Teflon membrane separating the first (e.g., cis) and second (e.g., trans) sides of the fluid chamber of the recording chamber was used. 登録商標A 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, purchased from Avanti Polar Lipids) lipid bilayer was formed at the membrane opening. After connecting to a patch-clamp amplifier (Axopatch 200B, Axon Instruments) using an Ag / AgCl electrode, both the trans side and the first side of the chamber were filled with electrolyte buffer: 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. A ClyA nanopore was added to the first side (i.e., the cis side) of the chamber, which was connected to a ground electrode. After pore insertion, excess ClyA was removed by buffer exchange. 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 (i.e., cis side) unless otherwise specified. All recordings were performed using a 2 kHz Bessel low-pass filter and a 10 kHz sampling rate. All electrical recording current traces were filtered with a Gaussian low-pass filter with a 1 kHz cutoff before analysis. The data analysis software used in this study was Clampfit.

[0262] Example 1: Functionalization of ClyA nanopore with nanobodies

[0263] To specifically detect proteins of various sizes, we designed a ClyA nanopore 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 ionic flux through the nanopore, thus inducing a distinguishable current signal indicative of protein detection. To enable site-specific attachment of the DNA linker to ClyA, we replaced the serine at position 110 with a cysteine ​​to create ClyA-CS. 41 The mutant was mutated (ClyA-S110C, Figure 1A). Next, a 16-nt DNA oligonucleotide bearing an azide group at its 3' end (f-azide) was attached to ClyA-S110C using a maleimide-PEG4-DBCO linker (Figure 1B). Addition of a 20-fold excess of the linker to ClyA-S110C resulted in a complete upshift of the product band compared to ClyA-S110C in an SDS-PAGE gel, indicating a high yield of the ClyA-DBCO product (Figure 1C). Subsequently, purified ClyA-DBCO was reacted with a 1.5-fold excess of f-azide, resulting in a full yield of the ClyA-f construct (Figure 1C). Furthermore, after self-assembly in the presence of detergent to form an oligomerized pore, the dodecamer of ClyA-S110C and ClyA-f was synthesized. 41 (Figure 1D, band I) is blue native polyacrylamide gel. 42 Based on the high conjugation efficiency and uniformity of oligomerization of the ClyA-f monomer, it can be estimated that there are approximately 12 oligonucleotides available on each ClyA-f dodecamer for nanobody attachment.

[0264] Amber codon suppression was used to anchor nanobodies onto the ClyA nanopore. 38Nanobodies bearing an N-terminal azide group were created by incorporating an unnatural amino acid and then conjugated to the complementary strand of oligo f, which contains a DBCO group (f'-DBCO) at the 5' end, via click chemistry. As a proof-of-concept, Ty1 nanobodies, which can reversibly bind to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, were conjugated to f'. The binding activity of the oligo-attached nanobodies was examined using a biolayer interferometry assay, which showed that oligo attachment did not affect the binding affinity of Ty1 nanobodies to the RBD (data not shown). Furthermore, to examine the potential binding of nanobodies to ClyA, ClyA-f monomers were incubated with a 5-fold excess of Ty1-f' conjugate and analyzed by SDS-polyacrylamide gel. The results showed that ClyA-f exhibited a clear mobility shift upon nanobody attachment, suggesting that the attachment efficiency was up to 100% (data not shown). Finally, nanobody-functionalized ClyA nanopores (ClyA-f-nb) were prepared by incubating ClyA-f dodecamer with individual nanobody-f' modules.

[0265] Example 2: Characterization of nanobody-functionalized ClyA nanopores

[0266] First, to investigate the effect of ssDNA and nanobody attachment, we performed electrical characterization of lyA-S110C, ClyA-f, and Ty1-modified ClyA (ClyA-f-Ty1) at different applied potentials using a C single-channel recording system. 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 oligo into the nanopore was observed (data not shown). However, the IV curves showed that the open-pore current of ClyA-f was slightly smaller than that of ClyA-S110C at positive potentials ranging from 10 mV to 90 mV (Figure 2B), indicating that the attached ssDNA, driven by the applied positive potential, partially blocked the pore. Nevertheless, the conductivity behavior of ClyA-f was not affected by the attachment of ssDNA at a negative bias (Fig. 2B). Conversely, attachment of the Ty1 nanobody had no effect on the ClyA-f-Ty1 pore at a positive potential (+35 mV), but the pore was partially blocked compared to ClyA-f when a negative potential (-35 mV) was applied. When the applied potential was reduced to -20 mV, we observed transient and reversible blockade signals (Fig. 2D). These signals consisted of two current levels (in and out), one of which was similar to that expected for an open pore current, while the other was consistent with the entry of a single nanobody into the nanopore. At the same applied potential, the current blockade percentage (I o -I b ) / I o× 100 (or ΔI / I o ×100,I o is the open pore current, and I bis the blocked pore current) was 14.2 ± 0.3% (n = 3), and the residence time of the blocking 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 percentage of area under the curve, we found that the open probability of ClyA-f-Ty1 at -20 mV was 51% (Figure 2E). Furthermore, by measuring the blocked pore current at different applied potentials, the I-V curves of ClyA-f-Ty1 were obtained, demonstrating that the current of ClyA-f-Ty1 was smaller than that of ClyA-f without nanobody attachment at negative biases ranging from -10 to -90 mV (Figure 2B). Consequently, the conductance of ClyA-f-Ty1 at -35 mV (1.71 ± 0.01 nS, n = 22) was smaller than that of ClyA-f (1.92 ± 0.01 nS, n = 22) (Figure 2C). These results indicated that Ty1 attachment resulted in voltage-dependent gating of the ClyA nanopore.

[0267] To further confirm that these blocking signals were caused by the translocation of attached nanobodies, we investigated the dependence of these blocking signals on the applied potential. By increasing the applied potential from -10 mV to -40 mV, we observed an increase in the probability of blocking the pore and the dwell time (t in ) significantly increased, while the interval time during which the pore remained open (t out) was significantly reduced (Figure 3). For example, at potentials above -50 mV, the ClyA pore was almost permanently blocked in the case of this specific nanobody. However, by reversing the applied potential, the ClyA-f-Ty1 nanopore could return to an unblocked state. These results indicate that the blocking signal is not caused by molecular translocation. Typically, molecules translocate faster within the nanopore at higher voltages (translocation is evidenced by a shorter residence time of the block at higher voltages). ClyA-AS is known to generate a strong electroosmotic flow, 43 The strong electroosmotic flow induces the capture of various proteins under negative applied voltage. 19 The small size of nanobodies (2.5 nm diameter and 4 nm height) 44 ) and the flexible tether of the 16-bp DNA duplex (approximately 5.5 nm long), the current blockade is the result of the bound Ty1 nanobody entering the nanopore in close proximity to the constriction region (Figure 2F).

[0268] In addition, the present inventors have found that Mg 2+ After adding 5 U of DNase I to the first side (e.g., the cis side) of the chamber in the presence of tRNA at -20 mV for approximately 30 min, we observed irreversible opening of the pore as a result of cleavage of the dsDNA linker (Figure 4). This result confirmed that the nanobody was successfully attached onto the ClyA nanopore by DNA duplex formation, thereby providing new evidence for interpreting the blocking signal. Herein, "in" and "out" are used to define the position of a nanobody either inside the nanopore vestibule or outside the nanopore vestibule, respectively, where t in and t outand represent the time the nanobody spends inside and outside the nanopore, respectively. These results demonstrate that attaching a nanobody to a ClyA nanopore via a flexible oligonucleotide linker allows the bound nanobody to dynamically move in and out of the nanopore and partially block the ionic current when inside the nanopore. Furthermore, the results demonstrate that the dynamics between the in and out states can be controlled through applied voltage.

[0269] Example 3: Real-time detection of SARS-CoV-2 spike protein

[0270] Bovine serum albumin (BSA) is a marker for ELISA-like sensing technologies. 45 In this case, non-specific interactions such as protein-protein or protein-surface interactions 46 In our case, by adding BSA to the first side (e.g., the cis side) of the ClyA-f-Ty1 nanopore, no additional blocking signal was observed due to the translocation of BSA. Surprisingly, we found that the t out Both the open probability (probability of being in the out state) and the open probability (probability of being in the out state) were found to decrease with increasing BSA concentration (Fig. 5, Fig. 6). For example, in the presence of 6 μM BSA and at a bias of -20 mV, the t out The time to release decreased from 42.9 ± 38.9 ms to 4.64 ± 0.38 ms, and the probability that ClyA-f-Ty1 was in the open state decreased from 14.2 ± 7.5% to 2.1 ± 0.8%. These results suggested that the presence of BSA dramatically reduced the time that the ClyA nanopore was unoccupied by the bound Ty1 nanobody. Given that BSA has dimensions of approximately 14 × 4 × 4 nm and a pI of 4.7 in aqueous solution, 47It is likely that BSA creates a crowded environment outside the nanopore, thus increasing the chances that nanobodies will enter the pore. Similar crowding effects have been reported in previous studies. 9,48,49 It has been used to enhance the capture of macromolecules in biosensors. Furthermore, the addition of BSA significantly reduced the pore-to-pore variance of ClyA-f-Ty1 (Figures 6E and 6G). Therefore, for further sensing applications, 6 μM BSA was added to the first side of the fluid chamber to minimize the background signal.

[0271] Multivalent interactions have been widely exploited to improve binding affinity and enhance sensing sensitivity. 50,51 It has been reported that the binding affinity between the SARS-CoV-2 spike protein and Ty1 was dramatically increased by nanobody multimerization. 39 Given the dodecameric structure and distinct distances, the ClyA nanopore is predicted to be an optimal scaffold for integrating nanobodies into close proximity with each other, allowing multiple nanobodies to simultaneously bind to a single protein, enhancing the sensitivity of spike protein recognition. To test the feasibility of this sensing system, ClyA-f-Ty1 was added to the first side (e.g., cis side) of the SARS-CoV-2 spike protein at a final concentration of 2.3 nM in the presence of 6 μM BSA in the fluid chamber of the nanopore system. Notably, after approximately 1 min, we observed that the frequency of blocking signals began to decrease, and t outWe observed an increase in the current (Figure 5, Figure 7A). Shortly thereafter, the current traces were almost completely locked in the "out" state, which corresponded to the restoration of the ClyA-f-Ty1 nanopore to an open-pore current state (approximately 38 pA) (Figure 5). Approximately 25 minutes into the recording period after spike protein addition, the probability that Ty1 was located outside the ClyA lumen (open-pore probability) increased from 3.9 ± 0.4% to 98.9 ± 0.6% (n = 3, Figure 7), suggesting that the Ty1 nanobody was retained outside the ClyA lumen in response to capture by the spike. Furthermore, the presence of the 2.3 nM spike increased the t out The logarithmic histogram of the interevent durations showed two peaks (Figure 7E), with mean interevent times of 5.03 ± 1.34 ms and 20.19 ± 1.95 ms, respectively (n = 3). The interevent duration of the first peak was very close to the time before spike protein addition (4.48 ± 1.32 ms, n = 3), suggesting that these events were due to nonspecific localization of unbound Ty1 nanobodies inside or outside the nanopore. Meanwhile, the events in the second peak were likely due to spike-nanobody binding. Compared to the first peak, the duration of the second peak increased by three orders of magnitude, suggesting a very strong binding interaction between spike trimers and multimerized Ty1 nanobodies.

[0272] To generate a calibration curve for spike detection and to further investigate the binding kinetics of trimeric spikes with multimerized Ty1 nanobodies, we tested the response of ClyA-f-Ty1 nanopores to various concentrations of spike protein. At low concentrations (0–460 pM), the open pore probability of ClyA-f-Ty1 increased with increasing spike concentration across the entire range (Figures 8A and 8B). We also measured the time it takes for Ty1 to be located outside the pore (t out ) increases almost linearly with increasing spike protein concentration, while the residence time inside the nanopore (t in) was found to be concentration-independent (100–500 pM, Figure 9). This confirmed that the increase in open pore probability was indeed caused by the spike protein associating with the Ty1 nanobody. Because the spike is a trimeric protein that can interact with three Ty1 nanobodies, 52 Occupation of any one of the 12 Ty1s on the ClyA nanopore by a single spike regulates ion flow. This therefore enables our platform to sensitively detect spikes at picomolar concentrations. With further increasing spike concentration, we found that the open-pore probability correlated positively with the concentration, reaching a plateau at approximately 2 nM (Figure 8C). The data could be approximated by the Hill-Langmuir equation, with a Hill coefficient greater than 1 (n = 1.31), indicating positive cooperativity between the trimeric spike and the multimerized Ty1. Furthermore, the previously observed long interevent duration and small dissociation constant (K) resulting from spike binding were consistent with the previously observed long interevent duration and small dissociation constant (K) resulting from spike binding. d =760.6 pM) reflects the fact that cooperative binding between multiple ligands and the same receptor can produce stronger binding affinity. 50,53 Therefore, we conclude that nanopores with multiple binding ligands to the same protein have great potential for highly sensitive detection.

[0273] Example 4: Detection of SARS-CoV-2 spike protein in blood

[0274] 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 influence of blood components using our ClyA-f-Ty1 pore sensor, 1 μL of defibrinated sheep blood (final concentration: 0.2 v / v%) in the presence of BSA was added to the first side (e.g., the cis side) of the fluid chamber (Figure 10A). Significantly, the conductive behavior of the ClyA-f-Ty1 nanopore was only slightly affected by blood, and the membrane remained stable (Figures 10B, 10C, and 10E). No obvious blood-induced blockade was observed, except for a slight, transient blocking signal with a current blockade of approximately 31.5% ± 0.1% (Figure 10E, level 2). However, the residence time of these events was very short (approximately 0.6 ms), suggesting that they may be due to transient collisions with blood proteins or platelets. Moreover, 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).

[0275] After the addition of 2.3 nM spike protein, the nanopore transitioned largely to an open state due to the binding of the Ty1 nanobody. Large blockage events lasting several seconds were observed in this state (Figures 11A and 11B). Presumably, in the absence of spike protein, steric hindrance of the nanobody on ClyA prevented blood components from entering the pore. In response to spike protein binding to the nanobody, the pore remained open, allowing some large proteins in the blood to occasionally enter the nanopore.

[0276] It is worth noting that, unlike other approaches, in this system, proteins do not need to enter the nanopore to be detected. This is important because the applied potential required for protein detection in this assay was only −20 mV, which was therefore much lower than the applied potential required to trap proteins within the nanopore. 22 The lower voltage reduces the likelihood of trapping unwanted background contaminants within the nanopore. Moreover, the bound nanobody at the nanopore entrance further prevents unwanted proteins and contaminants (e.g., background proteins in blood) from trapping and interfering within the nanopore, thereby greatly increasing the selectivity of the nanopore for the protein.

[0277] Example 5: General applicability of nanopores functionalized with nanobodies as protein sensors

[0278] This example demonstrates that the concepts illustrated for spike protein detection can be broadly applied to a variety of other proteins when suitable nanobodies are used. Nanobodies have similar characteristics in size and shape. 34 Therefore, it is expected that various nanobodies can induce similar transient blocking signals when immobilized on a ClyA nanopore, thus enabling the detection of proteins of various sizes. Taking advantage of the modularity of our approach, we functionalized the ClyA nanopore with the nanobodies 2Rs15d (ClyA-f-15d), 2Rb17c (ClyA-f-17c), and nb22 (ClyA-f-nb22). Among these nanobodies, 2Rs15d and 2Rb17c 54 Nanobody nb22 recognizes the N- and C-terminal halves of the human epidermal growth factor receptor 2 (HER2) protein, which is highly expressed in breast cancer. 55recognizes murine urokinase-type plasminogen activator (muPA), a biomarker associated with cancer progression.

[0279] All three nanobodies were successfully conjugated with oligo f', and these nanobodies could be functionalized onto ClyA with high attachment efficiency. Due to their similarity in size, shape, and surface charge, we hypothesize that these nanobodies affect the electrical behavior of ClyA in a similar manner to Ty1. Indeed, all of the nanobody-conjugated ClyA nanopores induced similar blocking signals at an applied potential of -20 mV (Figure 12).

[0280] In the presence of 6 μM BSA, the percentages of blockade produced by 2Rs15d, 2Rb17c, and nb22 were 11.7% ± 0.1%, 14.2% ± 0.4%, and 13.7% ± 0.1%, respectively. To verify the protein-sensing ability, the recombinant soluble protein Her2-hFc (96 kDa) was added to the pores of ClyA-f-15d and ClyA-f-17c. Similar to the phenomenon observed for the interaction between the spike and ClyA-f-Ty1, both nanopores functionalized with these nanobodies showed significantly increased open probability after the addition of Her2-hFc, as a result of protein binding to the bound nanobody (Figure 12).

[0281] Furthermore, we tested the protein-sensing potential of ClyA-f-nb22. Interestingly, after the protein muPA (48 kDa, pI 8.53, Figure 13A) was added to the first (i.e., cis) side of the ClyA-f-nb22 pore (Figure 13B), at a potential of -15 mV, we observed new classes of blockade events (Levels 2 and 3) in addition to the open-pore level (Level 0) and nanobody-induced events (Level 1). At 15 mV, the new Level 3 blockade exhibited a current block of 63.6 ± 0.1% and a relatively long duration of 45.45 ± 1.50 ms, whereas the Level 2 blockade was 34.1 ± 0.5% and a very short duration of 1.60 ± 0.43 ms. Level 3 blockade was not observed before muPA addition (left panel of Figure 13B) or when muPA was added to the ClyA-f or ClyA-f-Ty1 nanopores (data not shown), suggesting that level 3 events were not caused by nonspecific interactions between the nanobody and the protein, as well as free muPA protein itself. Presumably, level 3 blockade reflects entry of the nb22:muPA complex into the nanopore. Additionally, as the applied potential increased from -5 mV to -15 mV, the residence time of level 3 events increased by approximately 1.5 orders of magnitude (data not shown), consistent with the fact that the positively charged muPA:nb22 complex tends to reside in the nanopore for longer periods at more negative potentials. These results further confirmed that muPA complexed with nb22 enters the ClyA pore and triggers level 3 blockade events.

[0282] In comparison, the blockade at level 2 did not change significantly with applied voltage, and because the residence time was short and voltage-independent, the blockade at level 2 may reflect a transient collision of the nb22:muPA complex with the ClyA nanopore rather than complete entry into the nanopore.

[0283] Based on the above analysis, we constructed a model (Figure S13E) presenting the conformational transitions of ClyA-f-nb22 in response to muPA, which corresponded to the observed current levels. Together, these results demonstrate the ability to detect smaller analytes within the nanopore through binding to the bound nanobody.

[0284] References JPEG2026500150000001.jpg194170JPEG2026500150000002.jpg253170JPEG2026500150000003.jpg25317 0JPEG2026500150000004.jpg255168JPEG2026500150000005.jpg255170JPEG2026500150000006.jpg32170

Claims

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

2. 2. The method of claim 1, wherein the engineered nanopore is an oligomeric assembly comprising or consisting of monomers of the general formula NLR, where 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. 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 decreasing the frequency and / or magnitude of the current blockade events.

4. 4. The method of any one of claims 1 to 3, wherein the biological nanopore is functionalized with at least two different proteinaceous recognition elements, R' and R'', preferably wherein R' and R'' bind to distinct sites on the target analyte.

5. 5. The method of any one of claims 1 to 4, 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 virus capsid, a virus particle, a cell, a dendrimer, a polymer, or any combination thereof, preferably 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. 6. The method of any one of claims 1 to 5, 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, faeces, saliva, cerebrospinal fluid, breast milk and sputum.

7. 1. An engineered proteinaceous nanopore with a minimum pore diameter of 5 nm, the engineered proteinaceous nanopore functionalized via a flexible linker with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, that specifically reacts with a target analyte, preferably a target protein, and wherein R is capable of moving in and out of the pore to cause a blocking current.

8. 1. 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 an electric potential across the membrane, and at least one biological nanopore functionalized with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, capable of specifically binding to a target analyte, wherein R is positioned at the top of the nanopore via a flexible linker, allowing it to enter and exit the nanopore and trigger a transient current blockade event.

9. 1. A nanopore sensor system comprising a cis chamber containing a first conductive liquid medium in fluid communication with a trans chamber containing a second conductive liquid medium through a modified nanopore, wherein the modified nanopore is a biological nanopore functionalized with a proteinaceous recognition element R of 5-50 kDa, preferably 10-40 kDa, capable of specifically binding to a target analyte, wherein R is tethered to the top of the nanopore and is capable of being internalized into the pore and dynamically moving in and out of the nanopore lumen to trigger a transient current blockage event.

10. 10. The method, nanopore or sensor system of any one of claims 1 to 9, wherein R is an IgG-based moiety or a non-IgG-based moiety, preferably a nanobody, scFv fragment, Fab fragment, affimer, monobody, affibody, adnectin, DARPin or anticalin, more preferably R is a nanobody.

11. The biological nanopore is a pore-forming toxin, preferably having a maximum lumen diameter of 5 nm to 20 nm, more preferably cytolysin A (ClyA), pleurotolysin (PlyAB), YaxAB, perforin-2 (PFN2, PDB_ID 6SB3), tripartite alpha pore-forming toxin (AhlB, 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 6X6S), or the like. 6CB8), or a variant thereof that allows site-specific functionalization with a proteinaceous recognition element.

12. The method, nanopore or sensor system of any one of claims 1 to 11, wherein the biological nanopore is ClyA, preferably a mutant ClyA, more preferably a ClyA comprising the mutation S110C.

13. The method, nanopore or sensor system according to any one of claims 1 to 12, wherein the flexible linker is an oligonucleotide, preferably double-stranded DNA, or chemically modified RNA.

14. 14. The method, nanopore or sensor system of any one of claims 1 to 13, wherein the nanopore is (reversibly) functionalized with R via a flexible linker L, preferably by nucleic acid hybridization between a first oligonucleotide conjugated to the nanopore and a second oligonucleotide conjugated to R, the second oligonucleotide being complementary to the first oligonucleotide.

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

16. A kit for preparing an array as described in claim 14, 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 protector strand.

17. Use of the method, nanopore or sensor system, array, kit according to any one of claims 1 to 16 in single protein detection, preferably in combination with high-throughput analysis.

18. 18. The method of claim 17, wherein the sensor system is integrated into a portable device comprising multiple sensor systems.

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

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

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

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

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

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

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

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

27. The method of any one of claims 19 to 26, wherein the nanopore system further comprises a pair of electrodes.

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

29. 30. 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 results in a change in the current of the nanopore system.

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

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

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

34. 34. The method of claim 33, wherein the recognition element bound to the analyte results in movement of the recognition element.

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

36. 36. The method of claim 34 or 35, wherein the recognition element, when bound to the analyte, cannot move between an interior region of the nanopore and an exterior region of the nanopore.

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

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

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

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

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

42. 42. The method of any one of claims 19 to 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 virus capsid, a virus particle, a cell, a dendrimer, a polymer, or any combination thereof.

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

44. 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 to 44, wherein the analyte is from a sample.

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

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

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

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

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

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

52. 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 monobody, an affimer, an affibody, an adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

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

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

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

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

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

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

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

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

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

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

63. (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, and the nanopore is coupled to a recognition element; A system that includes:

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

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

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

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

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

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

71. The system of any one of claims 63 to 70, further comprising a pair of electrodes.

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

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

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

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

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

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

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

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

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

81. 81. The system of any one of claims 75 to 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 virus capsid, a virus particle, a cell, a dendrimer, a polymer, or any combination thereof.

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

83. 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 to 83, wherein the analyte is from a sample.

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

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

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

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

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

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

94. 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 monobody, an affimer, an affibody, an adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

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

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

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

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

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

100. 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 to 102, wherein the interior region of the nanopore comprises an interior diameter of about 5 nanometers to about 20 nanometers.

104. (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, the nanopore being coupled to a protein recognition element, the protein recognition element being configured to translocate between an interior region of the nanopore and an exterior region of the nanopore; A system comprising:

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

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

107. 107. The nanopore of claim 106, wherein the linker is between about 4 nanometers and about 8 nanometers in length.

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

109. A nanopore described in any one of claims 106 to 108, wherein the nanopore is attached to at least a portion of the linker.

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

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

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

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

114. 114. The nanopore of claim 113, wherein the recognition element bound to the analyte results in translocation of the recognition element.

115. 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 bound to the analyte.

116. 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 the recognition element is bound to the analyte.

117. 117. The nanopore of any one of claims 113 to 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 virus capsid, a virus particle, a cell, a dendrimer, a polymer, or any combination thereof.

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

119. 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 to 119, wherein the analyte is from a sample.

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

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

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

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

125. 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. A nanopore according to claims 105-125, wherein the nanopore is configured to be coupled to another recognition element.

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

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

130. 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 monobody, an affimer, an affibody, an adnectin, a designed ankyrin repeat protein (DARPin), an anticalin, or any combination thereof.

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

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

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

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

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

136. 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.

Images