Methods for determining the presence or absence of a target analyte comprising the use of a polynucleotide reporter molecule and a transmembrane pore

Abstract

The present invention relates to a method for determining the presence or absence of a target analyte in a sample. The method comprises immobilizing any target analyte present in the sample on a surface; contacting the surface with: (i) a first detection agent that specifically binds to the target analyte; and (ii) a polynucleotide reporter molecule, wherein the polynucleotide reporter molecule is bound to or associated with the first detection agent; and contacting a transmembrane pore with any polynucleotide reporter molecule immobilized on the surface, wherein the polynucleotide reporter molecule is immobilized on the surface by binding of the first agent to the target analyte, and detecting the polynucleotide reporter molecule using the transmembrane pore, thereby determining the presence or absence of the target analyte in the sample.

Description

[0001] This application is a divisional application with application date of May 3, 2018, application number of 201880029570.X, and invention name of “Method for determining the presence or absence of a target analyte comprising the use of a polynucleotide reporter molecule and a transmembrane pore”. Technical Field

[0002] The present invention relates to a novel assay for detecting molecular interactions such as protein-protein interactions. The method can be used to determine the presence, absence or amount of a target analyte in a sample. The method can be performed in a multiplex format and can be used to simultaneously determine the presence, absence or amount of multiple analytes. The method can be used to screen for binding partners of a target analyte. Background Art

[0003] There is a need for techniques that can detect the presence of low concentrations of analytes with high accuracy, particularly when multiple analytes are present in a single sample. The need to detect biomarkers and thereby diagnose the associated disease or condition is particularly acute.

[0004] Transmembrane pores (nanopores) have great potential as direct electronic biosensors for a variety of analytes, such as polymers and small molecules. When a potential is applied across a nanopore, a change in current occurs when a molecule, such as a nucleotide or polynucleotide, resides transiently in the barrel or channel of the nanopore for a period of time. Specific molecules, such as specific nucleotides and specific polynucleotides, give a change in current of known characteristics and duration. This change in current can be used to identify the nucleotide or polynucleotide present in the pore. Devices containing nanopore arrays can be used to detect and characterize polynucleotides, for example to sequence the polynucleotides. Summary of the invention

[0005] The inventors have demonstrated that a combination of antibodies and polynucleotides can be used in a nanopore-based immunosorbent assay to determine the presence, absence or concentration of an analyte. The assay is ideal for determining the presence, absence or concentration of one or more analytes. The assay can detect one target analyte, or can be a multiplex assay capable of detecting more than one target analyte.

[0006] More specifically, the inventors have demonstrated that the concentration of the target analyte can be determined in a monoplex assay using capture antibodies and detection antibodies and polynucleotide reporters fixed on the surface. The detection antibodies and polynucleotides used by the inventors are functionalized with biotin, and streptavidin is used to bind the polynucleotide reporters to the detection antibodies, but other means of connection can also be used. The amount of the polynucleotide reporter molecules bound to the detection antibody depends on the amount of the analyte fixed on the surface. The polynucleotide reporter molecules fixed on the surface by binding to the detection antibody bound to the captured analyte are directly detected using transmembrane pores (after release from the surface but without amplification). The inventors have shown that the amount of the polynucleotide reporter molecules detected is proportional to the amount of the analyte captured on the surface. By using a suitable calibration method, the concentration of the analyte in the sample can therefore be determined.

[0007] The inventor has also demonstrated that the concentration of more than one target analyte (i.e., two or more target analytes) can be determined in multiple determinations. In the method used by the inventor, each analyte is captured and labeled with different polynucleotide reporter molecules. The polynucleotide reporter molecules of the combination are washed out and merged to detect with a transmembrane hole. Each analyte of different known concentrations is used, wherein each concentration is detected using different polynucleotide reporter molecules to show that the determination can be used for simultaneously determining the concentration of multiple analytes. It is found that the detection sensitivity of each analyte exceeds the sensitivity required for detecting and measuring the concentration of the analyte at its physiological concentration. In an embodiment, determination is multiplexed at the sequencing level. However, by using specific attachment means (for example, different oligonucleotides (or multiple copies thereof) that are specifically hybridized with a polynucleotide reporter molecule are connected to each detection agent) or by preassembling detection agents / polynucleotide reporters, several analytes can be determined in a single reaction.

[0008] In the diagnosis and assessment of diseases, such as the measurement of disease progression or the response to treatment or the determination of possible responsiveness to treatment, it is usually necessary to detect and / or quantify at least one biomarker, more commonly two or more biomarkers. The inventors have determined that their method can be used to quantify a group of biomarkers simultaneously. The method can be easily performed without bulky equipment. For example, the method can be performed using standard immunoassay plates (e.g., ELISA plates) or beads, and a handheld device comprising a nanopore array (flow cell) can be used to detect polynucleotide reporters.

[0009] This method allows for balanced reporter readouts for all the different biomarkers in a panel, even though they may be present at very different levels in a sample (e.g., serum). The relatively few reporter readouts required for sensitive protein quantification means that large biomarker panels can be quantified on a single flow cell, while the same flow cell is used to simultaneously sequence the exome and / or transcriptome. Thus, this method can be used to provide a comprehensive assessment of an individual's health status.

[0010] The inventors have realised that this method can be used to identify binding partners of a target analyte, such as a drug target.Therefore, a method for drug screening is also provided.

[0011] Therefore, what is provided is:

[0012] - A method for determining the presence or absence of a target analyte in a sample, the method comprising:

[0013] (a) immobilizing any target analyte present in the sample on a surface;

[0014] (b) contacting the surface with: (i) a first detection agent that specifically binds to the target analyte; and (ii) a polynucleotide reporter molecule, wherein the polynucleotide reporter molecule is associated with or binds to the first detection agent; and

[0015] (c) contacting the transmembrane pore with any polynucleotide reporter molecules immobilized on the surface, wherein the polynucleotide reporter molecules are immobilized on the surface by binding of the first agent to the target analyte, and detecting the polynucleotide reporter molecules using the transmembrane pore to thereby determine the presence or absence of the target analyte in the sample;

[0016] - A method of determining the presence or absence of two or more target analytes in a sample, wherein each target analyte is detected using a different polynucleotide reporter molecule and the composition of the polynucleotide reporter molecule is determined using a transmembrane pore, and the method steps are as defined above;

[0017] - A method for identifying a binding partner of a target analyte, the method comprising:

[0018] (a) immobilizing a target analyte on a surface;

[0019] (b) contacting the surface with a candidate binding partner and a polynucleotide reporter molecule, wherein the polynucleotide reporter molecule is associated with or binds to the candidate binding partner; and

[0020] (c) using the transmembrane pore to detect any polynucleotide reporter molecules that have been immobilized on the surface, wherein the polynucleotide reporter molecules are immobilized on the surface by binding of the first agent to the target analyte, thereby determining whether the candidate molecule is a binding partner for the target analyte; and

[0021] - A kit for detecting a target analyte, comprising:

[0022] (a)(i) a polynucleotide attached to a first component of an affinity tag;

[0023] (ii) a second component of the affinity tag; and

[0024] (iii) a specific binding agent for the target analyte which is linked to a second component of an affinity tag or comprises an antibody and a first component of said affinity tag; or

[0025] (b)(i) polynucleotides; and

[0026] (ii) a specific binding agent for the target analyte, which is linked to an oligonucleotide that hybridizes to at least a portion of the polynucleotide; or

[0027] (c)(i) a specific binding agent for a target analyte; and

[0028] (ii) a polynucleotide, wherein the polynucleotide is bound or capable of binding to a specific binding agent and comprises an adaptor at the free end, which facilitates the interaction of a polypeptide reporter molecule with the transmembrane pore. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Sequencing results for two flow cell runs of barcoded polynucleotide reporter pools collected from PSA ELISA wells. The PSA protein concentration (pg / ml) added to each ELISA well is correlated to the number of reads for the associated barcoded polynucleotide reporter.

[0030] Figure 2 Figure 2: Sequencing results for two flow cell runs of barcoded polynucleotide reporter pools collected from PSA, RBP, and AAT ELISA well strips. The protein concentration (pg / ml) added to each ELISA well correlates to the number of reads in the sequencing of the barcoded polynucleotide reporter associated with that well. Concentration dependence was observed for each of the three proteins detected in the multiplex assay.

[0031] Figure 3 : Sequencing results for two flow cell runs of barcoded polynucleotide reporter pools collected from AAT (3.a.), CEA (3.b.), RBP (3.c.), and SCC (3.d.) ELISA well strips. The protein concentration (pg / ml) added to each ELISA well correlates with the number of reads in the sequencing of the barcoded polynucleotide reporter associated with that well. Concentration dependence was observed for each of the four proteins detected in the multiplex assay.

[0032] Figure 4 : Workflow diagram for nanopore protein quantitation. An adaptation of the sandwich ELISA using a secondary antibody that has a biotin group attached to the free end of one of the heavy chains. After the antibody / antigen complex is formed, streptavidin is added. Streptavidin is able to bind four biotin groups, which means that when a streptavidin molecule binds to a heavy chain, an average of three binding sites are still available. A polynucleotide reporter molecule is then added to these available binding sites.

[0033] Figure 5 : Combined testing of biomarker proteins, transcriptome, and exome on a single flow cell. The relatively small number of reporter reads required for sensitive protein quantification means that a large panel of biomarkers can be quantified on a single flow cell, while the same flow cell is used to simultaneously sequence the exome and transcriptome of that subject. This can be used to provide a comprehensive assessment of a subject's health status.

[0034] Figure 6 : Examples of different applications of this method:

[0035] 1. A capture agent that is an antibody that specifically binds to the target analyte is attached to the surface, the first detector is an antibody to the target analyte, and the polynucleotide reporter is directly bound to or directly bound to the first detector. The capture agent is preferably an antibody that binds to a different epitope on the analyte from the first detector. When the target analyte binds to the capture antibody, it can enter the epitope bound by the first detector.

[0036] 2. Attaching a capture agent that is an antibody that specifically binds to the target analyte to the surface, the first detection agent is an antibody (primary antibody) against the target analyte, the second detection agent is an antibody (secondary antibody) that binds to the first detection agent, and the polynucleotide reporter is bound to or associated with the second detection agent. The capture agent is preferably an antibody that binds to a different epitope on the analyte from the first detection agent. When the target analyte binds to the capture antibody, it becomes accessible to the epitope bound by the first detection agent.

[0037] 3. Non-specific binding of the target analyte to the surface. The first detection agent is an antibody (primary antibody) against the target analyte, the second detection agent is an antibody (secondary antibody) that binds to the first detection agent, and the polynucleotide reporter molecule is bound to or associated with the second detection agent.

[0038] 4. Attach a capture agent that is an antibody (target antibody) that binds to the target analyte to the surface. Antibodies can bind to a common epitope on a group of similar molecules, such as antibodies of the same isotype from a particular species, such as human IgG antibodies. The first detection agent is the antigen of the target antibody. The second detection agent is an antibody to the antigen, and the polynucleotide reporter molecule is bound to or associated with the second detection agent. The second detection agent is preferably an antibody that binds to a different epitope on the antigen from the target antibody. When the target analyte binds to the target antibody, the epitope to which the second detection agent binds is preferably accessible.

[0039] 5. Attach a capture agent that is an antibody (target antibody) that binds to the target analyte to the surface. Antibodies can bind to a common epitope on a group of similar molecules, such as antibodies of the same isotype from a particular species, such as human IgG antibodies. The first detection agent is an antigen of the target antibody, and the polynucleotide reporter molecule is bound to or associated with the second detection agent.

[0040] 6. A capture agent that is an antibody (target antibody) that binds to the antigen of the target analyte may be attached to the surface. The first detection agent is an antibody that binds to the target antibody, and the polynucleotide reporter is associated with or bound to the second detection agent. DETAILED DESCRIPTION

[0041] It will be understood that different applications of the disclosed products and methods can be tailored to the specific needs of the art.It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] Additionally, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, reference to a "well" includes two or more such wells, reference to a "surface" includes two or more such surfaces, reference to an "antibody" includes two or more such antibodies, reference to a "polynucleotide" includes two or more such polynucleotides, and so forth.

[0043] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0044] method

[0045] A method for determining the presence or absence of a target analyte is provided. The target analyte is typically present in a sample. The method may be a single-plex method for measuring one target analyte. The target analyte is typically predetermined. The method uses a component specifically designed to recognize the target analyte.

[0046] This method has several advantages. The method has high sensitivity, does not require large amounts of raw materials, has high yields, does not require amplification of target polynucleotides, and can be performed directly in blood, serum or plasma. The method can also be used to detect and quantify multiple target analytes simultaneously, i.e., the method can be a multiplex method. The method can be used to detect multiple target analytes from a single sample, thereby eliminating the need to test a single sample multiple times. The number of target analytes is unlimited. Each target analyte is specifically detected and associated with a unique polynucleotide reporter. There is no limit on the length of the polynucleotide. Therefore, a large number of different polynucleotide reporter sequences can be generated and distinguished by using transmembrane pore sequencing.

[0047] Compared to existing assays, the method is rapid and inexpensive, while still having high specificity. The output is analyzed in real time, allowing the assay to be stopped when enough information has been obtained. These methods can be performed by personnel with minimal training or certification.

[0048] The method may be based on a sandwich immunosorbent assay, which uses a capture antibody to specifically immobilize the target analyte to a surface, and a detection antibody that specifically binds to the target analyte to bind the captured target analyte. However, the method is not limited to a typical sandwich immunosorbent assay format and may be performed in other ways.

[0049] Methods for determining the presence or absence of a target analyte include:

[0050] (a) immobilizing any target analyte present in the sample on a surface;

[0051] (b) contacting the surface with: a first detection agent that specifically binds to the target analyte; and a polynucleotide reporter molecule, wherein the polynucleotide reporter molecule is associated with or bound to the first detection agent; and

[0052] (c) contacting the transmembrane pore with any polynucleotide reporter molecules immobilized on the surface, wherein the polynucleotide reporter molecules are immobilized on the surface by binding of the first agent to the target analyte, and detecting the polynucleotide reporter molecules using the transmembrane pore to determine the presence or absence of the target analyte in the sample.

[0053] Due to the specificity of the first detection agent to the target analyte, the polynucleotide reporter molecules are only bound to the surface, and then the target analyte is detected by the transmembrane pore whether it is present in the sample. If the polynucleotide reporter molecules are detected using the transmembrane pore, it is determined that the target analyte exists. If the polynucleotide reporter molecules are not detected using the transmembrane pore, the target analyte is not present. The method does not include an amplification step, such as PCR. The polynucleotide reporter molecules bound to the surface by the detection agent and the target analyte are the same polynucleotide molecules that interact with the transmembrane pore. The copy number (reporter molecule reading) of the polynucleotide reporter molecules detected using the hole is related to the concentration of the target analyte in the sample. The method is a quantitative method. Therefore, the reporter molecule reading can be used to determine the concentration of the target analyte in the sample. This can be achieved using standard methods, such as by comparing the reading with the correlation curve generated by the method using a target analyte of known concentration.

[0054] The target analyte can be fixed on the surface by any suitable method. The target analyte can be non-specifically bound to the surface. The non-specific binding of the target analyte to the surface can involve the capture of all molecules of a given type to the surface, such as all proteins present in the sample. In one embodiment, all proteins of a certain type can be captured on the surface, such as all antibodies or all antibodies of a given subtype, such as IgG, IgM, IgA or IgE. This can be achieved by coating the surface with a suitable antibody, such as an anti-human IgG antibody.

[0055] It is preferred that the target analyte specifically binds to the surface. This can be achieved by standard means. Typically, the target analyte is fixed to the surface by binding a capture agent that specifically binds to the target analyte, and the capture agent is fixed to the surface. Suitable capture agents are known in the art and include antibodies, aptamers, receptor molecules (wherein the target analyte is a ligand), ligand molecules (wherein the target analyte is a receptor) and antigens (wherein the target analyte is an antibody). Methods for coating surfaces with capture agents are known in the art and typically include a blocking step to reduce or prevent non-specific binding to the surface. Preferably, the capture agent is an antibody or an antigen. Antibodies preferably specifically bind to the target analyte.

[0056] The first detection agent can be any molecule that specifically binds to the target analyte. The detection agent is preferably an antibody, but can be an aptamer, a receptor molecule (where the target analyte is a ligand), a ligand molecule (where the target analyte is a receptor), or an antigen (where the target analyte is an antibody).

[0057] For example, in the case where the target analyte is an antibody, the first detection agent may be an antibody that binds to the target antibody, and the capture agent may be an antigen that the target antibody binds to; or, the first detection agent may be an antigen that the target antibody binds to, and the capture agent may be an antibody that binds to the target antibody. The first detection agent may be referred to as a primary antibody.

[0058] The polynucleotide reporter molecule can be attached directly or indirectly to the first detection agent.

[0059] The polynucleotide reporter molecule can be attached to the first detection agent before the step (b) of the claimed method, that is, the polynucleotide reporter molecule can be pre-bound to the first detection agent before the first detection agent is added to the surface fixed by the target analyte (if any). Alternatively, the polynucleotide reporter molecule can be added to the assay separately from the first detection agent. It can then be combined with the first detection agent near the surface. For example, the first detection agent can be added to the surface and bound to any target analyte on the surface. Any unbound first detection agent can be removed, usually by washing. The polynucleotide reporter molecule can then be added and bound to the fixed first detection agent.

[0060] In one embodiment, the polynucleotide reporter molecule is connected to a second detection agent. The second detection agent is a molecule that binds to the first detection agent but does not bind to any other component of the assay. The second detection agent is preferably an antibody, but can be an aptamer, a receptor molecule for which the first detection agent is a ligand, a ligand molecule for which the first detection agent is a receptor, or an antigen for which the first detection agent is an antibody. The second detection agent can be an antibody that binds to the first detection agent, such as an antibody that specifically binds to the constant region of an antibody of a particular species and / or isotype. For example, if the first detection agent is a mouse IgG antibody, the second detection agent can be a goat anti-mouse IgG antibody. The use of such first and second detection agents is conventional in the art, such as in ELISA assays. The second detection agent can be referred to as a secondary antibody.

[0061] When the polynucleotide reporter is bound to the first detector through a second detector, the second detector is typically added to the assay after allowing the first detector to bind to any target analyte on the surface and any unbound first detector to be removed, typically by washing. The polynucleotide reporter can be pre-bound to the second detector before the second detector is added to the surface on which the first detector (if any) is fixed. Alternatively, the polynucleotide reporter can be added to the assay separately from the second detector. It can then be bound to the second detector near the surface. For example, the second detector can be added to the surface and bound to any first detector on the surface. Any unbound second detector can be removed, typically by washing. The polynucleotide reporter can then be added and bound to the fixed second detector.

[0062] The polynucleotide reporter molecule can be directly or indirectly bound to or combined with the first detection agent or the second detection agent. The polynucleotide reporter molecule can be connected to the detection agent by any suitable means. Indirect binding includes binding through the second detection agent. Indirect binding also includes binding through a linker or linker molecule.

[0063] In one embodiment, an affinity tag can be used to connect the polynucleotide reporter to the first or second detection agent. Suitable affinity tags include streptavidin / biotin, his tags, glutathione and Ni-NTA. Preferably, the affinity tag is biotin / streptavidin. The detection agent and / or the polynucleotide reporter can be functionalized by adding biotin.

[0064] The polynucleotide reporter molecule can be associated with or bound to the first detection agent via a second detection agent that binds to the first agent. The second detection agent typically comprises an antibody.

[0065] In a specific embodiment of the method, the first detection agent or the second detection agent comprises a first component of an affinity tag and (a) a polynucleotide reporter molecule is linked to a second component of the affinity tag that specifically binds to the first component; or (b) a polynucleotide reporter molecule is linked to the first component of the affinity tag, and the second component of the affinity tag that specifically binds to the first component is used to link the first detection agent or the second detection agent to the polynucleotide reporter molecule. For example, the first component of the affinity tag can be biotin, and the second component of the affinity tag can be streptavidin.

[0066] In this method, the first detection agent or the second detection agent may comprise an oligonucleotide that hybridizes to at least a portion of the polynucleotide reporter molecule.

[0067] In this method, the polynucleotide reporter is typically about 10 to 5000, such as 20 to 1000, 30 to 500 or 50 to 100 nucleotides in length. The polynucleotide reporter may comprise DNA, RNA or a synthetic nucleic acid.

[0068] Two or more, possibly identical, polynucleotide reporters may be associated with or bound to the first detection agent.

[0069] The method can be a multiplex method for simultaneously detecting and / or quantifying two or more target analytes. Typically, the assay can be used for simultaneously detecting and / or quantifying two, ten, 20, 50 or more target analytes, such as 2 to 1000, 5 to 800, 10 to 500, 20 to 200 or 30 to 100 target analytes. Not all target analytes designed for detection can be present in a sample. Therefore, the multiplex method can determine the presence or absence of each member of a specific and predetermined group of two or more target analytes. The multiplex method can be used to determine the amount of the target analyte in the sample, such as the concentration. If the target analyte is not present in the sample, the amount is zero. Therefore, the method can be used to determine the amount of all target target analytes. In the multiplex method, multiple components as defined above are used. In order to make the multiplex method work effectively, each target analyte is typically specifically labeled with different polynucleotide reporter molecules. Each different reporter polypeptide typically has a different nucleotide sequence. Different polynucleotide reporter molecules can be distinguished from each other by their different sequences. The sequence of the polynucleotide reporter molecule can be easily determined using a nanopore sequencing method. The polynucleotide reporters can be mixed together and separated at the level of the nanopore, which can only interact with one polynucleotide at a time. All or part of the sequence of the polynucleotide reporter can be determined. It is sufficient to sequence only the different, preferably unique parts of the polynucleotide reporter to identify it.

[0070] Linking of polynucleotide reporter molecules to detection agents

[0071] The detection agent can be labeled with a polynucleotide reporter in any way. The detection agent is usually labeled with a polynucleotide reporter so that the polynucleotide reporter is not removed by any washing step discussed below, but can be removed from the surface when detected by a transmembrane hole. The labeling is preferably instantaneous, i.e. the polynucleotide reporter is not separated from the detection agent during the washing step, but can be separated from the detection agent during the detection step when interacting with the hole. The preferred instantaneous connection is by hybridization. The detection agent can be functionalized with an oligonucleotide or polynucleotide (e.g., any of the above-discussed) hybridized or specifically hybridized with a polynucleotide reporter.

[0072] Antibodies are typically functionalized at a location distal to the antigen binding site of the antibody, typically in the constant region, and preferably at the termini of the antibody molecule. An oligonucleotide or polynucleotide specifically hybridizes to a target polynucleotide when it preferentially or with high affinity hybridizes to the target polynucleotide, but does not substantially hybridize, does not hybridize, or hybridizes only with low affinity to other polynucleotides.

[0073] If the oligonucleotide or polynucleotide is bound to the target polynucleotide at a melting temperature (T m) is at least 2°C, such as at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C or at least 10°C higher than the T of the other sequences. m , then the oligonucleotide or polynucleotide will hybridize specifically. More preferably, the oligonucleotide or polynucleotide hybridizes with the target polynucleotide, wherein T m Higher than its T with other nucleic acids m At least 2° C., such as at least 3° C., at least 4° C., at least 5° C., at least 6° C., at least 7° C., at least 8° C., at least 9° C., at least 10° C., at least 20° C., at least 30° C. or at least 40° C. Preferably, for a sequence that differs from the target polynucleotide by one or more nucleotides, such as 1, 2, 3, 4 or 5 or more nucleotides, the oligonucleotide or polynucleotide is at a T greater than that of the target polynucleotide. m A T of at least 2°C, such as at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 20°C, at least 30°C or at least 40°C m The oligonucleotide or polynucleotide is typically hybridized to the target polynucleotide at a T of at least 90°C, such as at least 92°C or at least 95°C. m Hybridization. m This can be measured experimentally using known techniques, including the use of DNA microarrays, or can be measured using publicly available T m Calculator calculations, such as those available on the Internet.

[0074] Conditions that allow hybridization are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley-lnterscience, New York (1995)). Hybridization can be performed under low stringency conditions, such as at 37° C. in the presence of a buffer solution of 30% to 35% formamide, 1 M NaCl, and 1% sodium dodecyl sulfate (SDS), followed by 1X (0.1650 M Na) at 50° C. + ) to 2X(0.33M Na +) standard sodium citrate (SSC) washing 20 times. Hybridization can be carried out under moderate stringency conditions, for example, at 37°C in the presence of a buffer solution of 40% to 45% formamide, 1M NaCl and 1% SDS, and then at 55°C in 0.5X (0.0825M Na + ) to 1X(0.1650MNa + ) SSC. Hybridization can be performed under high stringency conditions, for example, at 37°C in the presence of a buffer solution of 50% formamide, 1 M NaCl, and 1% SDS, followed by washing at 60°C in 0.1X (0.0165 M Na + )SSC washing.

[0075] Oligonucleotide or polynucleotide can comprise part or district that is substantially complementary with part or district of polynucleotide reporter molecule.Therefore, compared with part or district in polynucleotide reporter molecule, district or part of oligonucleotide or polynucleotide can have 1,2,3,4,5,6,7,8,9,10 or more mispairings on the district of 5,10,15,20,21,22,30,40 or 50 nucleotides.

[0076] A portion of a region is typically 50 nucleotides or less, such as 40 nucleotides or less, 30 nucleotides or less, 20 nucleotides or less, 10 nucleotides or less, or 5 nucleotides or less.

[0077] Alternatively, both the detection agent and the polynucleotide reporter can be linked to oligonucleotides or polynucleotides that hybridize or are complementary to each other.

[0078] Other preferred transient ligations include, but are not limited to, the use of polyhistidine-tags (hexahistidine-tag, 6xHis-tag, His6-tag or ), Ni-NTA or streptavidin-biotin linkage.

[0079] A preferred method is to biotinylate the first or second detection agent and the polynucleotide reporter molecule and then bind them together using streptavidin. Other suitable labeling methods can be designed by a skilled person. The first or second detection agent can be functionalized with any of the groups discussed above with reference to the surface.

[0080] In some embodiments, the first and / or second detection agent is a chemically modified antibody. Antibodies can be chemically modified in any manner and at any site. Antibodies can preferably be chemically modified by attaching the molecule to one or more cysteines (cysteine ​​ligation), attaching the molecule to one or more lysines, attaching the molecule to one or more non-natural amino acids, enzymatic modification of epitopes, or modification of the ends.

[0081] Suitable methods for making such modifications are well known in the art. Suitable unnatural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz), and Liu CC and Schultz PG, Annu. Rev. Biochem., 2010, 79, 413-444. Figure 1 Any of the amino acids numbered 1-71 in the antibody. Antibodies can be chemically modified by attaching any molecule. For example, antibodies can be chemically modified by attaching polyethylene glycol (PEG), nucleic acids such as DNA, dyes, fluorophores, or chromophores.

[0082] In some embodiments, the antibody is chemically modified with a molecular adapter. The molecular adapter can be a cyclic molecule such as cyclodextrin, a species capable of hybridization, a DNA binder or an intercalator, a peptide or peptide analog, a synthetic polymer, an aromatic planar molecule, a positively charged small molecule, or a small molecule capable of hydrogen bonding. The adapter more preferably includes one or more chemical groups that can interact with one or more amino acids through non-covalent interactions, such as hydrophobic interactions, hydrogen bonding, van der Waals forces, cationic interactions, and / or electrostatic forces. The chemical group that can interact with one or more amino acids is typically a hydroxyl or an amine. The hydroxyl group can be attached to a primary, secondary, or tertiary carbon atom. The hydroxyl group can form a hydrogen bond with an uncharged amino acid.

[0083] The adaptor can be covalently attached to the antibody using any method known in the art. The adaptor is usually attached by chemical connection. If the molecular adaptor is attached by cysteine ​​connection, the one or more cysteines are preferably introduced into the mutant by substitution. The antibody can be chemically modified by attaching the molecular adaptor to one or two of these cysteines. Alternatively, the antibody can be chemically modified by attaching the molecule to one or more cysteines or non-natural amino acids such as FAZ introduced at other positions. The reactivity of cysteine ​​residues can be enhanced by modifying adjacent residues. For example, the basic groups of arginine, histidine or lysine residues on the side change the pKa of the cysteine ​​sulfhydryl group to the pKa of the more reactive S group. The reactivity of cysteine ​​residues can be protected by sulfhydryl protecting groups such as dTNB. Before attaching the linker, these can react with one or more cysteine ​​residues of the antibody.

[0084] Preferably, the molecule is attached to the antibody using a linker such as a chemical cross-linker or a peptide linker. Suitable chemical cross-linkers are well known in the art. Preferred cross-linkers include 2,5-dioxopyrrolidine-1-yl 3-(pyridine-2-yl disulfanyl) propionate, 2,5-dioxopyrrolidine-1-yl 4-(pyridine-2-yl disulfanyl) butyrate and 2,5-dioxopyrrolidine-1-yl 8-(pyridine-2-yl disulfanyl) octanoate. The most preferred cross-linker is 3-(2-pyridyl dithio) propionic acid succinimidyl ester (SPDP). Typically, before the molecule / cross-linker complex is covalently linked to the antibody, the molecule is covalently linked to a bifunctional cross-linker. Suitable linkers include but are not limited to iodoacetamide-based and maleimide-based linkers. In other embodiments, the antibody can be attached to a polynucleotide binding protein. Polynucleotide binding proteins are discussed below.

[0085] The polynucleotide binding protein can be covalently linked to the antibody using any method known in the art. The antibody and polynucleotide binding protein are chemically fused or genetically fused. Genetic fusion of proteins to polynucleotide binding proteins is discussed in WO 2010 / 004265.

[0086] The polynucleotide binding protein can be directly attached to the antibody through one or more linkers. The polynucleotide binding protein can be connected to the antibody using a hybridization linker as described in WO 2010 / 086602. Alternatively, a peptide linker can be used. A peptide linker is an amino acid sequence. The length, flexibility and hydrophilicity of the peptide linker are generally designed so that it does not interfere with the function of the antibody. Preferred flexible peptide linkers are 2 to 20, such as 4, 6, 8, 10 or serine and / or glycine stretches. More preferred flexible linkers include (SG)i, (SG)~, (SG)~(SG)4, (SG)q and (SG)8, wherein S is serine and G is glycine. Preferred rigid linkers are 2 to 30, such as 4, 6, 8, 16 or 24 proline stretches. More preferred rigid linkers include (P)iz, wherein P is proline.

[0087] Polynucleotides can be functionalized in the synthesis reaction using modified phosphoramidites that are easily compatible with the addition of reactive groups such as thiols, cholesterol, lipids, and biotin groups. These different attachment chemistries create a range of attachment options for polynucleotides. Each different modifying group tethers the polynucleotide in a slightly different manner, and the coupling is not always permanent, thus giving different residence times for the polynucleotide to couple to the bilayer.

[0088] Coupling of polynucleotides can also be achieved by a variety of other means, provided that a compatible group is added to the polynucleotide. Adding reactive groups to either end of DNA has been reported previously. Polynucleotide kinase and ATPyS can be used to add thiol groups to the 5' end of ssDNA (Grant, GP and PZ Qin (2007). "A facile method for attaching nitroxide spinlabels at the 5'terminus of nucleic acids." Nucleic Acids Research 35(10): e77). Terminal transferases can be used to add a wider range of chemical group choices, such as biotin, thiols, and fluorophores, to the 3' end of ssDNA to modify oligonucleotides (Kumar, A., P. Tchen et al. (1988). "Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase." Anal Biochem 169(2):376-82).

[0089] Alternatively, the reactive group can be considered as the addition of a short piece of DNA complementary to the DNA already coupled to the antibody, so that attachment can be achieved by hybridization. The connection of a short piece of ssDNA has been reported using T4 RNA ligase I (Troutt, AB, MG McHeyzer-Williams et al. (1992)). "Ligation-anchored PCR: a simple amplification technique with single-sided specificity". Proc Natl Acad Sci USA 89 (20): 98235). Alternatively, ssDNA or dsDNA can be connected to native dsDNA, and then the two chains can be separated by heat or chemical denaturation. A piece of ssDNA can be added to one or both of the ends of the native DNA duplex, or dsDNA can be added to one or both ends. Then, when the duplex is melted, if ssDNA is used for connection or modification at the 5' end, 3' end, each single strand will have a 5' modification or a 3' modification, or if dsDNA is used for connection, each single strand will have both 5' and 3' modifications. If the polynucleotide is a synthetic chain, coupling chemistry can be incorporated during the chemical synthesis of polynucleotides. For example, primers to which reactive groups are attached can be used to synthesize polynucleotides. A common technique for amplifying genomic DNA segments is to use polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, many copies of the same DNA segment can be generated, wherein for each copy, the 5' of each chain in the duplex will be a synthetic polynucleotide. By using antisense primers with reactive groups such as cholesterol, thiol, biotin or lipids, each copy of the target DNA amplification will contain reactive groups for coupling.

[0090] It may be possible to use a polynucleotide binding protein. It is very simple to determine in the art whether a protein binds to a polynucleotide. Proteins generally interact with polynucleotides and modify at least one of their properties. Proteins can modify polynucleotides by cleaving polynucleotides to form individual nucleotides or shorter nucleotide chains such as dinucleotides or trinucleotides. The polynucleotide binding protein is preferably a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide that can interact with a polynucleotide and modify at least one of its properties. The enzyme can modify a polynucleotide by cleaving a polynucleotide to form individual nucleotides or shorter nucleotide chains such as dinucleotides or trinucleotides. The enzyme can modify a polynucleotide by directing or moving the polynucleotide to a specific position. The polynucleotide handling enzyme does not need to show enzymatic activity, as long as it can bind to a target sequence. For example, the enzyme can be modified to remove its enzymatic activity or can be used under conditions that prevent it from acting as an enzyme. Such conditions are discussed in more detail below.

[0091] The polynucleotide handling enzyme is preferably derived from a nucleolytic enzyme. The polynucleotide handling enzyme used in the enzyme's construct is more preferably derived from a member of any of the following Enzyme Classification (EC) groups: 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30, and 3.1.31. The enzyme may be any enzyme disclosed in WO010 / 086603. Preferred enzymes are polymerases, exonucleases, helicases, and topoisomerases, such as gyrase.

[0092] sample

[0093] Each analyte is typically present in any suitable sample. The method is typically performed on two or more samples known to contain or suspected of containing the analyte. Alternatively, the method may be performed on two or more samples to confirm that the presence in the samples is a known or expected characteristic of two or more analytes.

[0094] The sample may be a biological sample. The method may be performed in vitro using a sample obtained or extracted from any organism or microorganism. The organism or microorganism is typically an archaebacterium, a prokaryotic or eukaryotic microorganism, and typically belongs to one of the following five kingdoms: Plant Kingdom, Animal Kingdom, Fungi Kingdom, Prokaryotes Kingdom, and Protist Kingdom. The method may be performed in vitro on a sample obtained or extracted from any virus. The sample is preferably a fluid sample.

[0095] The sample may comprise a body fluid of the patient. The sample may be urine, lymph, saliva, mucus or amniotic fluid, but is preferably blood, plasma or serum. Typically, the sample is of human origin; but alternatively it may be from another mammal, such as from a commercially farmed animal, such as a horse, cow, sheep, fish, chicken or pig; or alternatively may be a pet, such as a cat or dog.

[0096] Alternatively, the first sample and / or the second sample can be of plant origin, such as a sample obtained from a cash crop, such as a cereal, a legume, a fruit or a plant, for example wheat, barley, oats, canola, corn, soybeans, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugarcane, cocoa, or cotton.

[0097] The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids; water, such as drinking water, sea water or river water; and reagents for laboratory testing.

[0098] The sample is usually processed before use in the method, for example by centrifugation or by passing through a membrane that filters out unwanted molecules or cells (such as red blood cells). The sample can be measured immediately after acquisition. The sample can also be stored, preferably below -70°C, before determination.

[0099] analyze

[0100] The method is used to determine the presence or absence of one (single-plex method) or more than one (multiplex method) target analyte in a sample. In a single-plex method, the target analyte is present or absent. The method is quantitative and can be used to determine the amount of target analyte present in a sample. The concentration of the analyte can be determined using a suitable calibration curve. The use of calibration curves is standard in the art.

[0101] The method can be used to analyze any number of analytes simultaneously, such as 2, 5, 10, 15, 20, 30, 40, 50, 100, 500, 1000, 1500, 1750, 2000 or more analytes. Preferably, the method can be used to analyze about 2 to about 2000 analytes simultaneously, such as about 5 to about 1500 analytes, about 10 to about 1000 analytes, about 20 to about 500 analytes or about 50 to about 100 analytes.

[0102] The analyte can be any analyte that can be detected using an antibody. The analyte is preferably selected from metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, bleaches, drugs, diagnostic agents, recreational drugs, explosives and environmental pollutants. In a multiplex method, the group includes two or more analytes of the same type, such as two or more proteins, two or more nucleotides or two or more drugs. Alternatively, the group may include two or more analytes of different types, such as one or more proteins, one or more nucleotides and one or more drugs.

[0103] The analyte may be an analyte which is secreted from the cell. Alternatively, the analyte may be an analyte which is present inside the cell, such that the analyte must be extracted from the cell before the method can be performed.

[0104] The analyte is preferably selected from amino acids, peptides, polypeptides and / or proteins. The amino acids, peptides, polypeptides or proteins may be naturally occurring or non-naturally occurring. The polypeptides or proteins may contain synthetic or modified amino acids therein. A variety of different types of modifications to amino acids are known in the art. Suitable amino acids and modifications thereof are discussed below with reference to transmembrane pores. For the present purpose, it will be appreciated that the analyte may be modified by any method available in the art.

[0105] The protein may be selected from enzymes, antibodies, hormones, growth factors or growth regulatory proteins, such as cytokines. Cytokines may be selected from: interleukins, preferably IFN-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IL-13; interferons, preferably IL-γ; and other cytokines, such as TNF-α. The protein may be a bacterial protein, a fungal protein, a viral protein or a parasite-derived protein.

[0106] The analyte is preferably selected from oligonucleotides and / or polynucleotides. Nucleotides generally contain a nucleobase, a sugar and at least one phosphate group. Nucleobases are generally heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). Sugars are generally pentoses. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. Nucleotides are generally ribonucleotides or deoxyribonucleotides. Nucleotides generally contain monophosphate, diphosphate or triphosphate. Phosphoric acid can be attached to the 5' or 3' side of the nucleotide.

[0107] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (d deoxycytidine triphosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine monophosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate, and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotide is preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP. The nucleotide may be abasic (ie lacking a nucleobase). The nucleotide may contain additional modifications. Specifically, suitable modified nucleotides include, but are not limited to, 2'aminopyrimidines (such as 2'-aminocytidine and 2'-aminouridine), 2'-hydroxypurines (such as 2'-fluoropyrimidines (such as 2'-fluorocytidine and 2'-fluorouridine), hydroxypyrimidines (such as 5'-α-P-borane uridine), 2'-O-methyl nucleotides (such as 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine and 2'-O-methyluridine), 4'-thiopyrimidines (such as 4'-thiouridine and 4'-thiocytidine), and nucleotides having modifications to the nucleobases (such as 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0108] A nucleotide may be abasic (ie, lack a nucleobase). A nucleotide may also lack a nucleobase and a sugar (ie, be a C3 spacer).

[0109] Oligonucleotides are short nucleotide polymers, which typically have 50 or fewer nucleotides, such as 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer nucleotides. Oligonucleotides may include any of the nucleotides discussed below, including abasic and modified nucleotides.

[0110] Polynucleotides such as nucleic acids are macromolecules comprising two or more nucleotides. Polynucleotides or nucleic acids can include any combination of any nucleotides. Nucleotides can be naturally occurring or artificial. One or more nucleotides in the target polynucleotide can be oxidized or methylated. One or more nucleotides in the template polynucleotide can be damaged. For example, the polynucleotide can include pyrimidine dimers. Such dimers are generally associated with ultraviolet damage and are the main cause of skin melanoma. One or more nucleotides in the template polynucleotide can be modified, for example, with a label or tag. Suitable labels are described below. The template polynucleotide can include one or more spacers.

[0111] The nucleotides in a polynucleotide can be linked to each other in any manner. The nucleotides are usually attached via their sugar and phosphate groups, as in nucleic acids. The nucleotides can be linked via their nucleobases, as in pyrimidine dimers.

[0112] The polynucleotide may be double-stranded. The polynucleotide is preferably single-stranded. The polynucleotide may be one strand from a double-stranded polynucleotide.

[0113] A polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) morpholino nucleic acid, bridged nucleic acid (BNA) or other synthetic polymers with nucleotide side chains. A polynucleotide can include any of the nucleotides discussed below, including modified nucleotides.

[0114] The target polynucleotide is preferably about 15 to about 30 nucleotides in length, such as about 20 to about 25 nucleotides in length. For example, the polynucleotide can be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length.

[0115] The group of two or more target polynucleotides can be any group of polynucleotides. For example, the group can be associated with a particular phenotype. The group can be associated with a particular type of cell. For example, a group can indicate a bacterial cell. A group can indicate a virus, a fungus, or a parasite.

[0116] The target polynucleotide is preferably a microRNA (or miRNA). A group of two or more target polynucleotides is preferably a group of two or more miRNAs. Suitable miRNAs are well known in the art. For example, suitable miRNAs are stored in publicly available databases (Jiang Q., Wang Y., Hao Y., Juan L., Teng M., Zhang X., Li M., Wang G., Liu Y., (2009) miR2Disease: A manually curated database on microRNA dysregulation in human diseases. Nucleic Acids Res.).

[0117] miRNA can be preferably used for diagnosing or predicting a disease or condition. Disease or condition is preferably cancer, coronary heart disease, cardiovascular disease or sepsis. Disease or condition is more preferably abdominal aortic aneurysm, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myocardial infarction, acute promyelocytic leukemia (APL), adenoma, adrenocortical carcinoma, alcoholic liver disease, Alzheimer's disease, anaplastic thyroid cancer (ATC), anxiety, asthma, astrocytoma, atopic dermatitis, autism spectrum disorder (ASD), B cell chronic lymphocytic leukemia, B cell lymphoma, Becker muscular dystrophy (BMD), bladder cancer, brain tumor, breast cancer, Burkitt's lymphoma, myocardial hypertrophy, cardiomyopathy, cardiovascular disease, cerebellar neurodegeneration, cervical cancer, bile duct cancer, cholesteatoma, choriocarcinoma, chronic lymphocytic leukemia, leukemia, chronic myeloid leukemia, chronic pancreatitis, colon cancer, colorectal cancer, congenital heart disease, coronary artery disease, calf syndrome, dermatomyositis (DM), diabetic nephropathy, diarrhea-predominant irritable bowel syndrome, diffuse large B-cell lymphoma, dilated cardiomyopathy, Down syndrome (DS), Duchenne muscular dystrophy (DMD), endometrial cancer, endometrial endometrioid adenocarcinoma, endometriosis, epithelial ovarian cancer, esophageal cancer, esophageal squamous cell carcinoma, essential thrombocythemia (ET), facioscapulohumeral muscular dystrophy (FSHD), follicular lymphoma (FL), follicular thyroid cancer (FTC), frontotemporal dementia, gastric cancer (gastric cancer), glioblastoma, Glioblastoma multiforme (GBM), Glioma, Glomerular disease, Glomerulosclerosis, Hamartoma, HBV-related cirrhosis, HCV infection, Head and neck cancer, Head and neck squamous cell carcinoma (HNSCC), Hearing loss, Heart disease, Heart failure, Hepatitis B, Hepatitis C, Hepatocellular carcinoma (HCC), Hilar cholangiocarcinoma, Hodgkin's lymphoma, Homozygous sickle cell disease (HbSS), Huntington's disease (HD), Hypertension, Hypopharyngeal cancer, Inclusion body myositis (IBM), Insulinoma, Intrahepatic cholangiocarcinoma (ICC), Kidney cancer, Kidney disease, Laryngeal cancer, Advanced insomnia (sleep sickness), Pulmonary leiomyoma, Leukemia, Limb-girdle muscular dystrophy type 2A (LGMD2A), Lipoma, Lung Adenocarcinoma, lung cancer, lymphoproliferative diseases, malignant lymphoma, malignant melanoma, malignant mesothelioma (MM), mantle cell lymphoma (MCL), medulloblastoma, melanoma, meningioma, metabolic diseases, Miyoshi distal myopathy (MM), multiple myeloma (MM), multiple sclerosis, MYC rearranged lymphoma, myelodysplastic syndrome, myeloproliferative diseases, myocardial infarction, myocardial injury, myoma, nasopharyngeal carcinoma (NPC), nematode myopathy (NM), nephritis, neuroblastoma (NB), neutrophilia, Niemann-Pick type (NPC) disease, non-alcoholic fatty liver disease (NAFLD), non-small cell lung cancer (NSCLC), obesity, oral cancer,Ovarian cancer (OC), pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), pancreatic tumors, panic disorder, papillary thyroid carcinoma (PTC), Parkinson's disease, PFV-1 infection, pharyngeal disease, pituitary adenoma, polycystic kidney disease, polycystic liver disease, polycythemia vera (PV), polymyositis (PM), primary biliary cirrhosis (PBC), primary myelofibrosis, prion disease, prostate cancer, psoriatic arthritis, psoriasis, pulmonary hypertension, recurrent ovarian cancer, renal cell carcinoma, renal clear cell carcinoma, retinitis pigmentosa (RP), retinoblastoma , rhabdomyosarcoma, rheumatic heart disease and atrial fibrillation, rheumatoid arthritis, sarcoma, schizophrenia, sepsis, serous ovarian cancer, Sezary syndrome, skin diseases, small cell lung cancer, spinocerebellar ataxia, squamous cell carcinoma, T-cell leukemia, teratocarcinoma, testicular germ cell tumor, thalassemia, thyroid cancer, tongue squamous cell carcinoma, Tourette syndrome, type 2 diabetes, ulcerative colitis (UC), uterine leiomyoma (ULM), uveal melanoma, vascular disease, vesicular stomatitis, or Waldenstrom's macroglobulinemia (WM). Since the multiplex method can determine the presence or absence of two or more miRNAs, two or more of any of the diseases listed above can be predicted or diagnosed.

[0118] The method can be used to detect a group of two or more analytes, which can be any group of analytes. For example, the group can be associated with a specific phenotype. The group can be associated with a specific type of cell. For example, the group can indicate bacterial cells. The group can indicate viruses, fungi, or parasites. The group can be a specific group of recreational drugs (such as the SAMHSA 5-panel test), explosives, or environmental pollutants.

[0119] The group of two or more analytes is preferably a group of two or more biomarkers that can be used to diagnose or predict a disease or condition. Biomarkers can be any of the above analytes, such as proteins or polynucleotides. Suitable biomarker groups are known in the art, for example as described in the following: Edwards, AVG et al. (2008) Mol. Cell Proteomics 7, pp. 1824-1837; Jacquet, S. et al. (2009), Mol. Cell Proteomics 8, pp. 2687-2699; Anderson NL et al. (2010) Clin. Chem. 56, 177-185. The disease or condition is preferably cancer, coronary heart disease, cardiovascular disease or sepsis.

[0120] The group may contain two or more analytes in the same category. If the analytes have structural similarities, they belong to the same category. If the analytes are proteins, they belong to the same category if they belong to the same classification of protein structure classification (SCOP). If the analytes are functionally or phylogenetically related, they belong to the same category. For example, opioids, such as heroin, codeine and morphine, can be considered to belong to the same class of analytes. Similarly, different forms of interleukin 1, such as IL-1α, IL-1β and IL-1RA, can be considered to belong to the same class of analytes. In this article, a class of analytes is generally two or more analytes that are structurally different but can be bound by one aptamer. The method preferably includes the use of at least one probe that includes an aptamer that binds to a class of analyte members. For example, such an embodiment allows the determination of the presence or absence of one or more IL-1 analytes in a sample. The ability to detect the presence or absence of one or more analyte members in a particular class has its advantages. For example, initial multiple determinations can be performed on various classes of analytes. Once the presence of an additional class is determined, more specific multiplexed assays related to those classes can be performed to determine the presence or absence of one or more analyte members of each class.

[0121] The inventor has recognized that the method can be used for identifying the binding partner of target analyte such as drug target. Therefore, a drug screening method is also provided. In this embodiment, the drug target is fixed on the surface, for example, by non-specific binding (such as absorption) to the surface or by combining a suitable capture agent. A group of candidate agents can be added under the conditions suitable for combining the drug target, and each agent is associated with different reporter genes. Before adding the assay, the candidate agent can be combined with its respective polynucleotide reporter molecules, or a detection agent specific to each candidate agent can be used, wherein the polynucleotide reporter molecules are combined or combined with the detection agent in a specific manner. Then the nucleotide reporter molecules are sequenced to identify the polynucleotide reporter molecules, thereby identifying the candidate agents combined in the assay.

[0122] surface

[0123] Any surface can be used in the method. Suitable examples include, but are not limited to, probes, plates, columns, pins, beads, and dipsticks. The probe can be any probe discussed in MA Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and WO 2006 / 100484.

[0124] The surface can be any surface used in an enzyme-linked immunosorbent assay (ELISA). The surface can be a standard 96 or 384 well plate. Such plates are commercially available from Fisher scientific, VWR suppliers, Nunc, Starstedt or Falcon.

[0125] The binding strength of the capture agent to its target is typically sufficient so that the target remains bound to the antibody after any wash steps discussed below.

[0126] The capture agent, which may be an antibody, may be attached to the surface in any manner. Attachment may be direct. Attachment may be indirect through one or more linkers or functionalized molecules. Attachment may be covalent. The capture agent is typically attached to the surface so that it is not removed by any of the washing steps discussed below. Methods for attaching capture agents to surfaces (e.g., microparticles) are known in the art.

[0127] The surface is preferably the surface of one or more microparticles. Any number of microparticles can be used in the present method. For example, the method can use a single microparticle or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 1,000, 5,000, 10,000, 100,000, 500,000 or 1,000,000 or more microparticles. If two or more microparticles are used, the microparticles can be identical. Alternatively, a mixture of different microparticles can be used.

[0128] Each microparticle can be attached with a capture agent. Alternatively, each microparticle can be connected with two or more capture agents, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, 5,000 or more, 10,000 or more, 100,000 or more, 1000,000 or more, or 5000,000 or more capture agents. Microparticles can be coated or covered with analyte substantially or completely. Microparticles can have capture agents connected to substantially all or all of their surfaces.

[0129] Microparticles are microscopic particles whose size is usually measured in micrometers (μm). Microparticles may also be referred to as microspheres or microbeads. Microparticles may be nanoparticles. Nanoparticles are microscopic particles whose size is usually measured in nanometers (nm).

[0130] The microparticles typically have a particle size of about 0.001 μm to about 500 μm. For example, the microparticles can have a particle size of about 0.01 μm to about 200 μm or about 0.1 μm to about 100 μm. More commonly, the microparticles have a particle size of about 0.5 μm to about 100 μm, or, for example, about 1 μm to about 50 μm. The microparticles can have a particle size of about 1 nm to about 1000 nm, such as about 10 nm to about 500 nm, about 20 nm to about 200 nm, or about 30 nm to about 100 nm.

[0131] Microparticles can be spherical or non-spherical. Spherical microparticles can be referred to as microspheres. Non-spherical particles can be, for example, plate-shaped, needle-shaped, irregular or tubular. The term "particle size" as used herein means the diameter of a particle if the particle is spherical, or if the particle is non-spherical, means the particle size based on volume. The particle size based on volume is the diameter of a sphere having the same volume as the non-spherical particle in question.

[0132] If two or more microparticles are used in the method, the average particle size of the microparticles can be any size discussed above, such as about 0.5 μm to about 500 μm. The population of two or more microparticles preferably has a coefficient of variation (ratio of standard deviation to mean) of 10% or less, such as 5% or less or 2% or less.

[0133] Any method can be used to determine the size of the microparticles. Suitable methods include, but are not limited to, flow cytometry (see, e.g., Chandler et al., J Thromb Haemost. 2011 Jun;9(6):1216-24).

[0134] The microparticles may be formed from any material. The microparticles are preferably formed from ceramics, glass, silica, polymers or metals. The polymer may be a natural polymer such as polyhydroxyalkanoate, dextran, polylactide, agarose, cellulose, starch or chitosan, or a synthetic polymer such as polyurethane, polystyrene, poly(vinyl chloride), silane or methacrylate. Suitable microparticles are known in the art and are commercially available. Ceramic and glass microspheres can be purchased from Silica and polymer microparticles are available from EPRUI Nanoparticles & Microspheres Co. Ltd. Microparticles are also available from Polysciences Inc., Bangs Laboratories Inc., and Life Technologies.

[0135] The microparticles may be solid. The microparticles may be hollow. The microparticles may be formed from polymer fibers.

[0136] The surface of the microparticle can interact with and attach to the capture agent. The surface can interact with the capture agent naturally without being functionalized. The surface of the microparticle is usually functionalized to facilitate the attachment of the capture agent. Suitable functionalizations are known in the art. For example, the surface of the microparticle can be labeled with a polyhistidine tag (hexahistidine tag, 6xHis-tag, His6 tag or ), Ni-NTA, streptavidin, biotin, oligonucleotides, polynucleotides (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxyl groups, quaternary amine groups, thiol groups, azide groups, alkyne groups, DIBO, lipids, FLAG-tags (FLAG octapeptide, polynucleotide binding proteins (including any of those discussed below), peptides, proteins, antibodies or antibody fragments. Antibody fragments are discussed in more detail below. Microparticles can also be functionalized with other linkers or groups.

[0137] The microparticles are preferably paramagnetic or magnetic. The microparticles preferably comprise paramagnetic or superparamagnetic materials or paramagnetic or superparamagnetic metals, such as iron. Any suitable magnetic microparticles can be used. For example, magnetic beads available from, for example, Clontech, Promega, Invitrogen ThermoFisher Scientific, and NEB can be used. In some embodiments, the microparticles comprise magnetic particles to which organic groups are attached, such as metal chelating groups, such as nitrilotriacetic acid (NTA). The organic component can, for example, comprise a group selected from the following: -C(=O)O-, -COC-, -C(=O)-, -NH-, -C(=O)-NH, -C(=O)-CH2-I, -S(=O)2-, and -S-. The organic component can comprise a metal chelating group, such as nitrilotriacetic acid (NTA). Typically, metals such as gold, iron, nickel, or cobalt are also attached to the metal chelating group. Such magnetic beads are typically used to capture His-tagged proteins, but are also suitable for the present method.

[0138] The microparticles are most preferably His-Tag microparticles available from Life Technologies. MagStrep beads from IBA, Streptavidin magnetic beads from NEB, Solid Phase Reversible Immobilization (SPRI) beads from Beckman Coulter, or Agencourt AMPure XP beads or MyOne TM Streptavidin C1 (ThermoFisher Scientific).

[0139] The method may be performed using any suitable capture and / or detection agents. The skilled person will be able to readily select suitable reagents. The format of the method may be adapted to the nature of the target analyte, the purpose of the method, the level of the target analyte expected, including the relative levels of multiple target analytes, the capture and detection agents used, the surface used, etc. Some examples of suitable assay formats are shown in Figure 6 middle.

[0140] Antibody

[0141] The antibody used in the method can be a monoclonal antibody or a polyclonal antibody. For the present purpose, unless otherwise specified, the term "antibody" includes fragments that specifically bind to the relevant target. Such fragments include Fv, F(ab') and F(ab')2 fragments, and single-chain antibodies. In addition, the antibody or its fragment can be a chimeric antibody, a CDR-grafted antibody or a humanized antibody. Antibodies can be produced by any suitable method. Methods for preparing and characterizing antibodies are well known in the art, see, for example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, antibodies can be produced by generating antibodies against the entire polypeptide or its fragment (e.g., its antigenic epitope, hereinafter referred to as "immunogen") in a host animal. The fragment can be any fragment mentioned herein (usually at least 10 or at least 15 amino acids long). The method for producing polyclonal antibodies includes immunizing a suitable host animal, such as an experimental animal, with an immunogen, and separating immunoglobulins from the serum of the animal. Therefore, the animal can be inoculated with an immunogen, and then blood is taken from the animal and the IgG fraction is purified. Methods for producing monoclonal antibodies involve immortalizing cells that produce the desired antibody.Hybridoma cells can be produced by fusing spleen cells from vaccinated experimental animals with tumor cells (Kohler and Milstein (1975) Nature 256, 495-497).

[0142] Immortalized cells that produce the desired antibodies can be selected by conventional methods. Hybridomas can be grown in culture or injected intraperitoneally to form ascites fluid or into the bloodstream of an allogeneic or immunocompromised host. Human antibodies can be prepared by immunizing human lymphocytes in vitro and then transforming the lymphocytes with Epstein-Barr virus.

[0143] For the production of monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse. If desired, the immunogen can be administered as a conjugate, wherein the immunogen is coupled to a suitable carrier, for example, via the side chain of one of the amino acid residues. The carrier molecule is typically a physiologically acceptable carrier. The antibodies obtained can be separated and, if desired, purified.

[0144] Antibodies are also commercially available.

[0145] An antibody or other molecule or group "specifically binds" a target when it specifically binds to the target with preferential or high affinity but does not bind substantially, does not bind, or binds only with low affinity to other analytes. Various protocols for competitive binding or immunoradiometric analysis to determine the specific binding ability of an antibody are well known in the art (see, e.g., Maddox et al., J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of a complex between a specific target and its antibody and the measurement of complex formation.

[0146] If 1×10 -6 M or less, more preferably 1×10 -7 M or smaller, 5×10 -8 M or less, more preferably 1×10 -8 M or less or more preferably 5×10 -9 M or less, then the antibody or other molecule or group binds preferentially or with high affinity. -6 M or more, more preferably 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, even more preferably 1×10 -2 If a molecule or group binds with a Kd of M or greater, then the molecule or group binds with low affinity.

[0147] Preferably, the affinity of the antibody to the target is at least 10 times greater than its affinity for other analytes or other molecules, such as at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 1000 times, or at least 10,000 times. Affinity can be measured using known binding assays, such as binding assays using fluorescence and radioisotopes. Competitive binding assays are also known in the art. Binding strength can be measured using nanopore force spectroscopy, as described in Hornblower et al., Nature Methods. 4: 315-317. (2007).

[0148] Polynucleotide reporter

[0149] The polynucleotide reporter can be any type of polynucleotide described above. The detector can be labeled with any number of polynucleotide reporters, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500 or 1000 or more polynucleotide reporters. The more polynucleotide reporters attached to a single detector molecule, the greater the amplification of the signal and the lower the concentration of the detectable target analyte. All polynucleotide reporters attached to a single detector molecule are preferably identical, or at least comprise identical nucleotide sequences.

[0150] The method may include specifically labeling two or more target analytes (if present), each analyte having a specific polynucleotide reporter. This means that the method involves labeling the target analytes (if present) with polynucleotide reporters specific for the analyte. The assay is designed so that a first detection agent that specifically binds to a first target analyte (directly or through a second detection agent) is labeled with a polynucleotide reporter specific for the target analyte.

[0151] The transmembrane pore is able to determine any differences between polynucleotides, such as differences in (i) polynucleotide length, (ii) polynucleotide identity, (iii) polynucleotide sequence, (iv) polynucleotide secondary structure, and (v) whether a polynucleotide is modified. Any combination of (i) to (v) can be used to design specific polynucleotide reporters, such as: {i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii}, {ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv}, {i,ii,v}, {i,iii,iv}, {i,iii,v}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v}, {ii,iv,v}, {iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v} or {i,ii,iii,iv,v}. Preferably, each polynucleotide reporter has a unique sequence that can be used to detect the corresponding target analyte.

[0152] The detector is preferably labeled with two or more identical polynucleotides as described above. In such embodiments, each detector in the group will be labeled with two or more identical polynucleotides, and the polynucleotides are different between each detector. Based on any combination of (i) to (v) discussed above, the polynucleotides can be different. Specific polynucleotides generally have different sequences. This ensures that each target analyte in the group is labeled with different, i.e., specific, polynucleotides.

[0153] The polynucleotide reporter can be single-stranded. The polynucleotide reporter is preferably double-stranded. One or more double-stranded polynucleotides are preferably connected at one end using a hairpin loop or a hairpin loop adapter. When one or more double-stranded polynucleotides are detected by a hole, two chains can be interrogated through the hole. Connecting and interrogating two chains on a double-stranded construct in this way improves the efficiency and accuracy of characterization. This is well known in the art and described in WO2013 / 014451.

[0154] Suitable hairpin loops and adapters can be designed using methods known in the art. The hairpin loop can be any length. The length of the hairpin loop is typically 110 or less nucleotides, such as 100 or less nucleotides, 90 or less nucleotides, 80 or less nucleotides, 70 or less nucleotides, 60 or less nucleotides, 50 or less nucleotides, 40 or less nucleotides, 30 or less nucleotides, 20 or less nucleotides, or 10 or less nucleotides. The length of the hairpin loop is preferably about 1 to 110, 2 to 100, 5 to 80 or 6 to 50 nucleotides. If the ring is related to the differential selectivity of the adapter, a hairpin loop of a longer length such as 50 to 110 nucleotides is preferred. Similarly, if the ring is irrelevant to the selectable combination as discussed below, a hairpin loop of a shorter length such as 1 to 5 nucleotides is preferred.

[0155] The hairpin adapter can be provided at either end of the polynucleotide, i.e., the 5' end or the 3' end. The hairpin adapter can be connected to the polynucleotide using any method known in the art. A ligase such as T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, Tma DNA ligase, and 9°N DNA ligase can be used to ligate the hairpin adapter.

[0156] Any method known in the art can be used to separate the two strands of the polynucleotide. For example, they can be separated by polynucleotide binding proteins or using conditions that are conducive to dehybridization (examples of conditions that are conducive to dehybridization include, but are not limited to, high temperature, high pH, ​​and the addition of agents that can disrupt hydrogen bonding or base pairing, such as formamide and urea).

[0157] The hairpin adapter preferably comprises a selectable binding moiety. This allows the polynucleotide to be purified or separated. The selectable binding moiety is a moiety that can be selected based on its binding properties. Therefore, the selectable binding moiety is preferably a moiety that specifically binds to the surface. If the selectable binding moiety binds to the surface at a much higher degree than any other moiety used in the method, then it specifically binds to the surface. In a preferred embodiment, the moiety is bound to a surface that is not bound to the other moieties used in the method.

[0158] Suitable selective binding moieties are known in the art. Preferred selective binding moieties include, but are not limited to: biotin; polynucleotide sequences; antibodies; antibody fragments, such as Fab and ScFv; antigens; polynucleotide binding proteins; polyhistidine tails and GST tags. The most preferred selective binding moieties are biotin and a selectable polynucleotide sequence. Biotin specifically binds to a surface coated with avidin. A selectable polynucleotide sequence specifically binds (i.e., hybridizes) to a surface coated with a homologous sequence. Alternatively, a selectable polynucleotide sequence specifically binds to a surface coated with a polynucleotide binding protein.

[0159] The hairpin adapter and / or the selectable binding moiety may comprise a region that can be sheared, cut, cracked or hydrolyzed. Such regions may be designed to allow the polynucleotide to be removed from the surface to which it is bound after purification or separation. Suitable regions are known in the art. Suitable regions include, but are not limited to, RNA regions, regions comprising desthiobiotin and streptavidin, disulfide bonds, and photocleavable regions.

[0160] The polynucleotide reporter molecule may also have a leader sequence that preferentially penetrates the hole. The leader sequence assists the method. The leader sequence is designed to preferably penetrate the hole and thus assist the movement of the polynucleotide analyte through the hole. The leader sequence may also be used to connect the polynucleotide to one or more anchors as discussed below.

[0161] The leader sequence typically comprises a polymer. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA; a modified polynucleotide (such as abasic DNA); PNA; LNA; polyethylene glycol (PEG) or a polypeptide. The leader preferably comprises a polynucleotide, and more preferably comprises a single-stranded polynucleotide. The leader sequence may comprise any of the polynucleotides discussed above. The single-stranded leader sequence most preferably comprises a single strand of DNA, such as a poly dT segment. The leader sequence preferably comprises one or more spacers.

[0162] The leader sequence may be of any length, but is typically 10 to 150 nucleotides in length, for example 20 to 150 nucleotides in length. The length of the leader sequence will typically depend on the transmembrane pore used in the method.

[0163] If one or more double-stranded polynucleotides comprise a hairpin loop or a hairpin loop adapter at one end, they may comprise a Y adapter at the other end. A Y adapter typically comprises (a) a double-stranded region and (b) a single-stranded region or a region that is not complementary at the other end. If a Y adapter comprises a single-stranded region, it may be described as having an overhang. The presence of a non-complementary region in a Y adapter gives the adapter a Y shape because the two chains do not typically hybridize to each other, unlike the double-stranded portion. A Y adapter may include one or more anchors discussed in more detail above. A Y adapter preferably comprises a leader sequence that is preferably screwed into a hole. Leader sequences are discussed above. As discussed above with respect to hairpin loops and hairpin loop adapters, a Y adapter may be connected to a polynucleotide reporter molecule.

[0164] Transmembrane pores are typically present in the membrane, as discussed in more detail below. The polynucleotide reporter preferably comprises one or more anchors capable of coupling to the membrane. The method preferably further comprises coupling the polynucleotide reporter to the membrane using the one or more anchors.

[0165] The anchor includes a group coupled (or bound) to the polynucleotide reporter and a group coupled (or bound) to the membrane. Each anchor may be covalently coupled (or bound) to the polynucleotide reporter and / or the membrane.

[0166] Any number of anchors can be used to couple the polynucleotide reporter to the membrane, such as 2, 3, 4 or more anchors. For example, two anchors can be used to couple the polynucleotide reporter to the membrane, each anchor being coupled (or bound) to the polynucleotide reporter and the membrane, respectively.

[0167] The one or more anchors may include one or more polynucleotide binding proteins. Each anchor may include one or more polynucleotide binding proteins. The polynucleotide binding protein may be any of the following discussions.

[0168] If the membrane is an amphiphilic layer such as a triblock copolymer membrane, the one or more anchors preferably include a polypeptide anchor present in the membrane and / or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, a fatty acid, a sterol, a carbon nanotube, a polypeptide, a protein or an amino acid, such as cholesterol, palmitic acid or tocopherol. In a preferred embodiment, the one or more anchors are not pores.

[0169] Membrane components, such as amphiphilic molecules, copolymers or lipids, can be chemically modified or functionalized to form the one or more anchors. Examples of suitable chemical modifications and suitable ways to functionalize membrane components are discussed in more detail below. Any proportion of the membrane components can be functionalized, for example, at least 0.01%, at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or 100%.

[0170] The polynucleotide reporter can be directly coupled to the membrane. One or more anchors for coupling the polynucleotide reporter to the membrane preferably comprise a joint. One or more anchors can comprise one or more, for example 2, 3, 4 or more joints. A joint can be used to couple more than one, such as 2, 3, 4 or more polynucleotides to the membrane.

[0171] Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides, and polypeptides. These linkers can be linear, branched, or cyclic. For example, the linker can be a cyclic polynucleotide. The polynucleotide reporter can hybridize to a complementary sequence on a cyclic polynucleotide linker.

[0172] One or more anchors or one or more linkers may comprise a component that can be cleaved or degraded, such as a restriction site or a photolabile group.

[0173] Functionalized linkers and their coupling modes to molecules are known in the art. For example, a linker functionalized with a maleimide group will react with and be connected to a cysteine ​​residue in a protein. In this context, the protein may be present in a membrane, or may be used for coupling (or combining) to a polynucleotide reporter molecule.

[0174] A "lock and key" arrangement can be used to avoid cross-linking of polynucleotides. Each joint has only one end that can react together to form a longer joint, and the other end of the joint is each separately reacted with a polynucleotide reporter molecule or a membrane. Such joints are described in WO 2010 / 086602.

[0175] In the sequencing embodiments discussed below, the use of adapters is preferred. If the polynucleotide reporter is permanently coupled directly to the membrane in the sense that it will not decouple when interacting with the hole, some sequence data will be lost because the round of sequencing cannot continue to the end of the polynucleotide reporter due to the distance between the membrane and the hole. If an adapter is used, the polynucleotide reporter can be fully processed.

[0176] The coupling may be permanent or stable. In other words, the coupling may allow the polynucleotide reporter to remain coupled to the membrane while interacting with the pore.

[0177] The coupling can be temporary. In other words, the coupling can make the polynucleotide reporter molecule decoupled from the membrane when interacting with the hole. For certain applications such as aptamer detection and polynucleotide sequencing, the coupling of temporary nature is preferred. If a permanent or stable joint is directly connected to the 5' end or 3' end of the polynucleotide, and the joint is shorter than the distance between the membrane and the channel of the transmembrane hole, then when the sequencing operation cannot continue to the end of the polynucleotide, some sequence data will be lost. If the coupling is temporary, then when the end of the coupling randomly becomes free of membrane, the polynucleotide can be processed completely. The chemical groups that form permanent / stable or temporary connections are discussed in more detail below. Using cholesterol or fatty acyl chains, polynucleotide reporters can be temporarily coupled to amphiphilic layers or triblock copolymer membranes. Any fatty acyl chain with a length of 6 to 30 carbon atoms, such as hexadecanoic acid, can be used.

[0178] The polynucleotide reporter is preferably coupled to an amphiphilic layer, such as a triblock copolymer membrane or a lipid bilayer. The coupling of nucleic acids to synthetic lipid bilayers has been previously performed using a variety of different tethering strategies. These are summarized in Table 1 below.

[0179] Table 1

[0180]

[0181] Synthetic polynucleotide and / or joint can be functionalized using modified phosphoramidites in building-up reactions, and the modified phosphoramidites are easily compatible with suitable anchoring groups directly added, such as cholesterol, tocopherol, palmitate, thiol, lipid and biotin groups. These different connection chemistries provide a set of selections for the connection of polynucleotide. Each different modifying group couples polynucleotide in a slightly different manner, and coupling may not always be permanent, therefore giving polynucleotide reporter molecules different residence times on the film. The advantage of temporary coupling is discussed above.

[0182] The coupling of polynucleotides to joints or to functionalized membranes can also be achieved by a variety of other means, with the limiting condition that complementary reactive groups or anchoring groups can be added to the polynucleotides. It has been previously reported that reactive groups are added to either end of a polynucleotide. T4 polynucleotide kinase and ATPγS can be used to add thiol groups to the 5' of ssDNA or dsDNA (Grant, GP and PZQin (2007). "A facile method for attaching nitroxide spinlabels at the 5'terminus of nucleic acids." Nucleic Acids Res 35 (10): e77). T4 polynucleotide kinase and γ-[2-azidoethyl]-ATP or γ-[6-azidohexyl]-ATP can be used to add azido groups to the 5'-phosphate of ssDNA or dsDNA. Using thiol or click chemistry, a tether containing any of thiol, iodoacetamide OPSS or maleimide groups (reactive to thiol) or dibenzocyclooctyne (DIBO) or alkynyl (reactive to azide) can be covalently attached to a polynucleotide reporter. Terminal transferases can be used to increase the selection of more diverse chemical groups, such as biotin, thiol and fluorophore, so that the modified oligonucleotides are incorporated into the 3' (Kumar, A., P. Tchen et al. (1988) of ssDNA. "Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase." Anal Biochem 169 (2): 376-82). Streptavidin / biotin and / or streptavidin / desulfobiotin coupling can be used. It is also possible that a terminal transferase with appropriately modified nucleotides (e.g. cholesterol or palmitate) can be used to directly add anchors to polynucleotides.

[0183] One or more anchors are preferably coupled to the polynucleotide reporter molecules on the film by hybridization.Hybridization can be present in any part of one or more anchors, such as between one or more anchors and the polynucleotide reporter molecules, in one or more anchors or between one or more anchors and the film.As discussed above, hybridization in one or more anchors allows coupling in a temporary manner.For example, a joint can include two or more polynucleotides hybridized together, such as 3,4 or 5 polynucleotides.One or more anchors can directly hybridize with the polynucleotide reporter molecules, or directly hybridize with Y adapters and / or the leader sequence attached to the polynucleotide reporter molecules, or directly hybridize with the hairpin loop adapter attached to the polynucleotide reporter molecules (as discussed below).Alternately, one or more anchors can hybridize with one or more (such as 2 or 3) intermediate polynucleotides (or "splints"), and the intermediate polynucleotides hybridize with the polynucleotide reporter molecules, hybridize with the Y adapters and / or the leader sequence attached to the polynucleotide reporter molecules, or hybridize with the hairpin loop adapter attached to the polynucleotide reporter molecules (as discussed above).

[0184] The one or more anchors may comprise single-stranded or double-stranded polynucleotides. A portion of the anchor may be joined to a single-stranded or double-stranded polynucleotide. It has been reported that short pieces of ssDNA are connected using T4 RNA ligase I (Troutt, AB, MG McHeyzer-Williams et al. (1992). "Ligation-anchored PCR: a simple amplification technique with single-sided specificity." Proc Natl Acad Sci USA 89 (20): 9823-5). Alternatively, a single-stranded or double-stranded polynucleotide may be connected to a double-stranded polynucleotide, and then the two chains may be separated by heat or chemical denaturation. For double-stranded polynucleotides, a piece of single-stranded polynucleotide may be added to one or both ends of the duplex, or a double-stranded polynucleotide may be added to one or both ends. In order to add a single-stranded polynucleotide to a double-stranded polynucleotide, T4 RNA ligase I may be used to achieve connection to other regions of the single-stranded polynucleotide. To add a double-stranded polynucleotide to a double-stranded polynucleotide, the ligation can be "blunt-ended" by adding a 3' dA / dT tail to the polynucleotide and the added polynucleotide, respectively (routinely performed for many sample preparation applications to prevent concatemer or dimer formation) or using "sticky ends" generated by restriction digestion of one or more polynucleotides and ligation-compatible adapters. Then, when the duplex is melted, each single strand will have a 5' or 3' end modification if a single-stranded polynucleotide is used for ligation; or each single strand will have a modification at the 5' end, 3' end, or both ends if a double-stranded polynucleotide is used for ligation.

[0185] If the polynucleotide reporter is a synthetic strand, one or more anchors may be incorporated during chemical synthesis of the polynucleotide. For example, a polynucleotide may be synthesized using a primer to which a reactive group is attached.

[0186] Adenylated polynucleotides are intermediates in the ligation reaction, where adenosine monophosphate is attached to the 5'-phosphate of the polynucleotide. Various kits are available for generating such intermediates, such as the 5' DNA Adenylation Kit from NEB. Reactive groups (such as thiols, amines, biotin, azides, etc.) can be added to the 5' of the polynucleotide by replacing the modified nucleotide triphosphates with ATP in the reaction. Anchors can also be added directly to the polynucleotide using a 5' DNA Adenylation Kit with appropriately modified nucleotides (such as cholesterol or palmitate).

[0187] A common technique for amplifying genomic DNA segments is to use polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, many copies of the same DNA segment can be generated, wherein for each copy, the 5' of each strand in the duplex will be a synthetic polynucleotide. Single or multiple nucleotides can be added to the 3' end of single or double-stranded DNA by using a polymerase. Examples of polymerases that can be used include (but are not limited to) terminal transferase, Klenow, and E. coli Poly (A) polymerase). By replacing ATP for the modified nucleotide triphosphate in the reaction, an anchor (such as cholesterol, sulfhydryl, amine, azide, biotin or lipid) can be incorporated into the double-stranded polynucleotide. Therefore, each copy of the amplified polynucleotide will contain an anchor.

[0188] Ideally, the polynucleotide reporter molecules are coupled to the membrane without having to functionalize the polynucleotide reporter molecules. This can be achieved by coupling one or more anchors, such as polynucleotide binding proteins or chemical groups, to the membrane and interacting the one or more anchors with the polynucleotide reporter molecules or by functionalizing the membrane. The one or more anchors can be coupled to the membrane by any method described herein. Specifically, the one or more anchors can include one or more joints, such as maleimide functionalized joints.

[0189] The one or more anchors may comprise any group that couples, binds or interacts with a single-stranded or double-stranded polynucleotide, a specific nucleotide sequence within a polynucleotide, or a pattern of modified nucleotides within a polynucleotide, or any other ligand present on a polynucleotide.

[0190] Binding proteins suitable for use in anchors include, but are not limited to, E. coli single-stranded binding protein, P5 single-stranded binding protein, T4 gp32 single-stranded binding protein, TOPO V dsDNA binding region, human histone protein, E. coli HU DNA binding protein, and other archaeal, prokaryotic or eukaryotic single-stranded or double-stranded polynucleotide (or nucleic acid) binding proteins, including those listed below.

[0191] The specific nucleotide sequence may be a sequence recognized by a transcription factor, a ribosome, an endonuclease, a topoisomerase, or a replication initiation factor. The pattern of the modified nucleotide may be a methylation pattern or a damage pattern.

[0192] The one or more anchors can include any group coupled, combined, inserted or interacted with polynucleotide reporter molecules. The group can be inserted into the polynucleotide reporter molecules or interact with the polynucleotide reporter molecules by static electricity, hydrogen bond or Van der Waals interaction. Such groups include lysine monomers, polylysine (which will interact with ssDNA or dsDNA), ethidium bromide (which inserts into dsDNA), universal bases or universal nucleotides (which can be hybridized with any polynucleotide) and osmium complexes (which can react with methylated bases). Therefore, the polynucleotide reporter molecules can be coupled to the membrane using one or more universal nucleotides connected to the membrane. Each universal nucleotide can be coupled to the membrane using one or more joints. The universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formyl indole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl (C6 aromatic ring). The universal nucleotide more preferably comprises one of the following nucleosides: 2'-deoxyinosine, inosine, 7-deaza-2'-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-O'-methylinosine, 4-nitroindole 2'-deoxynucleoside, 4-nitroindole nucleoside, 5-nitroindole 2'-deoxynucleoside, 5-nitroindole nucleoside, 6-nitroindole 2'-deoxynucleoside, 6-nitroindole nucleoside, 3-nitropyrrole 2'-deoxynucleoside, 3-nitropyrrole nucleoside, acyclic sugar analogs of hypoxanthine, nitroimidazole 2'-deoxynucleoside, nitroimidazole nucleoside, 4-nitropyrazole 2'-deoxynucleoside. '-deoxynucleoside, 4-nitropyrazole nucleoside, 4-nitrobenzimidazole 2'-deoxynucleoside, 4-nitrobenzimidazole nucleoside, 5-nitroindazole 2'-deoxynucleoside, 5-nitroindazole nucleoside, 4-aminobenzimidazole 2'-deoxynucleoside, 4-aminobenzimidazole nucleoside, 5-nitroindazole 2'-deoxynucleoside, 5-nitroindazole nucleoside, 4-aminobenzimidazole 2'-deoxynucleoside, 4-aminobenzimidazole nucleoside, phenyl C-nucleoside, phenyl C-2'-deoxyribosyl nucleoside, 2'-deoxyfumocylindole, 2'-deoxyisoguanosine, K-2'-deoxynucleoside, P-2'-deoxyribose and pyrrolidine. Universal nucleotides more preferably include 2'-deoxyinosine. Universal nucleotides are more preferably IMP or dIMP. The universal nucleotide is most preferably dPMP (2'-deoxy-P-nucleoside monophosphate) or dKMP (N6-methoxy-2,6-diaminopurine monophosphate).

[0193] One or more anchors can be coupled (or combined) to a polynucleotide reporter molecule by Hoogsteen hydrogen bonding (wherein two nucleobases are held together by hydrogen bonding) or reverse Hoogsteen hydrogen bonding (wherein one nucleobase is rotated 180° relative to another nucleobase). For example, one or more anchors can comprise one or more nucleotides, one or more oligonucleotides, or a reporter polynucleotide that form Hoogsteen hydrogen bonding or reverse Hoogsteen hydrogen bonding with a polynucleotide reporter molecule. These types of hydrogen bonding allow a third polynucleotide strand to wrap around a double-stranded helix and form a triplex. One or more anchors can be coupled (or combined) to a double-stranded polynucleotide by forming a triplex with a double-stranded duplex.

[0194] In this embodiment, at least 1%, at least 10%, at least 25%, at least 50%, or 100% of the membrane components may be functionalized.

[0195] When the one or more anchors comprise proteins, the one or more anchors may be able to anchor directly into the membrane without further functionalization, for example when it already has an external hydrophobic region that is compatible with the membrane. Examples of such proteins include, but are not limited to, transmembrane proteins, intramembrane proteins, and membrane proteins. Alternatively, the protein may be expressed with a hydrophobic region genetically fused to it that is compatible with the membrane. Such hydrophobic protein regions are known in the art.

[0196] The one or more anchors are preferably mixed with the polynucleotide reporter molecules prior to delivery to the membrane, but the one or more anchors may be contacted with the membrane and subsequently contacted with the polynucleotide reporter molecules.

[0197] On the other hand, the polynucleotide reporter can be functionalized using the methods described above so that it can be recognized by a specific binding group. Specifically, the polynucleotide reporter can be functionalized with a ligand such as biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or fusion protein), Ni-NTA (for binding to polyhistidine or polyhistidine-tagged proteins) or a peptide (such as an antigen).

[0198] The polynucleotide reporter may comprise one or more polynucleotide binding proteins. The polynucleotide binding protein may be any protein capable of binding to a polynucleotide and controlling its movement through a transmembrane pore. The protein facilitates detection of the polynucleotide reporter using a pore.

[0199] It is simple to determine in the art whether a protein is bound to a polynucleotide. Proteins typically interact with polynucleotides and modify at least one of their properties. Proteins can modify polynucleotides by cleaving polynucleotides to form individual nucleotides or shorter nucleotide chains such as dinucleotides or trinucleotides. The moieties can modify polynucleotides by positioning or moving the polynucleotide to a specific location (i.e., controlling its movement).

[0200] The polynucleotide binding protein is preferably derived from a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide that can interact with a polynucleotide and modify at least one of its properties. The enzyme can modify a polynucleotide by cleaving the polynucleotide to form a single nucleotide or a shorter nucleotide chain such as a dinucleotide or trinucleotide. The enzyme can modify a polynucleotide by directing or moving the polynucleotide to a specific position. The polynucleotide handling enzyme does not need to exhibit enzymatic activity, as long as it can bind to the polynucleotide and control its movement through the hole. For example, the enzyme can be modified to remove its enzymatic activity or can be used under conditions that prevent it from acting as an enzyme. This condition is discussed in more detail below.

[0201] The polynucleotide handling enzyme is preferably derived from a nucleolytic enzyme. The polynucleotide handling enzyme used in the construct of the enzyme is more preferably derived from a member of any group in the following enzyme classification (EC) group: 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The enzyme can be any of those disclosed in International Application PCT / GB10 / 000133 (published as WO 2010 / 086603).

[0202] Preferred enzymes are polymerases, exonucleases, helicases, translocases, and topoisomerases, such as gyrase. Suitable enzymes include, but are not limited to, exonuclease I from E. coli, exonuclease III from E. coli, RecJ from T. thermophilus, and bacteriophage lambda exonucleases, TatD exonucleases, and variants thereof. The polymerase may be 3173 DNA polymerase (which can be purchased from Company), SD polymerase (available from ) or a variant thereof. The enzyme is preferably a Phi29 DNA polymerase or a variant thereof. The topoisomerase is preferably a member of any of the partial classification (EC) groups 5.99.1.2 and 5.99.1.3.

[0203] The enzyme is most preferably derived from a helicase. The helicase may be or be derived from a Hel308 helicase, a RecD helicase, such as a TraI helicase or a TrwC helicase, an XPD helicase or a Dda helicase. The helicase may be or be derived from Hel308 Mbu, Hel308 Csy Hel308 Tga, Hel308 Mhu, TraI Eco, XPD Mbu or a variant thereof.

[0204] The helicase may be any helicase, modified helicase or helicase construct disclosed in WO 2013 / 057495; WO 2013 / 098562; WO 2013 / 098561; WO 2014 / 013260; WO 2014 / 013259; WO 2014 / 013262 and WO / 2015 / 055981.

[0205] The helicase is preferably Trwc Cba or a variant thereof, Hel308 Mbu or a variant thereof, or Dda or a variant thereof. The variant may differ from the native sequence in any of the ways discussed below for transmembrane pores. Preferred variants of Dda include: (a) E94C and A360C; or (b) E94C, A360C, C109A and C136A, and then optionally (ΔM1)G1 (i.e., M1 is deleted and then G1 is added). It may also be referred to as M1G. Any variant discussed above may further include M1G.

[0206] Any number of helicases can be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases can be used. In some embodiments, different numbers of helicases can be used.

[0207] The method preferably comprises contacting the polynucleotide with two or more helicases. The two or more helicases are typically the same helicase. The two or more helicases can be different helicases.

[0208] The two or more helicases can be any combination of the helicases mentioned above. The two or more helicases can be two or more Dda helicases. The two or more helicases can be one or more Dda helicases and one or more TrwC helicases. The two or more helicases can be different variants of the same helicase.

[0209] Two or more helicases are preferably attached to each other. Two or more helicases are more preferably covalently attached to each other. The helicases may be attached in any order and using any method. Preferred helicase constructs are described in WO 2014 / 013260; WO 2014 / 013259; WO 2014 / 013262 and WO2015 / 055981.

[0210] A variant of any of the above enzymes is an enzyme having an amino acid sequence that is different from any of the above enzymes and that retains polynucleotide binding ability. This can be measured using any method known in the art. For example, a variant can be contacted with a polynucleotide and its ability to bind to and move along the polynucleotide can be measured. The variant can contain modifications that facilitate polynucleotide binding and / or facilitate its activity at high salt concentrations and / or room temperature. The variant can be modified so that it binds to a polynucleotide (i.e., retains polynucleotide binding ability) but does not act as a helicase (i.e., when all the necessary components (e.g., ATP and Mg) are present to facilitate movement. 2+ ) does not move along the polynucleotide. Such modifications are known in the art. For example, Mg in helicase 2+ Modification of the binding domain generally produces variants that do not act as helicases. These types of variants can act as molecular brakes (see below).

[0211] The enzyme may be covalently attached to the pore.Any method may be used to covalently attach the enzyme to the pore.

[0212] The preferred molecular brake is TrwC Cba-Q594A. The variant does not function as a helicase (i.e., when all the necessary components for movement (e.g., ATP and Mg) are present). 2+ ), binds to the polynucleotide but does not move along it).

[0213] In chain sequencing, polynucleotides are translocated through nanopores by an applied potential or by resisting an applied potential. Exonucleases that gradually or stepwise work on double-stranded polynucleotides can be used on the cis side of the hole to supply the remaining single strand under an applied potential, or on the trans side to supply the remaining single strand under a reverse potential. Similarly, helicases that unwind double-stranded DNA can also be used in a similar manner. Polymerases can also be used. Sequencing applications that require chain translocation against an applied potential are also possible, but DNA must first be "captured" by enzymes at opposite potentials or without potential. As the potential is subsequently converted after binding, the chain will pass through the hole in a cis-to-trans manner and be in an extended configuration by current retention. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can serve as molecular motors, and the molecular motors resist the applied potential to pull the recently translocated single strand back to the trans through the hole in a stepwise controlled manner (trans to cis).

[0214] Any helicase can be used in the method. The helicase can work relative to the two modes of the hole. First, the method is preferably carried out using a helicase so that the helicase moves the polynucleotide through the hole under the action of the field caused by the applied voltage. In this mode, the 5' end of the polynucleotide is first captured in the hole, and the helicase moves the polynucleotide into the hole so that it passes through the hole under the action of the field until it finally translocates and passes through, arriving at the reverse side of the membrane. Alternatively, the method is preferably carried out in this way, and the helicase moves the polynucleotide through the hole against the field caused by the applied voltage. In this mode, the 3' end of the polynucleotide is first captured in the hole, and the helicase moves the polynucleotide through the hole so that it is pulled out of the hole against the applied field until it is finally pushed back to the cis side of the membrane.

[0215] The method can also be performed in the reverse direction. The 3' end of the polynucleotide can first be captured in the pore and the helicase can move the polynucleotide into the pore so that it is moved through the pore by the field until it eventually translocates through to the opposite side of the membrane.

[0216] When the helicase does not possess the essential components that are convenient to move or is modified to stop or prevent it from moving, the helicase can be combined with the polynucleotide and serve as a brake to slow down the movement of the polynucleotide when the polynucleotide is pulled into the hole by the external field. In the inactive mode, it is not important whether the 3' or 5' of the polynucleotide is captured, and it is the external field that pulls the polynucleotide into the hole towards the opposite side under the effect of the enzyme that serves as the brake. When in the inactive mode, the control of the movement of the polynucleotide by the helicase can be described in a variety of ways, including toothing, sliding and braking. The helicase variant lacking helicase activity can also be used in this way.

[0217] The polynucleotide may be contacted with the polynucleotide binding protein and the pore in any order. Preferably, when the polynucleotide is contacted with a polynucleotide binding protein such as a helicase and the pore, the polynucleotide first forms a complex with the protein. When a voltage is applied across the pore, the polynucleotide / protein complex will form a complex with the pore and control the movement of the polynucleotide through the pore.

[0218] Any step in the detection method using a polynucleotide binding protein is usually carried out in the presence of free nucleotides or free nucleotide analogs and enzyme cofactors that promote the action of the polynucleotide binding protein. The free nucleotides can be one or more of any of the individual nucleotides discussed above. Free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyribonucleic acid monophosphate (DGP), deoxyribonucleic acid monophosphate (DGP), deoxyribonucleic acid monophosphate (DTP ...MP), deoxyribonucleic acid monophosphate (DTP), deoxyribonucleic acid monophosphate (DTP), deoxyribonucleic acid monophosphate (D The free nucleotide is preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotide is preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that allows the construct to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg. 2+ , Mn 2+ , Ca 2+ or Co 2+ The enzyme cofactor is most preferably Mg 2+ .

[0219] Washing steps

[0220] Between the various steps of the method, the surface is typically washed to remove reagent molecules that are not bound to the surface and retain only those molecules that have been bound to the surface. For example, the method may involve one or more washing steps. For example, the method may include one or more washing steps: (i) after contacting the surface with a first capture agent attached to a specific binding target analyte, but before contacting the surface with a first detection agent that specifically binds, (ii) after contacting the surface with a second first detection agent that specifically binds to the target analyte, but before contacting the surface with a detection agent that specifically binds to the first detection agent and / or with a polynucleotide reporter molecule, (iii) after contacting the surface with a second detection agent that specifically binds to the first detection agent, but before contacting the surface with a polynucleotide reporter molecule, and / or (iv) after contacting the surface with a polynucleotide reporter molecule, but before eluting the polynucleotide reporter molecule or before delivering the surface to a membrane comprising a transmembrane pore.

[0221] The washing steps are typically performed using the same buffer used throughout the method, but without the associated components. Any suitable buffer may be used, and suitable buffers are known in the art.

[0222] Transmembrane pore

[0223] A transmembrane pore is a structure that penetrates the membrane to some extent. It allows hydrated ions to flow through or into the membrane, driven by an applied potential. A transmembrane pore usually penetrates the entire membrane, allowing hydrated ions to flow from one side of the membrane to the other side of the membrane. However, a transmembrane pore does not necessarily have to penetrate the membrane. One end of it can be closed. For example, a pore can be a well in the membrane along which hydrated ions can flow or flow into it.

[0224] Any transmembrane pore can be used in the present method. The pore can be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores.

[0225] Any membrane can be used in the present method. Suitable membranes are well known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed by amphiphilic molecules such as phospholipids having at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles forming a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers generally have properties contributed by each monomer subunit. However, block copolymers may have unique properties that polymers formed by separate subunits do not have. Block copolymers may be engineered so that one of the monomer subunits is hydrophobic (i.e., lipophilic) in an aqueous medium, while one or more other subunits are hydrophilic. In this case, the block copolymers can possess amphiphilic properties and can form structures that mimic biological membranes. Block copolymers can be diblock (composed of two monomer subunits), but can also be built from more than two monomer subunits, forming more complex arrangements that behave as amphiphiles. Copolymers can be triblock, tetrablock or pentablock copolymers.

[0226] The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is typically a planar lipid bilayer or a supported bilayer.

[0227] The amphiphilic layer is usually a lipid bilayer. The lipid bilayer is a model of a cell membrane and serves as an excellent platform for a series of experimental studies. For example, the lipid bilayer can be used for in vitro studies of membrane proteins by single channel recording. Alternatively, the lipid bilayer can be used as a biosensor for detecting the presence of a series of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include but are not limited to planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International Application No. PCT / GB08 / 000563 (published as WO2008 / 102121), International Application No. PCT / GB08 / 004127 (published as WO2009 / 077734) and International Application No. PCT / GB2006 / 001057 (published as WO2006 / 100484).

[0228] Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the Examples. Lipid bilayers are typically formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA, 1972; 69: 3561-3566), in which a lipid monolayer is carried on an aqueous solution / air interface on either side of an opening that is perpendicular to the interface.

[0229] The method of Montal and Mueller is popular because it is a cost-effective and relatively simple method for forming good quality lipid bilayers suitable for protein pore insertion. Other common methods for bilayer formation include tip dipping of liposomal bilayers, brushing of bilayers, and patch clamping.

[0230] In a preferred embodiment, the lipid bilayer is formed as described in WO 2009 / 077734.

[0231] In another preferred embodiment, the membrane is a solid-state layer. The solid-state layer is not of biological origin. In other words, the solid-state layer is not derived from or isolated from a biological environment, such as an organism or a cell or a synthetically manufactured version of a biologically obtainable structure. The solid-state layer can be formed of both organic and inorganic materials, including but not limited to: microelectronic materials; insulating materials, such as Si3N4, A12O3 and SiO; organic and inorganic polymers, such as polyamides; plastics, such as or elastomers, such as two-component addition-cured silicone rubber; and glass. The solid layer may be formed from a single atomic layer, such as graphene, or a layer that is only a few atoms thick. Suitable graphene layers are disclosed in WO 2009 / 035647.

[0232] The method is typically performed using: (i) an artificial amphiphilic layer comprising a pore; (ii) an isolated naturally occurring lipid bilayer comprising a pore; or (iii) a cell inserted with a pore. The method is typically performed using an artificial amphiphilic layer such as an artificial lipid bilayer. The layer may include other transmembrane and / or intramembrane proteins and other molecules in addition to the pore. Suitable equipment and conditions are discussed below. The method is typically performed in vitro.

[0233] The transmembrane pore is preferably a transmembrane protein pore. A transmembrane protein pore is a polypeptide or a collection of polypeptides that allow hydrated ions such as analytes to flow from one side of the membrane to the other side of the membrane. Transmembrane protein pores can form holes that allow hydrated ions driven by an applied potential to flow from one side of the membrane to the other side. Transmembrane protein pores preferably allow analytes such as polynucleotides to flow from one side of a membrane, such as a lipid bilayer, to the other side. Transmembrane protein pores allow polynucleotides such as DNA or RNA to move through the hole.

[0234] The transmembrane protein pore may be a monomer or an oligomer. The pore preferably consists of several repeating subunits, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 subunits. The pore preferably consists of 6, 7, 8 or 9 subunits. The pore is preferably a hexamer, heptamer, octamer or nonamer pore. The pore may be a homo-oligomer or a hetero-oligomer.

[0235] Transmembrane protein pores typically contain a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and provide strands for a transmembrane beta barrel or channel or a transmembrane alpha-helical bundle or channel.

[0236] The barrel or channel of the transmembrane protein hole usually comprises an amino acid that promotes interaction with an analyte such as a nucleotide, a polynucleotide or a nucleic acid. These amino acids are preferably located near the contraction of the barrel or channel. The transmembrane protein hole usually comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids usually promote the interaction between the hole and the nucleotide, polynucleotide or nucleic acid.

[0237] Transmembrane protein pores can be derived from β-barrel pores or α-helical bundle pores. β-barrel pores include barrels or channels formed by β-strands. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin, anthrax toxin and leukocidin, and bacterial outer membrane proteins / porins, such as Mycobacterium smegmatis porins (Msp), such as MspA, MspB, MspC or MspD, CsgG, outer membrane porins F (OmpF), outer membrane porins G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP) and other pores, such as lysenin. α-helical bundle pores include barrels or channels formed by α-helices. Suitable α-helical bundle pores include, but are not limited to, inner membrane proteins and α outer membrane proteins, such as WZA, ClyA toxins and FraC. Transmembrane pores can be derived from lysenin. Suitable pores derived from lysin are disclosed in WO 2013 / 153359. Suitable pores derived from CsgG are disclosed in WO 2016 / 034591. The transmembrane pore may be derived from Msp or α-hemolysin (α-HL). The transmembrane protein pore is preferably derived from Msp, preferably from MspA.

[0238] Methods for inserting pore subunits into membranes such as amphiphilic layers are known in the art. For example, the subunits can be suspended in a purified form in a solution containing a lipid bilayer so that they diffuse into the lipid bilayer and are inserted by binding to the lipid bilayer and assembling into a functional state. Alternatively, the subunits can be inserted directly into the membrane using the "pick and place" method described in MA Holden, H. Bayley. Journal of the American Chemical Society (J. Am. Chem. Soc.) 2005, 127, 6502-6503 and WO 2006 / 100484.

[0239] The transmembrane pore may be any pore disclosed in WO 2015 / 166275.

[0240] The monomers of the well can be modified, for example, by the addition of histidine residues (his-tags), aspartic acid residues (asp-tags), streptavidin tags or flag tags, or by the addition of signal sequences for promoting secretion of mutant monomers from cells in which the polypeptide does not naturally contain such sequences, to aid in their identification or purification. An alternative to introducing a genetic tag is to chemically react the tag to a native or engineered position on the well. An example of such a procedure is to react a gel shift reagent with an engineered cysteine ​​outside the well. This has been shown to be a method for separating hemolysin isooligomers (Chem Biol. 1997 Jul; 4(7): 497-505).

[0241] The monomer may be labeled with a revealing label. The revealing label may be any suitable label that allows the pore to be detected. Suitable labels are described below.

[0242] D-amino acids can also be used to generate monomers. For example, a monomer may include a mixture of L-amino acids and D-amino acids. This is conventional in the art for generating such proteins or peptides.

[0243] The monomer contains one or more specific modifications to facilitate nucleotide identification. The monomer may also contain other non-specific modifications, as long as the modification does not interfere with pore formation. Various non-specific side chain modifications are known in the art and can be performed on the side chains of the monomer. Such modifications include, for example, reductive alkylation of amino acids by reaction with aldehydes followed by NaBH4, amidination with methylacetimidate, or acylation with acetic anhydride.

[0244] Monomers can be produced using standard methods known in the art. Monomers derived from Msp can be prepared synthetically or by recombinant means. For example, holes can be synthesized by in vitro translation and transcription (IVTT). WO2010 / 004273, WO2010 / 004265 and WO2010 / 086603 discuss suitable methods for producing holes and inserting holes into membranes. Methods for inserting holes into membranes are also discussed.

[0245] The pores can be produced on a large scale from protein producing organisms after purification by any protein liquid chromatography system or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA system, Bio-Cad system, Bio-Rad BioLogic system, and Gilson HPLC system.

[0246] Using hole detection

[0247] Methods for detecting and characterizing polynucleotide reporters using transmembrane pores are well known in the art. Step (c) of the method may preferably include (i) delivering the surface to a membrane comprising a transmembrane pore, or eluting the polynucleotide reporter from the surface after removing unbound polynucleotide reporters and delivering the eluted polynucleotide reporter to a membrane comprising a transmembrane pore, (ii) allowing the polynucleotide reporter (if present) to interact with the transmembrane pore so that the polynucleotide reporter moves through the pore, and (ii) performing one or more measurements as the polynucleotide reporter (if present) moves relative to the pore, wherein the measured value represents the characteristics of the polynucleotide reporter.

[0248] The polypeptide reporter molecule can be eluted from the surface by any suitable method. For example, by enzymatically cleaving the linker between the polynucleotide and the first or second detection agent or the first or second digestion agent from the polynucleotide reporter molecule at a site close to the attachment point of the polynucleotide reporter molecule, digesting the protein on the surface and recovering the polynucleotide.

[0249] The characteristics (e.g., sequence) of the polynucleotide reporter molecules are used to determine whether the target analyte is present in the sample, and in the case of multiple target analytes, determine which target analytes are present. The amount of the polynucleotide reporter molecules corresponding to the detected target analyte can be used to determine the amount or abundance of the target analyte in the sample. For example, the concentration of the target analyte can be determined by using a calibration curve.

[0250] Some examples of alternative types of measurements include, but are not limited to, electrical measurements and optical measurements. A suitable optical method involving fluorescence measurements is disclosed in the Journal of the American Chemical Society 2009, 131 1652-1653. Possible electrical measurements include current measurements, impedance measurements, tunneling measurements (e.g., as disclosed in Ivanov AP et al., Nano Lett. 2011 Jan 12; 11(1): 279-85) and FET measurements (e.g., as disclosed in International Application WO2005 / 124888). Optical measurements can be combined with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan; 81(1): 014301). The measurement can be a transmembrane current measurement, such as measuring the ionic current through a transmembrane pore. The ionic current can typically be a DC ionic current, but in principle an alternative is to use an AC current (i.e., the magnitude of the AC current flowing under an applied AC voltage).

[0251] Step (c) is preferably performed with an applied potential across the pore. The applied potential typically causes the polynucleotide reporter molecule to translocate or move through the pore. The applied potential may be a voltage potential. Alternatively, the applied potential may be a chemical potential. An example is the use of a salt gradient across the amphiphilic layer. Salt gradients are disclosed in Holden et al., J Am Chem Soc. 2007 Jul 11; 129(27):8650-5.

[0252] The method preferably comprises measuring the current flowing through the pore to detect the polynucleotide reporter molecule.

[0253] Any method known in the art can be used to measure the current. The specific method is described below.

[0254] The method includes delivering the surface (e.g., one or more microparticles) toward the film. The surface (e.g., one or more microparticles) delivers the polynucleotide reporter (if present) to the transmembrane pore in the film. The surface can be delivered toward the film in any way. The method preferably includes positioning the surface near the film or adjacent to the film and allowing the surface to move toward the film. The surface can be located at any distance from the film, for example, about 500 μm or closer from the film, about 200 μm or closer from the film, about 100 μm or closer from the film, about 50 μm or closer from the film or about 30 μm or closer from the film.

[0255] The surface moves toward the membrane. The surface usually moves onto the membrane. The surface can contact the membrane. The surface does not have to contact the membrane. For example, if the surface is substantially or completely coated with polynucleotide reporters or if substantially all or all of the surface is attached to polynucleotide reporters, the surface may not contact the membrane. In some embodiments, the polynucleotide reporters may be larger or longer than the size of the surface or one or more microparticles. The polynucleotide reporters can serve as a cushion between the surface and the membrane. The surface moves close enough to the membrane to deliver the polynucleotide reporters to the hole. The technician can design the system so that the polynucleotide reporters are delivered to the hole.

[0256] The one or more microparticles can move toward the membrane in any manner. The method preferably includes allowing one or more microparticles to move along an electrochemical gradient, a diffusion gradient, a hydrophilic gradient, or a hydrophobic gradient. A gradient is an increase or decrease in the magnitude of a property observed from one point or one moment to another. A skilled person will understand how to generate any of the above gradients and how to move one or more microparticles along them. For example, charged microparticles will typically move along an electrochemical gradient. Microparticles will typically diffuse toward the membrane. Microparticles will typically flow in a solution along a pressure gradient. Hydrophilic or hydrophobic microparticles will typically move along a hydrophilic or hydrophobic gradient. Polynucleotide reporter molecules, antibodies, and any associated molecules, such as one or more anchors, can affect the charge and / or hydrophilicity / hydrophobicity of one or more microparticles.

[0257] The method preferably includes allowing one or more particles to move within a magnetic field. The method preferably includes using a magnetic field to deliver one or more particles to the film. Magnetic particles are discussed above. Suitable methods known for generating a magnetic field include, but are not limited to, magnetic materials or electromagnets.

[0258] The method preferably includes allowing one or more particles to move within the electric field. The method preferably includes delivering one or more particles to the membrane using the electric field. Charged particles are known in the art and discussed above. Suitable methods for generating an electric field are also known.

[0259] The method preferably comprises allowing one or more microparticles to move under pressure. The method preferably comprises using pressure or flow to deliver one or more microparticles to the membrane. The pressure may be physical pressure or osmotic pressure. Suitable methods for generating such pressure are known.

[0260] The method preferably includes allowing one or more microparticles to move in a gravitational field or under gravity. The method preferably includes using gravity to deliver one or more microparticles to film. The dense microparticles positioned above the film in the solution will move toward the film under the influence of gravity. The method can include allowing one or more microparticles to advance, move, slide or roll along the surface toward the film. The surface is usually tilted away from the vertical wall of the chamber (wherein the wall is roughly perpendicular to the plane of the film) comprising the film at an angle of about 45 ° to about 69 °. Compared with the plane of the film, the surface is usually tilted toward the film at an angle of about 21 °, 22 °, 23 °, 24 °, 25 °, 26 °, 27 °, 28 °, 29 °, 30 °, 31 °, 32 °, 33 °, 34 °, 35 °, 36 °, 37 °, 38 °, 39 °, 40 °, 41 °, 42 °, 43 °, 44 ° to 45 °. In a preferred embodiment, the sloped surface is formed by pre-treating one or more surfaces of the chamber containing the membrane with a suitable pre-treatment such as silicone oil, AR20 or hexadecane. Suitable equipment including a chamber for use in the method is discussed below.

[0261] If one or more particles contact the membrane, the method may include allowing the particles to travel, move, slide or roll along the membrane. If one or more particles do not contact the membrane, the method may include allowing the particles to travel, move, slide or roll parallel to the membrane.

[0262] Equipment and conditions

[0263] Electrical measurements can be made using standard single channel recording equipment, for example, as described in Stoddart, DS et al., (2009), Proceedings of the National Academy of Sciences of the United States of America 106, p7702-7707, Lieberman KR et al., J Am Chem Soc, 2010; 132(50): 17961-72 and International Application WO-2000 / 28312. Alternatively, electrical measurements can be made using a multi-channel system, for example, as described in WO2009 / 077734 and WO2011 / 067559.

[0264] The method can be performed using any device suitable for studying a membrane / pore system in which a pore is inserted into a membrane. The method can be performed using any device suitable for transmembrane pore sensing. For example, the device includes a chamber containing an aqueous solution and a barrier that divides the chamber into two sections. The barrier has an opening in which a membrane containing a pore is formed.

[0265] The method may be carried out using the apparatus described in WO 2008 / 102120.

[0266] The method involves measuring the current flowing through the hole. Therefore, the device may also include a circuit capable of applying a potential across the membrane and the hole and measuring the electrical signal. The method can be performed using a patch clamp or a voltage clamp. The method preferably involves using a voltage clamp.

[0267] The method may be performed on silicon-based arrays of wells, wherein each array comprises 128, 256, 512, 1024 or more wells.

[0268] The present method may be related to measuring the current flowing through the hole. Suitable conditions for measuring the ionic current through the transmembrane pore are known in the art and disclosed in the examples. The method is usually carried out when the transmembrane and hole apply voltage. The voltage used is usually +2V to -2V, usually -400mV to +400mV. The voltage used is preferably in the range with a lower limit and an upper limit, and the lower limit is selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV, and the upper limit is independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV and +400mV. The voltage used is more preferably in the range of 100mV to 240mV, and most preferably in the range of 120mV to 220mV. Discrimination of different nucleotides by the pore can be increased by using increased applied potential.

[0269] The method is typically carried out in the presence of any charge carrier, such as a metal salt, for example an alkali metal salt; a halogen salt, for example a chloride salt, such as an alkali metal chloride salt. The charge carrier may include an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus discussed above, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) is typically used. KCl is preferred. The salt concentration may be saturated. The salt concentration may be 3M or less, and is typically 0.1M to 2.5M, 0.3M to 1.9M, 0.5M to 1.8M, 0.7M to 1.7M, 0.9M to 1.6M, or 1M to 1.4M. The salt concentration is preferably 150mM to 1M. The method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio and allow identification of currents indicative of binding / no binding against the background of normal current fluctuations.

[0270] The method is typically performed in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in an aqueous solution in the chamber. Any buffer may be used in the method. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The method is typically performed at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0271] The method can be carried out at a temperature of 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. The method is typically carried out at room temperature. The method is optionally carried out at a temperature that supports enzyme function, such as at about 37°C.

[0272] Methods for measuring concentration

[0273] For those target analytes present, the method preferably also includes measuring the number of polynucleotide reporter molecules detected by the transmembrane pore. This helps determine the concentration of the analyte present in the sample, which is usually referenced to a calibration curve, using equilibrium constants or reference control data. Methods for calculating concentrations are well known in the art.

[0274] Also provided is a method of determining the concentration of a target analyte in a sample, the method comprising:

[0275] (i) performing steps (a) to (c) of the method for determining the presence or absence of a target analyte in a sample; and

[0276] (ii) if the target analyte is shown to be present in the sample, comparing the number of polynucleotide reporters detected using the wells to control or reference data for the target analyte, thereby determining the concentration of the target analyte in the sample.

[0277] Control or reference data can be generated by performing a control experiment in which known concentrations of the analyte members are used to calibrate the assay. The calibration experiment is typically performed using the same components and under the same conditions (particularly component concentrations) as the experimental assay.

[0278] Products and Kits

[0279] Also provided are antibodies labeled with polynucleotide reporters, wherein the polynucleotide reporters are suitable for analysis using transmembrane pores. Preferably, the polynucleotide reporters are double-stranded and contain a double-stranded polynucleotide adapter at one end. For example, the adapter can contain a site in one chain that can be cut with an enzyme to release the polynucleotide reporter from the antibody so that the single-stranded end can be used to interact with the transmembrane pore. The antibody can be any of those discussed above. Antibodies typically specifically bind to a target analyte or an antibody that specifically binds to a target analyte. Streptavidin can be used to label antibodies with polynucleotide reporters, or reporters can be attached to antibodies by any method disclosed herein. As described above, antibodies can be labeled with any number of polynucleotides. The polynucleotide reporter can be any of those discussed above.

[0280] Also provided are panels of two or more antibodies as disclosed herein.

[0281] Also provided is a kit for implementing the disclosed method. The kit comprises a capture agent that specifically binds to a target analyte, a detection agent that specifically binds to a target analyte, and a polynucleotide reporter. The polynucleotide reporter can be attached to a first detection agent. The kit can further comprise streptavidin and / or a polynucleotide reporter, and the first detection agent can be biotinylated.

[0282] Also provided is a kit comprising a set of two or more detection agents, such as antibodies, each detection agent specifically binds to a different target analyte, and a set of polynucleotide reporters, wherein each nucleotide reporter is different, such as comprising a unique nucleotide sequence, and optionally, two or more second detection agents, such as antibodies, which specifically bind to one of the first detection agents in the set. The polynucleotide reporter can be linked to the first detection agent, or optionally to the second detection agent.

[0283] The kit may further comprise (a) a set of two or more capture agents, each capture agent specifically binding to a different target analyte, which capture agents may be attached to one or more surfaces. The kit may further comprise a surface.

[0284] If the surface comprises one or more microparticles, the different capture agents in the set can be combined on the same microparticle. Alternatively, the different capture agents in the set can be on different microparticles.

[0285] The sets of capture agents and first detector agents are preferably different, eg, are distinct. Preferably, each set comprises antibodies against each target analyte, wherein the antibodies against the target analytes in the capture agent set are different from the antibodies against the same target analytes in the detector agent set.

[0286] The kit preferably further comprises a surface, such as one or more microparticles. The capture agent is preferably attached to the surface.

[0287] Any of the embodiments discussed above with reference to the methods apply equally to the sets and kits.

[0288] The kit may further include components of membranes, such as phospholipids required to form an amphiphilic layer, such as a lipid bilayer.

[0289] The test kit may additionally include one or more other reagents or instruments that enable any embodiment mentioned above to be performed. Such reagents or instruments include one or more of the following: suitable buffer (aqueous solution), a device for obtaining a sample from a subject (such as a container or an instrument comprising a needle), a device for amplifying and / or expressing polynucleotides, a film as defined above, a transmembrane pore or a voltage clamp or a patch clamp device. Reagent may be present in the test kit in a dry state so that the fluid sample resuspends the reagent. The test kit may also optionally include the instructions that enable the test kit to be used in the method disclosed herein or the details of which patients the method may be used for. The test kit may optionally include nucleotides.

[0290] Example 1

[0291] This example illustrates how a combination of enzyme-linked immunosorbent assay (ELISA) and sequencing of barcoded polynucleotides on the MinION can be used to indicate the presence and concentration of the protein prostate specific antigen (PSA) in a sample.

[0292] Materials and methods

[0293] The Human PSA ELISA kit (KLK3) (Abcam, ab113327) was selected for detection of PSA. A protein standard dilution series was used as an indicator of whether the barcoded biotinylated polynucleotide reporter could be used for concentration-dependent protein detection. The initial ELISA protocol steps differed little from the Abcam kit protocol. An 8-well strip was prepared. Protein standards were prepared following the instructions of the Abcam kit protocol. This resulted in the following concentrations of protein, each of which was assigned a barcoded polynucleotide reporter (see polynucleotide sequences; all oligonucleotides synthesized by IDT) to be ready for reporter binding at the end of the protocol:

[0294] Table 1: Concentrations (pg / ml) of protein standards added to each ELISA well.

[0295] hole Prostate-specific antigen (PSA) protein standard concentration (pg / ml) Polynucleotide reporter sequence 1 100000 SEQ ID NO:1 2 50000 SEQ ID NO:2 3 25000 SEQ ID NO:3 4 12500 SEQ ID NO:4 5 6250 SEQ ID NO:5 6 3125 SEQ ID NO:6 7 1563 SEQ ID NO:7 8 0 SEQ ID NO:8

[0296] PSA is measured using the following protocol:

[0297] 1. Prepare reagents as described in detail in the Abcam protocol.

[0298] 2. Prepare PSA standards as described in detail in the Abcam protocol. Here, dilute standards in diluent A are assayed.

[0299] 3. Add standard products:

[0300] a. Add 100 μl of standard to each well of an 8-well ELISA plate and one standard dilution to each well. Cover the wells and incubate at room temperature for 2.5 hours with gentle agitation.

[0301] 4. Washing:

[0302] a. Discard the solution.

[0303] b. Add 300 μl of 1x washing solution (Human PSA ELISA Kit (KLK3), Abcam, ab113327). Discard the washing solution.

[0304] c. Repeat (b) for a total of 4 washes.

[0305] d. Remove any remaining detergent.

[0306] 5. Add biotinylated PSA detection antibody:

[0307] a. Dilute 80x stock biotinylated PSA detection antibody (Human PSA ELISA Kit (KLK3), Abcam, ab113327) to 1x with 1x Assay Diluent B (Human PSA ELISA Kit (KLK3), Abcam, ab113327).

[0308] b. Add 100 μl of 1x detection antibody to each well, cover the wells and incubate for 1 hour at room temperature with gentle agitation.

[0309] 6. Wash (as in step 5).

[0310] 7. Add streptavidin:

[0311] a. Dilute streptavidin (Streptavidin from Streptavidin avidin, Sigma-Aldrich, S4762) to 40 ng / μl with 1x PBS (Phosphate Buffered Saline 10x, Sigma-Aldrich, P5493).

[0312] b. Add 50 μl of 40 ng / μl streptavidin in PBS to each well. Cover the wells and agitate gently at room temperature for 50 minutes.

[0313] 8. Wash (as in step 5).

[0314] 9. Add barcode reporter (See the polynucleotide sequence of the barcode polynucleotide reporter assigned to each well. All oligonucleotides synthesized by IDT.):

[0315] a. Assign one barcode reporter to each well. Dilute each to 80 ng in 50 μl of 1xTE (TE buffer (20x), ThermoFisher Scientific, T11493).

[0316] b. Add one 50 μl barcode polynucleotide reporter sample to each appropriate well. Cover the wells and incubate for 30 minutes with gentle agitation.

[0317] 10. Wash (as in step 5).

[0318] 11. Elution of barcoded reporter molecules:

[0319] a. Add 50 μl of 1xPBS containing 1 μl of USER TM Enzyme (USER TM enzyme, NEB, M5505).

[0320] b. Add 50 μl of USER to each well. TM / TE solution, cover the wells and incubate at 37°C with agitation for 20 min.

[0321] c. Pool all eluted samples in 1.5 ml DNA LoBind tubes ( LoBind microcentrifuge tubes, Sigma-Aldrich, Z666548) to form a 400 μl sample.

[0322] d.SPRI cleaning:

[0323] i. Add 400 μl of Agencourt AMPure XP DNA SPRI beads (Agencourt AMPure XP, Beckman-Coulter, A63880) and incubate at room temperature for 5 minutes with agitation. Pellet the beads by placing the tube on a magnetic stand.

[0324] ii. Wash twice with 200 μl of 70% ethanol.

[0325] iii. Spin down and re-pellet the beads, remove any remaining wash solution, and dry the pellet at room temperature for 5-10 minutes.

[0326] iv. Elute with 51 μl of Nuclease-Free Water (Thermo Fisher Scientific, AM9937) at room temperature for 5 minutes. The beads were pelleted and the eluted samples were collected.

[0327] 12. Ligate adapters and prepare for sequencing:

[0328] a. Mix the following in a 1.5 ml DNA LoBind tube (Eppendorf):

[0329] 50 μl barcoded reporter pool

[0330] 20 μl of 1D Barcode Adapter Mix (BAM) (Native Barcoding Kit 1D, Oxford Nanopore Technologies, EXP-NBD103)

[0331] 20 μl NEBNext T4 Quick Ligation Buffer ( Quick Ligation Module, NEB, E6056)

[0332] 10 μl of NEBNext T4 Quick Ligase ( Quick Ligation Module, NEB, E6056)

[0333] b. Incubate at room temperature for 10 minutes.

[0334] c. Clean up with SPRI beads:

[0335] i. Add 40 μl of Agencourt AMPure XP DNA SPRI beads and incubate at room temperature with agitation for 5 minutes. Pellet the beads by placing the tube on a magnetic rack. Remove the supernatant.

[0336] ii. Add 140 μl of Adapter Bead Binding Buffer (ABB) (Ligation Sequencing Kit ID, Oxford Nanopore Technologies, SQK-LSK108) and flick the tube to mix. Pellet the beads and remove the buffer.

[0337] iii. Add 25 μl of Elution Buffer (ELB) (Ligation Sequencing Kit 1D, Oxford Nanopore Technologies, SQK-LSK108) and mix. Incubate at room temperature for 10 minutes. Allow the beads to settle and collect the eluted library.

[0338] 13. Prepare the ligated library for loading into the flow cell:

[0339] a. Combine the following materials in duplicate:

[0340] 12 μl eluted adapter-ligated library

[0341] 25.5 μl library loading beads (LLB) (Library Loading Bead Kit, Oxford Nanopore Technologies, EXP-LLB001)

[0342] 37.5 μl running buffer (RBF) (ligation sequencing kit 1D, Oxford Nanopore Technologies, SQK-LSK108)

[0343] b. Each SpotON Mk I (R9.4) flow cell (Oxford Nanopore Technologies, FLO-MIN106) was loaded with one 75 μl sample, set up on MinIONs (MinION Mk 1B, Oxford Nanopore Technologies) and primed as described in protocol 1D Native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) (Oxford Nanopore Technologies).

[0344] c. Sequencing for 19 hours. Basecall sequencing data was performed using the EPI2ME platform (Metrichor Ltd).

[0345] discuss

[0346] The sequencing data showed that higher PSA concentrations correlated with higher reads of the associated barcoded polynucleotide reporter. This suggests that this combination of ELISA and barcode sequencing allows for concentration-dependent protein detection.

[0347] PSA is used as a biomarker for screening prostate cancer; there is some disagreement about the cut-off PSA level for referral recommendations, but the National Institute for Health and Care Excellence (NICE) recommends an age-dependent threshold starting at 3 ng / ml. This suggests that protein detection by sequencing is within the desired range for this test.

[0348] Example 2

[0349] This example illustrates that a combination of enzyme-linked immunosorbent assay (ELISA) and sequencing of barcoded polynucleotides on the MinION can be used to indicate the presence and concentration of several proteins in a multiplex approach.

[0350] Materials and methods

[0351] The following three ELISA kits are used for concentration-dependent multiplex protein detection testing of specific three proteins:

[0352] Human PSA ELISA Kit (KLK3) (Abcam, ab113327)

[0353] Human Retinol Binding Protein ELISA Kit (RBP) (Abcam, ab108899)

[0354] Human α1 antitrypsin ELISA kit (SERPINA1) (Abcam, ab108799)

[0355] The protein standard dilution series included with each kit serves as an indicator of whether the barcoded biotinylated polynucleotide "reporters" can be used for concentration-dependent protein detection in multiplex methods.

[0356] Each kit prepares one 8-well strip. For each, prepare the protein standards as indicated in each protocol (nb diluents and concentrations vary from protocol to protocol). This results in the following concentrations of protein, each of which is assigned a barcoded polynucleotide reporter (see polynucleotide sequences; all oligonucleotides synthesized by IDT) ready to bind the reporter at the end of the protocol:

[0357] Table 2: Concentrations (pg / ml) of protein standards added to each ELISA well.

[0358]

[0359]

[0360] PSA, RBP, and AAT were measured using the following protocol:

[0361] 1. Prepare reagents as described in detail in the individual kit protocols.

[0362] 2. Prepare standards as described in detail in the individual kit protocols. Here, diluted PSA standards in assay diluent A (option of A or B) and RBP / AAT standards in diluent N are provided in each kit.

[0363] 3. Add standard products:

[0364] a. Add standard to each well of an 8-well ELISA plate (100 μl for PSA, 50 μl for RBP and AAT). Cover the wells and incubate overnight at 4°C, then with gentle agitation at room temperature for 2.5 hours.

[0365] 4. Washing:

[0366] a. Discard the solution.

[0367] b. Add (300 μl for PSA, 200 μl for RBP and AAT) 1x appropriate wash solution provided in each ELISA kit. Discard the wash solution.

[0368] c. Repeat (b) in total (4 washes for PSA, 5 washes for RBP and AAT).

[0369] d. Remove any remaining detergent.

[0370] 5. Add designated biotinylated detection antibodies:

[0371] a. Dilute each desired protein-specific detection antibody as described in the relevant kit protocol.

[0372] b. Add (100 μl for PSA, 50 μl for RBP / AAT) 1x detection antibody to each well, cover wells and incubate for 1 hour at room temperature with gentle agitation.

[0373] 6. Wash (as in step 5).

[0374] 7. Add streptavidin:

[0375] a. Dilute streptavidin (Streptavidin from Streptavidin avidin, Sigma-Aldrich, S4762) to 40 ng / μl with 1x PBS (Phosphate Buffered Saline 10x, Sigma-Aldrich, P5493).

[0376] b. Add 50 μl of 40 ng / μl streptavidin to each well. Cover the wells and agitate gently at room temperature for 50 minutes.

[0377] 8. Wash (as in step 5).

[0378] 9. Add barcode reporter (See the polynucleotide sequence of the barcode polynucleotide reporter assigned to each well. All oligonucleotides synthesized by IDT.):

[0379] a. Assign one barcode reporter to each well. Dilute each to 80 ng in 50 μl of 1xTE (TE buffer (20x), ThermoFisher Scientific, T11493).

[0380] b. Add one 50 μl barcode reporter sample to each appropriate well. Cover the wells and incubate overnight at 4°C, then at room temperature for 30 minutes with gentle agitation.

[0381] 10. Wash (as in step 5).

[0382] 11. Elution of barcoded reporter molecules:

[0383] a. Add 50 μl of 1xPBS containing 1 μl of USER TM Enzyme (USER TM enzyme, NEB, M5505).

[0384] b. Add 50 μl of USER to each well. TM / TE solution, cover the wells and incubate at 37°C with agitation for 20 min.

[0385] c. Pool all eluted samples in 1.5 ml DNA LoBind tubes ( LoBind microcentrifuge tubes, Sigma-Aldrich, Z666548) to form one 1.2 ml pooled sample. Split between two 1.5 ml DNA LoBind tubes to form two 600 μl samples.

[0386] d.SPRI cleaning:

[0387] i. Add 600 μl of Agencourt AMPure XP DNA SPRI beads (Agencourt AMPure XP, Beckman-Coulter, A63880) to each sample and incubate at room temperature for 5 minutes with agitation. Pellet the beads by placing the tubes on a magnetic stand.

[0388] ii. Wash twice with 200 μl of 70% ethanol.

[0389] iii. Spin down and re-pellet the beads, remove any remaining wash solution, and dry the pellet at room temperature for 5-10 minutes.

[0390] iv. Elute each sample with 26 μl of Nuclease-Free Water (Thermo Fisher Scientific, AM9937) at room temperature for 5 minutes. Pellet the beads, collect and combine the eluted samples to form a barcode reporter pool.

[0391] 12. Ligate adapters and prepare for sequencing:

[0392] a. Mix the following in a 1.5 ml DNA LoBind tube (Eppendorf):

[0393] 50 μl barcoded reporter pool

[0394] 20 μl of 1D Barcode Adapter Mix (BAM) (Native Barcoding Kit 1D, Oxford Nanopore Technologies, EXP-NBD103)

[0395] 20 μl NEBNext T4 Quick Ligation Buffer ( Quick Ligation Module, NEB, E6056)

[0396] 10 μl of NEBNext T4 Quick Ligase ( Quick Ligation Module, NEB, E6056)

[0397] b. Incubate at room temperature for 10 minutes

[0398] c. Clean up with SPRI beads:

[0399] i. Add 40 μl of Agencourt AMPure XP DNA SPRI beads and incubate at room temperature with agitation for 5 minutes. Pellet the beads by placing the tube on a magnetic rack. Remove the supernatant.

[0400] ii. Add 140 μl of Adapter Bead Binding Buffer (ABB) (Ligation Sequencing Kit ID, Oxford Nanopore Technologies, SQK-LSK108) and flick the tube to mix. Pellet the beads and remove the buffer.

[0401] iii. Add 25 μl of Elution Buffer (ELB) (Ligation Sequencing Kit 1D, Oxford Nanopore Technologies, SQK-LSK108) and mix. Incubate at room temperature for 10 minutes. Allow the beads to settle and collect the eluted library.

[0402] 13. Prepare the ligated library for loading into the flow cell:

[0403] a. Combine the following materials in duplicate:

[0404] 12 μl eluted adapter-ligated library

[0405] 25.5 μl library loading beads (LLB) (Library Loading Bead Kit, Oxford Nanopore Technologies, EXP-LLB001)

[0406] 37.5 μl running buffer (RBF) (ligation sequencing kit 1D, Oxford Nanopore Technologies, SQK-LSK108)

[0407] b. Each SpotON Mk I (R9.4) flow cell (Oxford Nanopore Technologies, FLO-MIN106) was loaded with one 75 μl sample, set up on MinIONs (MinION Mk 1B, Oxford Nanopore Technologies) and primed as described in protocol 1D Native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) (Oxford Nanopore Technologies).

[0408] c. Sequencing for 48 hours. Basecall sequencing data was performed using the EPI2ME platform (Metrichor Ltd).

[0409] discuss

[0410] Sequencing data ( Figure 2 ) showed that higher protein concentrations were associated with higher reads of the associated barcode polynucleotide reporter. There was concentration-dependent detection for all three proteins. This suggests that this protocol can be used for concentration-dependent detection of a panel of proteins in a multiplex approach.

[0411] Example 3

[0412] This example demonstrates that a combination of enzyme-linked immunosorbent assay (ELISA) and sequencing of barcoded polynucleotides on the MinION can be used to determine the presence and concentration of a panel of proteins of clinical interest in a multiplex approach.

[0413] Materials and methods

[0414] A four-protein lung cancer biomarker panel identified in a serum biomarker panel for lung cancer diagnosis (Patz et al., J Clin Oncol. December 2007) was selected for multiplex testing.

[0415] Select the following ELISA kits to detect four specific target proteins:

[0416] AAT: Abcam Human α1 Antitrypsin ELISA Kit (SERPINA1) (ab108799)

[0417] CEA: Abcam Human Carcino embryonic antigen ELISA kit (CEA) (ab99992)

[0418] ·RBP: Abcam Human Retinol Binding Protein ELISA Kit (RBP) (ab108899)

[0419] ·SCC: RayBio Human Serpin B3 / SCCA1 ELISA Kit (ELH-SERPINB3-1)

[0420] For each, protein standards were prepared following the instructions in each ELISA kit protocol (nb diluents and concentrations varied from protocol to protocol). This resulted in the following concentrations of protein, each of which was assigned a barcoded polynucleotide reporter (see polynucleotide sequences; all oligonucleotides synthesized by IDT) ready to bind the reporter at the end of the protocol:

[0421] Table 3: Concentrations (pg / ml) of protein standards added to each ELISA well.

[0422]

[0423]

[0424] AAT, CEA, RBP, and SCC were measured using the following protocol:

[0425] 1. Prepare reagents as described in detail in the individual kit protocols.

[0426] 2. Prepare standards as described in detail in the individual kit protocols. Here, diluted PSA standards in assay diluent A (option of A or B), RBP / AAT standards in diluent N, and SCC standards in diluent E2 are provided in each kit.

[0427] 3. Add standard products:

[0428] a. Add 50 μl (AAT / RBP) or 100 μl (CEA / SCC) of standard to each well of an 8-well ELISA plate as specified in the appropriate kit protocol, and add one standard dilution to each well. Cover the wells and incubate at room temperature for 2.5 hours with gentle agitation.

[0429] 4. Washing:

[0430] a. Discard the solution.

[0431] b. Add (300 μl for CEA and SCC, 200 μl for RBP and AAT) 1x appropriate wash solution provided in each ELISA kit. Discard the wash solution.

[0432] c. Repeat (b) in total (4 washes for CEA and SCC, 5 washes for RBP and AAT).

[0433] d. Remove any remaining detergent.

[0434] 5. Add designated biotinylated detection antibodies:

[0435] a. Dilute each desired protein-specific detection antibody as described in the relevant kit protocol.

[0436] b. Add 50 μl (AAT / RBP) or 100 μl (CEA / SCC) 1x detection antibody per well as specified in the appropriate kit protocol, cover the wells and incubate with gentle agitation at room temperature for 1 hour.

[0437] 6. Wash (as in step 5).

[0438] 7. Add streptavidin:

[0439] a. Dilute streptavidin (Streptavidin from Streptavidin avidin, Sigma-Aldrich, S4762) to 40 ng / μl with 1x PBS (Phosphate Buffered Saline 10x, Sigma-Aldrich, P5493).

[0440] b. Add 50 μl of 40 ng / μl streptavidin to each well, cover the wells and incubate with gentle agitation at room temperature for 50 minutes.

[0441] 8. Wash (as in step 5)

[0442] 9. Add barcode reporter (See the polynucleotide sequence of the barcode polynucleotide reporter assigned to each well. All oligonucleotides synthesized by IDT.):

[0443] a. Assign one barcode reporter molecule to each well and dilute each to 80 ng in 50 μl of 1xTE (TE buffer (20x), ThermoFisher Scientific, T11493).

[0444] b. Add one 50 μl barcode reporter sample to each appropriate well. Cover the wells and incubate overnight at 4°C, then at room temperature for 30 minutes with gentle agitation.

[0445] 10. Wash (as in step 5).

[0446] 11. Elution of barcoded reporter molecules:

[0447] a. Add 2 μl of USER to each well in 50 μl of 1xPBS TM Enzyme (USER TM enzyme, NEB, M5505).

[0448] b. Add 50 μl of USER / PBS solution to each well, cover the wells and incubate at 37°C with agitation for 90 minutes.

[0449] c. Pool each set of 8 eluted samples in separate 1.5 ml DNA LoBind tubes ( LoBind microcentrifuge tubes, Sigma-Aldrich, Z666548).

[0450] d.SPRI cleaning:

[0451] i. Add 400 μl of Agencourt AMPure XP DNA SPRI beads (Agencourt AMPure XP, Beckman-Coulter, A63880) to each sample and incubate for 5 minutes with agitation. Pellet the beads by placing the tubes on a magnetic stand.

[0452] ii. Wash twice with 200 μl of 70% ethanol.

[0453] iii. Spin and re-pellet the beads, remove residual wash solution and dry the pellet at room temperature for 5-10 minutes.

[0454] iv. Elute each sample with 41 μl of Nuclease-Free Water (Thermo Fisher Scientific, AM9937) at room temperature for 5 minutes. Allow the beads to settle and collect the eluted samples.

[0455] v. Pool all four eluted samples to form one barcoded reporter pool.

[0456] 12. Ligate adapters and prepare for sequencing:

[0457] a. Mix the following in a 1.5 ml DNA LoBind tube (Eppendorf):

[0458] 160 μl barcoded reporter pool

[0459] 64 μl of 1D Barcode Adapter Mix (BAM) (Native Barcoding Kit 1D, Oxford Nanopore Technologies, EXP-NBD103)

[0460] 64 μl NEBNext T4 Quick Ligation Buffer ( Quick Ligation Module, NEB, E6056)

[0461] 32 μl of NEBNext T4 Quick Ligase ( Quick Ligation Module, NEB, E6056)

[0462] b. Incubate at room temperature for 10 minutes

[0463] c. Clean up with SPRI beads:

[0464] i. Add 128 μl of Agencourt AMPure XP DNA SPRI beads and incubate at room temperature with agitation for 5 minutes. Pellet the beads by placing the tube on a magnetic rack. Remove the supernatant.

[0465] ii. Add 448 μl of Adapter Bead Binding Buffer (ABB) (Ligation Sequencing Kit ID, Oxford Nanopore Technologies, SQK-LSK108) and flick the tube to mix. Pellet the beads and remove the buffer.

[0466] iii. Add 25 μl of Elution Buffer (ELB) (Ligation Sequencing Kit 1D, Oxford Nanopore Technologies, SQK-LSK108) and mix. Incubate at room temperature for 10 minutes. Allow the beads to settle and collect the eluted library.

[0467] 13. Prepare the ligated library for loading into the flow cell:

[0468] a. Combine the following materials in duplicate:

[0469] 12 μl eluted adapter-ligated library

[0470] 25.5 μl library loading beads (LLB) (Library Loading Bead Kit, Oxford Nanopore Technologies, EXP-LLB001)

[0471] 37.5 μl running buffer (RBF) (ligation sequencing kit 1D, Oxford Nanopore Technologies, SQK-LSK108)

[0472] b. Each SpotON Mk I (R9.4) flow cell (Oxford Nanopore Technologies, FLO-MIN106) was loaded with one 75 μl sample, set up on MinIONs (MinION Mk 1B, Oxford Nanopore Technologies) and primed as described in protocol 1D Native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) (Oxford Nanopore Technologies).

[0473] c. Sequencing for 48 hours. Basecall sequencing data was performed using the EPI2ME platform (Metrichor Ltd).

[0474] discuss

[0475] There was concentration-dependent detection for all four proteins, with high similarity in the relative abundance of each barcode reporter molecule seen between flow cells. As seen in Example 2, the sequencing data ( Figure 3 ) showed that higher protein concentrations were associated with higher readouts of the associated barcoded polynucleotide reporter. This suggests that this protocol can be used for concentration-dependent detection of a panel of protein biomarkers of clinical interest.

Claims

1. A method for determining the presence or absence of a target analyte in a sample, the method comprising: (a) immobilizing any target analyte present in the sample on a surface; (b) contacting the surface with: (i) a first detection agent that specifically binds to a target analyte; and (ii) a polynucleotide reporter molecule, wherein the polynucleotide reporter molecule is associated with or binds to the first detection agent; and (c) contacting the transmembrane pore with any polynucleotide reporter molecules immobilized on the surface through binding of the first detection agent to the target analyte, and detecting the polynucleotide reporter molecules using the transmembrane pore to thereby determine the presence or absence of the target analyte in the sample.

2. The method of claim 1, comprising determining the concentration of the target analyte in the sample.

3. The method according to claim 1 or 2, wherein: (i) immobilizing the target analyte on the surface by binding a capture agent that specifically binds to the target analyte, the capture agent being immobilized on the surface; (ii) immobilizing the target analyte on the surface by binding to an antibody immobilized on the surface, optionally wherein the antibody specifically binds to the target analyte.

4. The method according to claim 3, wherein: (i) the target analyte is a target antibody; and / or (ii) The first detection agent comprises an antibody.

5. The method according to claim 4, wherein: (a) the first detection agent is an antibody that binds to the target antibody, and the capture agent is an antigen that the target antibody binds to; or (b) the first detection agent is an antigen that the target antibody binds to, and the capture agent is an antibody that binds to the target antibody.

6. The method according to claim 1 or 2, wherein: (i) the polynucleotide reporter is indirectly associated with or binds to the first detection agent, optionally wherein the polynucleotide reporter is associated with or binds to the first detection agent via a second detection agent that binds to the first agent, optionally wherein the second detection agent comprises an antibody; and / or (ii) the first detection agent or the second detection agent comprises: (A) a first component of an affinity tag and (I) the polynucleotide reporter is linked to a second component of an affinity tag that specifically binds to the first component; or (II) the polynucleotide reporter is linked to a first component of an affinity tag, and a second component of an affinity tag that specifically binds to the first component is used to link a first detection agent or a second detection agent to the polynucleotide reporter, optionally, wherein the first component of the affinity tag is biotin and the second component of the affinity tag is streptavidin; or (B) An oligonucleotide that hybridizes to at least a portion of the polynucleotide reporter.

7. The method of claim 1 or 2, wherein two or more polynucleotide reporter molecules are associated with or bound to the first detection agent, optionally, wherein the two or more polynucleotide reporter molecules are the same.

8. The method of claim 1 or 2, comprising determining the presence or absence of two or more target analytes in the sample, wherein each target analyte is detected using a different polynucleotide reporter molecule, and the composition of the polynucleotide reporter molecule is determined using the transmembrane pore.

9. The method of claim 8, wherein the two or more target analytes: (i) is fixed to a separate surface; or (ii) are immobilized on the same surface; optionally, wherein (A) each of the polynucleotide reporters is bound to only one first detection agent that specifically binds to one target analyte, or (B) the polynucleotide reporters are bound to the first detection agent prior to step (b).

10. The method according to claim 1 or 2, wherein: (i) the sample is from an individual, and in step (c), the transmembrane pore is used to simultaneously analyse one or more polynucleotides from the individual, optionally wherein the one or more polynucleotides are part of the exome or transcriptome of the individual; and / or (ii) The target analyte is a disease biomarker.

11. A method for identifying a binding partner of a target analyte, the method comprising: (a) immobilizing the target analyte on a surface; (b) contacting the surface with a candidate binding partner and a polynucleotide reporter molecule, wherein the polynucleotide reporter molecule is associated with or binds to the candidate binding partner; and (c) using the transmembrane pore to detect any polynucleotide reporter molecules immobilized on the surface through binding of the candidate binding partner to the target analyte, thereby determining whether the candidate molecule is a binding partner for the target analyte.

12. The method according to claim 11, wherein: (i) step (b) comprises contacting the surface with two or more candidate molecules, wherein each candidate molecule is associated with or binds to a different polynucleotide reporter molecule, and determining the composition of the polynucleotide reporter molecules using the transmembrane pore; (ii) the target analyte is a drug target; (iii) immobilizing the target analyte on the surface by binding a capture agent that specifically binds to the target analyte, the capture agent being immobilized on the surface, optionally wherein the capture agent is an antibody; (iv) the candidate molecule is linked to a first component of an affinity tag, and (a) the polynucleotide reporter is linked to a second component of an affinity tag that specifically binds to the first component; or (b) the polynucleotide reporter is linked to a first component of an affinity tag, and a second component of an affinity tag that specifically binds to the first component is used to link the candidate molecule to the polynucleotide reporter, optionally wherein the first component of the affinity tag is biotin and the second component of the affinity tag is streptavidin; (v) the candidate molecule is linked to an oligonucleotide which hybridizes to at least a portion of the polynucleotide reporter molecule; and / or (vi) Two or more polynucleotide reporter molecules are associated with or bind to the candidate molecule.

13. The method according to claim 1 or 11, wherein: (i) the target analyte is a peptide, protein, glycoprotein, carbohydrate or hormone; (ii) the target analyte is a post-translationally modified protein; (iii) the polynucleotide reporter molecule has a length of 10 to 5000 nucleotides; (iv) the polynucleotide reporter comprises DNA, RNA or synthetic nucleic acid; (v) the surface is the surface of one or more microparticles, optionally wherein the microparticles are delivered to the transmembrane pore; (vi) the polynucleotide reporter comprises an adaptor at the free end, which facilitates the interaction of the polynucleotide reporter with the transmembrane pore; and / or (vii) determining at least a partial sequence of the polynucleotide reporter using the transmembrane pore.

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