Methods and compositions for detecting and analyzing analytes

Abstract

Nanopore-based methods, compositions, and systems for assessing analyte-ligand interactions and analyte concentration in a fluid solution are provided. The composition includes an analyte detection complex bound to a nanopore to form a nanopore assembly, the analyte detection complex including an analyte ligand. Upon application of a first voltage across the nanopore assembly, the analyte ligand is presented to the analyte in solution. Upon application of a second voltage across the nanopore assembly having a polarity opposite to the first voltage, the analyte binds to the analyte. By comparing the total number of analyte-ligand binding pairs with a control binding count, the concentration of the analyte can be determined. In other embodiments, further increasing the second voltage can cause the analyte-ligand pair to dissociate, thereby determining a dissociation voltage and, therefore, a dissociation constant.

Description

[0001] The present disclosure relates generally to methods, compositions, and systems for detecting target analytes, and more particularly to methods, compositions, and systems for determining analyte concentrations and assessing analyte-ligand interactions using biochips.

[0002] Nanopore-based methods, compositions, and systems for evaluating analyte-ligand interactions and analyte concentrations in fluid solutions are provided. The composition includes an analyte detection complex that is bound to a nanopore to form a nanopore assembly, the analyte detection complex including an analyte ligand. As a first voltage is applied across the nanopore assembly, the analyte ligand is presented to the analyte in the solution. As a second voltage having a polarity opposite to the first voltage is applied across the nanopore assembly, the analyte binds to the analyte. The concentration of the analyte can be determined by comparing the total number of analyte-ligand binding pairs with a control binding count. In other embodiments, further increasing the second voltage can cause the analyte-ligand pair to dissociate, thereby determining the dissociation voltage and, therefore, the dissociation constant. Background of the Invention

[0004] Biologically active components (such as small molecules, proteins, antigens, immunoglobulins, and nucleic acids) are involved in numerous biological processes and functions. Therefore, any disturbance in the levels of such components can lead to disease or accelerate the progression of disease. For this reason, much effort has been spent on developing reliable methods to quickly detect and identify biologically active components for patient diagnosis and treatment. For example, detecting proteins or small molecules in blood or urine samples can be used to assess a patient's metabolic state. Similarly, detecting antigens in blood or urine samples can be used to identify pathogens to which a patient has been exposed, thereby facilitating appropriate treatment. It would be further beneficial to be able to determine the concentration of an analyte in a solution. For example, determining the concentration of a blood or urine component can allow the component to be compared with a reference value, thereby facilitating further evaluation of the patient's health status.

[0005] However, although numerous detection and identification methods are available, many methods are expensive and can be quite time-consuming. For example, many diagnostic tests can take several days to complete and require a large amount of laboratory resources. And in some cases, diagnostic delays can have a negative impact on patient care, such as when analyzing markers associated with myocardial infarction. In addition, the complexity of many diagnostic tests intended to identify biologically active components makes them prone to error, thereby reducing accuracy. Moreover, many detection and identification methods can only analyze one or several biologically active components at a time, and they cannot determine the concentration of a given component in a test sample.

[0006] In addition to identifying the biologically active components in the test sample, it is also desirable to screen for novel binding pairs in the biological sample, such as small molecule-protein binding pairs or protein-protein binding pairs. For example, determining that a specific protein binds to a small molecule may result in the small molecule being developed as a new therapeutic drug or diagnostic reagent. Similarly, the identification of new protein-protein interactions may result in the development of new drugs or diagnostic reagents. However, although many traditional methods can be used to examine the interactions between different biologically active components, such methods are often designed to examine one or several candidate binding pairs at a time. Such methods are also expensive and may be time-consuming.

[0007] Therefore, need additional method, composition and system, they can detect and identify biologically active components rapidly, especially in an effective and cost-effective manner.Also need can measure multiple biologically active components simultaneously thereby reduce the method, composition and system of cost.In addition, need method, composition and system to determine the concentration of biologically active components in the fluid solution.Also need fast and cost-effective method to assess the binding interaction between the biologically active components, thereby further promote the development of new drugs and treatment plans. SUMMARY OF THE INVENTION

[0009] In certain embodiments, an analyte detection complex is provided, comprising an analyte ligand, a threading element, a signaling element, and an anchor tag. The analyte ligand is located on the proximal end of the analyte detection complex, and the signaling element is bound within the threading element. The analyte detection complex may further comprise an anchor tag on the distal end of the threading element. In certain embodiments, the analyte detection complex also comprises a second signaling element.

[0010] In certain aspects of the present invention, a nanopore assembly comprising an analyte detection complex is provided. For example, the nanopore assembly can be a heptameric α-hemolysin nanopore assembly. The analyte detection complex is inserted into a nanopore to form the nanopore assembly.

[0011] In certain embodiments, a method for evaluating the binding strength between an analyte and an analyte ligand is provided. The method comprises providing a chip in the presence of a first voltage, the chip comprising a nanopore assembly as described herein. The nanopore assembly is, for example, disposed within a membrane. A sensing electrode is positioned adjacent to or near the membrane. The method further comprises contacting the chip with a fluid solution comprising an analyte, the analyte having a binding affinity for the analyte ligand of the analyte detection complex. Thereafter, a gradually increasing second voltage is applied across the membrane, the polarity of the second voltage being opposite to that of the first voltage. In response to the gradually increasing second voltage applied across the membrane, a binding signal is determined by means of the sensing electrode, the binding signal providing an indication of the binding of the analyte to the analyte ligand. And as the second voltage further increases, a dissociation signal is determined by means of the sensing electrode, the dissociation signal providing an indication of the binding strength between the analyte and the analyte ligand.

[0012] In certain aspects, the method further comprises detecting a transit signal using a sensing electrode, the transit signal providing an indication that the transit element is within the pore of the nanopore assembly. In certain aspects, the transit signal is compared to the binding signal. The comparison can, for example, provide an indication of binding of the analyte to the analyte ligand.

[0013] In certain embodiments, the method further comprises determining a dissociation voltage associated with the dissociation of the analyte from the analyte ligand from the dissociation signal. By comparing the determined dissociation voltage with a reference dissociation voltage, a dissociation constant of the analyte and analyte ligand binding pair can be determined.

[0014] In certain embodiments, a method for determining the concentration of an analyte in a fluid solution is provided. The method includes, for example, providing a chip in the presence of a first voltage, the chip comprising a plurality of nanopore assemblies as described herein. The nanopore assemblies are, for example, disposed within a membrane, and at least a first subset of the nanopore assemblies comprises a first analyte ligand. The method also includes positioning a plurality of sensing electrodes adjacent to or near the membrane and contacting the chip with the fluid solution. The fluid solution comprises a first analyte having a binding affinity for the first analyte ligand. A binding count is then determined using the sensing electrodes and a computer processor. The binding count, for example, provides an indication of the number of binding interactions between the first analyte ligand and the first analyte. The concentration of the analyte in the fluid solution can be determined by then comparing the determined binding count with a reference count.

[0015] In certain aspects, determining a binding count includes determining a transit signal for each nanopore assembly of a first subset of nanopore assemblies using a sensing electrode. The transit signal, for example, provides an indication that a transit element is within a nanopore of the nanopore assembly. Thereafter, a second voltage is applied across the membrane, gradually increasing, the second voltage having a polarity opposite to the first voltage. In response to applying the second voltage gradually increasing across the membrane, a binding signal is determined for each nanopore assembly of the first subset of nanopore assemblies using the sensing electrode. The method then includes, for each nanopore assembly of the first subset of nanopore assemblies, comparing the determined transit signal with the determined binding signal. The comparison provides, for example, an indication of binding of the first analyte to the first analyte ligand. From the comparison of each determined transit signal with the determined binding signal, a total number of indications of binding of the first analyte to the first analyte ligand can be determined, the total number of indications corresponding to the binding count. In certain aspects, the binding count is compared to a reference binding count.

[0016] These and other aspects, objects, features and advantages of example embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the illustrative example embodiments. DETAILED DESCRIPTION

[0018] The embodiments described herein may be more readily understood by reference to the following detailed description, examples, and claims, and their preceding and subsequent descriptions. Before disclosing and describing the present systems, devices, compositions, and / or methods, it should be understood that, unless otherwise indicated, the embodiments described herein are not limited to the specific systems, devices, and / or compositions and methods disclosed, as these may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0019] In addition, the following description is provided as an implementation teaching of each embodiment in its best currently known aspects. Those skilled in the relevant art will recognize that many changes can be made to the described aspects while still obtaining the beneficial results of the present disclosure. It will also be apparent that by selecting some features of the various embodiments and not utilizing other features, some desired benefits of the present invention can be obtained. Therefore, those skilled in the art will recognize that many modifications and changes to the various embodiments described herein are possible, and in some cases may even be desirable, and are part of the present disclosure. Therefore, the following description is provided as an example of the principle of the embodiments described herein, rather than a limitation thereof.

[0020] Overview

[0021] As described herein, nanopore-based methods, compositions, and systems for determining the concentration of an analyte in a fluid solution are provided. Also provided are nanopore-based methods, compositions, and systems for evaluating analyte-ligand binding interactions in a fluid solution. The composition includes, for example, an analyte detection complex that is bound to a nanopore to form a nanopore assembly, the analyte detection complex comprising an analyte ligand. Upon application of a first voltage across a membrane comprising the nanopore assembly, the analyte ligand is presented to the cis side of the nanopore, where it can bind to an analyte in the fluid solution. Upon application of a second voltage across the membrane having a polarity opposite to the initial voltage, a signal indicative of binding between the analyte and the analyte ligand can be determined. By determining the total number of analyte-ligand binding pairs across multiple nanopore assemblies and comparing this value to a known reference value, the concentration of the analyte in the solution can be determined. In other embodiments, further increasing the second voltage can cause the analyte-ligand pair to dissociate, thereby determining a dissociation voltage and, therefore, a dissociation constant.

[0022] More specifically, the analyte ligand of the analyte detection complex can be any ligand that targets the analyte. For example, the analyte ligand can be an antibody or functional fragment thereof that targets a specific antigen, thereby providing an immunoassay-type method for identifying the antigen. In certain embodiments, the analyte is a blood antigen or other biological fluid antigen. In other embodiments, the analyte is a polypeptide, amino acid, polynucleotide, carbohydrate, or small organic or inorganic compound for which the analyte ligand of the analyte detection complex has affinity.

[0023] In addition to the analyte ligand, the analyte detection complex also includes a crossing element connected to the analyte ligand. The crossing element can be, for example, a single-stranded or double-stranded nucleic acid sequence or other molecular polymers that can pass through the hole of the nanopore. The analyte ligand is connected to the proximal end of the crossing element, and the distal end of the crossing element is combined with an anchor tag. The anchor tag can be used to prevent the distal end of the crossing element from moving to the cis side of the nanopore assembly through the nanopore assembly. Combined with the crossing element are one or more signal elements, which can be used to change the electronic signal passing through the hole. The signal element of the analyte detection complex can be any entity that can be positioned in the hole of the nanopore assembly, such as an oligonucleotide, a peptide or a polymer. In certain embodiments, one or more signal elements can be used to determine the position of the crossing element in the hole of the nanopore assembly.

[0024] When assembled into a membrane of a chip, a nanopore assembly comprising an analyte detection complex as described herein can be used to assess binding interactions between an analyte and an analyte ligand. The nanopore can, for example, be any protein nanopore, such as an α-hemolysin (α-HL) nanopore, an OmpG nanopore, or other protein nanopores. Without wishing to be bound by any particular theory, when a first voltage is applied across the membrane comprising the nanopore assembly, the proximal end of the analyte detection complex penetrates the pore, thereby positioning the transmissive element and its one or more signaling elements within the pore. Further, as the analyte detection complex penetrates the pore, the analyte ligand of the analyte detection complex can be presented to the cis side of the nanopore assembly, where it can interact with (and bind to) the analyte. In certain embodiments, an electrode associated with the nanopore assembly can be used to determine a transmissive signal corresponding to the presence of the transmissive element within the pore. For example, in response to applying a first voltage across the membrane, a first signaling element associated with the transmissive element can be positioned within the pore in such a manner that the position of the transmissive element within the pore can be determined via a sensing electrode.

[0025] Once the transmissive element is within the pore of the nanopore assembly and the analyte ligand has had an opportunity to bind to the analyte, a second voltage having a polarity opposite to the first voltage can be gradually applied across the membrane. The second voltage acts, for example, to pull the analyte detection complex toward the trans side of the nanopore assembly. Without wishing to be bound by any particular theory, in the absence of analyte, the pulling force pulls the analyte detection complex through the pore to the trans side of the pore. However, in the presence of analyte, the binding of the analyte ligand to the analyte on the cis side of the nanopore assembly prevents the analyte detection complex from moving through the pore to the trans side of the nanopore assembly. In certain embodiments, the pulling force generated by the second voltage positions the second signal element within the pore, thereby enabling a binding signal to be determined from an electrode bound to the nanopore assembly. The binding signal can, for example, provide an indication of binding of the analyte to the analyte ligand.

[0026] To assess the binding interaction between the analyte and the analyte ligand, such as the binding strength, the second voltage can be further increased until a dissociation signal is obtained from the nanopore assembly via the associated electrode. The dissociation signal corresponds, for example, to the point at which the increased voltage forces the analyte to separate from the analyte ligand, thereby allowing the analyte detection complex to be drawn through the pore to the opposite side of the membrane. Based on the dissociation signal, a dissociation voltage can be determined, which corresponds to the voltage at which dissociation occurs between the analyte and the analyte ligand. In certain embodiments, the dissociation voltage can be compared to one or more reference voltages of known analyte-ligand pairs, thereby allowing the determination of the dissociation constant of the analyte and the analyte ligand.

[0027] In certain embodiments, the binding between the analyte and the analyte ligand can be so strong that the analyte does not separate from the analyte ligand. In contrast, even when the second voltage is further increased, the analyte remains bound to the analyte ligand. In such embodiments, when evaluating the binding properties of multiple different analytes to different analyte ligands, the analyte with the strongest binding property can be easily identified. In other embodiments, multiple analytes are analyzed to determine their relative binding strengths to one or more analyte ligands. For example, for different analyte-ligand interactions on the same chip, the binding strength can be determined as weak, strong, or very strong.

[0028] In certain embodiments, the methods, compositions, and systems described herein can also be used to determine the concentration of a test analyte in a fluid solution. For example, as described herein, a plurality of nanopore assemblies can be formed on a chip in the presence of a first voltage, thereby presenting a plurality of analyte ligands to the cis-lateral test analyte of each nanopore assembly. The fluid sample can then be applied to the cis-side of the membrane. When a test analyte is present in the fluid sample, the test analyte can bind to the analyte ligand. Thereafter, as described herein, a second voltage having a polarity opposite to the first voltage can be gradually applied across the membrane, thereby pulling each analyte detection complex toward the reverse side of the membrane. However, as described herein, the binding of the analyte to the analyte ligand can prevent the analyte detection complex from moving through the hole to the reverse side of the hole. Further, the movement of the signal transfer element into the hole of the nanopore assembly can allow determination of the binding signal.

[0029] By counting the number of monomers, it is possible to determine the binding count corresponding to the total number of analyte-ligand interactions, and thereby determine the number of the test analytes of combination. The binding count can then be compared with a reference count to determine the concentration of the test analyte in the solution. For example, a second analyte of known quantity can be included in the fluid sample as a control, and the number of the combination between the second analyte and the second analyte ligand can be determined as a reference count as described herein. The binding count can then be compared with a reference count to determine the concentration of the test analyte.

[0030] In certain example embodiments, the methods described herein can be repeated on the chip to increase the confidence of the assessment. For example, if multiple nanopore assemblies are used to assess the binding strength between different analyte-ligand pairs, the second voltage can be increased until the ligand pair dissociates. Then, the first voltage can be reapplied to reposition the analyte detection complex within the hole and allow the analyte-ligand to bind. After binding, the second voltage (whose polarity is opposite to the initial voltage) can be reapplied until the analyte-ligand pair dissociates, thereby providing an additional measurement of the binding strength described herein. Similarly, for concentration determination, once the binding count for the analyte-ligand pair is determined as described herein, a second voltage can be applied to force the analyte-ligand binding pair to dissociate. The steps of concentration determination can be repeated to re-determine the concentration of the analyte. In certain example embodiments, the method is repeated multiple times to further increase the confidence level of the binding strength and / or concentration assessment.

[0031] Terminology Summary

[0032] The present invention will now be described in detail using the following definitions and examples only by reference. Unless otherwise defined in this article, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. It should be understood that the present invention is not limited to the described concrete methods, procedures and reagents, as these may vary.

[0033] The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be obtained by reference to the specification as a whole.Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0034] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0035] Range or value can be expressed as from "about" a specific value and / or to "about" another specific value in this article.When expressing such a range, another aspect includes from a specific value of this range and / or to another specific value of this range.It is further understood that the endpoints of each range are significant relative to the other endpoints and are independent of the other endpoints.Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another aspect.In certain example embodiments, the term "about" is understood to be within the normal tolerance range of this art of a given measurement, for example, such as within 2 standard deviations of the mean value.In certain example embodiments, depending on measurement, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the value.Unless otherwise understood from the context, all numerical values ​​provided herein can be modified by the term "about". Furthermore, the use of terms such as "embodiment," "exemplary," or "illustrative" herein is not meant to indicate a preference, but rather to explain that the aspects discussed thereafter are merely examples of the aspects presented.

[0036] The term "antibody" as used herein broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, namely two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof that retains the basic epitope binding characteristics of the Ig molecule. Such mutant, variant or derivative antibody entities are known in the art. Functional fragments of antibodies, for example, include any part of an antibody that, when separated from the antibody as a whole, retains the ability to bind or partially bind to the antigen to which the antibody is directed. "Nanobodies" are, for example, single domain antibody fragments.

[0037] As used herein, the term "amino acid" refers to an organic compound containing an amino group and a carboxylic acid group. A peptide or polypeptide comprises two or more amino acids. For the purposes of this document, amino acids include the twenty naturally occurring amino acids, unnatural amino acids, and amino acid analogs (i.e., amino acids in which the α-carbon has a side chain).

[0038] As used herein, "polypeptide" refers to any polymer chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymer chain of amino acids. The term "polypeptide" includes polypeptide analogs of natural or artificial proteins, protein fragments, and protein sequences. Polypeptides can be monomeric or polymeric and can include many modifications. Typically, a peptide or polypeptide has a length greater than or equal to 2 amino acids, and typically has a length less than or equal to 40 amino acids.

[0039] As used herein, "α-hemolysin," "α-hemolysin," "α-HL," "a-HL," and "hemolysin" are used interchangeably and refer to monomeric proteins that self-assemble into heptameric, water-filled transmembrane channels (i.e., nanopores). Depending on the context, the term may also refer to a transmembrane channel formed by seven monomeric proteins. In certain example embodiments, the α-hemolysin is a "modified α-hemolysin," meaning that the α-hemolysin is derived from another (i.e., parent) α-hemolysin and contains one or more amino acid changes (e.g., amino acid substitutions, deletions, or insertions) compared to the parent α-hemolysin. In some example embodiments, the modified α-hemolysin of the present invention is derived from or modified from a naturally occurring or wild-type α-hemolysin. In some example embodiments, the modified α-hemolysin is derived from or modified from a recombinant or engineered α-hemolysin, including, but not limited to, a chimeric α-hemolysin, a fusion α-hemolysin, or another modified α-hemolysin. Typically, the modified α-hemolysin has at least one altered phenotype compared to the parent α-hemolysin. In certain exemplary embodiments, the α-hemolysin is derived from a "variant hemolysin gene" or is a "variant hemolysin," which means that the nucleic acid sequence of the α-hemolysin gene from Staphylococcus aureus has been altered by removal, addition, and / or manipulation of the coding sequence, or the amino acid sequence of the expressed protein has been modified consistent with the invention described herein.

[0040] As used herein, the terms "analyte" or "target analyte" broadly refer to any compound, molecule, or other substance of interest to be detected, identified, or characterized. For example, the terms "analyte" or "target analyte" include any physiological molecule or reagent of interest, which is a specific substance or component that is detected and / or measured. In certain example embodiments, the analyte is a physiological analyte of interest. Additionally or alternatively, the analyte can be a chemical substance with a physiological effect, such as a drug or pharmacological agent. Additionally or alternatively, the analyte or target analyte can be an environmental factor or other chemical agent or entity. The term "reagent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biomacromolecule, or an extract prepared from a biological material. For example, a reagent can be a cytotoxic agent.

[0041] In certain example embodiments, example "analytes" or "target analytes" include toxins, organic compounds, proteins, peptides, microorganisms, amino acids, carbohydrates, nucleic acids, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes and those administered for illicit purposes), lipids, viral particles, and metabolites or antibodies to any of the foregoing. For example, such analytes may include ferritin; creatinine kinase MIB (CK-MIB); digoxin; phenytoin; phenobarbital; carbamazepine; vancomycin; gentamicin; theophylline; valproic acid; quinidine; luteinizing hormone (LH); follicle-stimulating hormone (FSH); estradiol, progesterone; IgE antibodies; vitamin B2 microglobulin; glycated hemoglobin (Gly. Hb); cortisol; digitoxin; N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, such as rubella-IgG and rubella-IgM; antibodies to toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylates; acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies to hepatitis B core antigen, such as anti-hepatitis B core antigen IgG and IgM (anti-HBC); human immunodeficiency viruses 1 and 2 (HTLV); hepatitis B antigen (HBeAg); antibodies to hepatitis B antigen (anti-Hbe); thyroid stimulating hormone (TSH); thyroxine (T4); total triiodothyronine (total T3); free triiodothyronine (free T3); carcinoembryonic antigen (CEA); and alpha-fetoprotein (AF); and drugs of abuse and controlled substances, including but not limited to amphetamines; methamphetamine; barbiturates such as Amobarbital, seobarbital, pentobarbital, phenobarbital, and barbital; benzodiazepines such as librium and valium; cannabinoids such as hashish and marijuana; cocaine; fetanyl; LSD; methapualone; opiates such as diamorphine, morphine, codine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone, and opium; phencyclidine; and propoxyphene. The term analyte also includes any antigenic substance, hapten, antibody, macromolecule, and combinations thereof.

[0042] Other example analytes or target analytes include folate, folate RBC, iron, soluble transferrin receptor, transferrin, vitamin B12, lactate dehydrogenase, bone calcium, N-MID osteocalcin, PINP, phosphorus, PTH, PTH (1-84), b-CrossLaps, vitamin D, cardiac apolipoprotein A1, apolipoprotein B, cholesterol, CK, CK-MB, CK-MB (mass), CK-MB (mass) STAT, CRP hs, cystatin C, D-dimer, cardiac digitoxin, digoxin, GDF-154, HDL cholesterol direct, homocysteine, hydroxybutyrate dehydrogenase, LDL cholesterol direct, lipoprotein (a), myoglobin, myoglobin STAT, NT-proBNP, NT-proBNP STAT, 1 troponin I, 1 troponin I STAT, troponin T hs, troponin T hs STAT, coagulation AT III, D-Dimer, Drugs of Abuse Tests Amphetamines (Ecstasy), Benzodiazepines, Benzodiazepines (Serum), Cannabinoids, Cocaine, Ethanol, Methadone, Methadone Metabolite (EDDP), Methaqualone, Opiates, Oxycodone, 3, Phencyclidine, Dextropropoxyphene, Amylase, ACTH, Anti-Tg, Anti-TPO, Anti-TSH-R, Calcitonin, Cortisol, C-Peptide, FT3, FT4, hGH, Hydroxybutyrate Dehydrogenase, IGF-14, Insulin, Lipase, PTH STAT, T3, T4, Thyroglobulin (TG II), Thyroglobulin Confirmed, TSH, T-Uptake, Fertility Anti-Muellerian Hormone, DHEA-S, Estradiol, FSH, hCG, hCG+ Beta, LH, progesterone, prolactin, SHBG, testosterone, Hepatology AFP, alkaline phosphatase (IFCC), alkaline phosphatase (opt.), 3, ALT / GPT (with Pyp), ALT / GPT (without Pyp), ammonia, anti-HCV, AST / GOT (with Pyp), AST / GOT (without Pyp), bilirubin - direct, bilirubin - total, acetylcholinesterase, 3, butyrylcholinesterase, gamma glutamyltransferase, glutamate dehydrogenase, HBeAg, HBsAg, lactate dehydrogenase, Infectious Diseases Anti-HAV, anti-HAV IgM, anti-HBc, anti-HBc IgM, anti-HBe, HBeAg, anti-HBsAg, HBsAg, HBsAg confirmation, HBsAg quantification, anti-HCV, Chagas 4, CMV IgG, CMV IgG avidity, CMV IgM, HIVcombi PT, HIV-Ag, HIV-Ag confirmed, HSV-1 IgG, HSV-2 IgG, HTLV-I / II, Rubella IgG, Rubella IgM, Syphilis, Toxo IgG, ToxoIgG avidity, Toxo IgM, TPLA (syphilis), anti-CCP, ASLO, C3c, C4, ceruloplasmin, CRP (latex), haptoglobin, IgA, IgE, IgG, IgM, immunoglobulin A CSF, immunoglobulin M CSF, interleukin-6, kappa light chain, kappa light chain free 6, 2, 3, lambda light chain, lambda light chain free 6, 2, 3, prealbumin, procalcitonin, rheumatoid factor, alpha-1-acid glycoprotein, alpha-1-antitrypsin, bicarbonate (CO2), calcium, chloride, fructosamine, glucose, HbA1c (hemolysate), HbA1c (whole blood), insulin, lactate, LDL cholesterol direct, magnesium, potassium, sodium, total protein, triglycerides, triglycerides deglycerolated, total vitamin D, acid phosphatase, AFP, CA 125, CA 15-3, CA 19-9, CA 72-4, calcitonin, Cyfra 21-1, hCG + β, HE4, kappa light chain free6, 2, 3, lambda light chain free6, 2, 3, NSE, proGRP, free PSA, total PSA, SCC, S-100, thyroglobulin (TG II), thyroglobulin confirmed, b2-microglobulin, albumin (BCG), albumin (BCP), albumin immunological, creatinine (enzymatic), creatinine (Jaffe), cystatin C, potassium, PTH, PTH (1-84), total protein, total protein, urine / CSF, urea / BUN, uric acid, α1-microglobulin, β2-microglobulin, acetaminophen (paracetamol), amikacin, carbamazepine, cyclosporine, digitoxin, digoxin, everolimus, gentamicin, lidocaine, lithium, ISE mycophenolic acid, NAPA, phenobarbital, phenytoin, primidone, procainamide, quinidine, salicylates, sirolimus, tacrolimus, theophylline, tobramycin, valproic acid, vancomycin, anti-Muellerian hormone, AFP, b-Crosslaps, DHEA-S, estradiol, FSH, free ßhCG, hCG, hCG + β, hCG STAT, HE4, LH, N-MID osteocalcin, PAPP-A, PlGF, sFIt-1, P1NP, progesterone, prolactin, SHBG, testosterone, CMV IgG, CMV IgG avidity, CMV IgM, Rubella IgG, Rubella IgM, Toxo IgG, Toxo IgG avidity, and / or Toxo IgM.

[0043] As used herein, the terms "complementary" or "complementarity" are used to refer to polynucleotides (i.e., nucleotide sequences) that bind according to conventional base pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," where only some of the bases of the nucleic acids match according to the base pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0044] As used herein, the term "homology" refers to the degree of complementarity. Homology includes partial homology or complete homology (i.e., identity). For example, a partially complementary sequence is a sequence that at least partially inhibits hybridization of a completely complementary sequence to a target nucleic acid, and is denoted by the functional term "substantially homologous." Inhibition of hybridization of a completely complementary sequence to a target sequence can be examined using hybridization assays (Southern or Northern blotting, solution hybridization, etc.) under low stringency conditions. Substantially homologous sequences or probes will compete with and inhibit binding (i.e., hybridization) to a completely homologous target under low stringency conditions. However, low stringency conditions exist and therefore allow for nonspecific binding; low stringency conditions require that the binding of two sequences to each other is a specific (i.e., selective) interaction. The absence of nonspecific binding can be tested by using a second target that lacks even partial complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding, the probe will not hybridize to the second, non-complementary target.

[0045] As used herein, the term "ligand" or "analyte ligand" broadly refers to any compound, molecule, molecular group, or other substance that binds to another entity (e.g., a receptor) to form a larger complex. For example, an analyte ligand is an entity that has binding affinity for an analyte, as that term is understood in the art and broadly defined herein. Examples of analyte ligands include, but are not limited to, peptides, carbohydrates, nucleic acids, antibodies, or any molecule that binds to a receptor. In certain embodiments, a ligand forms a complex with an analyte for a biological purpose. As will be understood by those skilled in the art, the relationship between a ligand and its binding partner (e.g., analyte) is a function of charge, hydrophobicity, and / or molecular structure. Binding can occur through a variety of intermolecular forces, such as ionic bonds, hydrogen bonds, and van der Waals forces. In certain embodiments, a ligand or analyte ligand is an antibody or a functional fragment thereof that has binding affinity for an antigen.

[0046] As used herein, the term "DNA" refers to a molecule comprising at least one deoxyribonucleotide residue. A "deoxyribonucleotide" is a nucleotide having a hydrogen instead of a hydroxyl group at the 2' position of the β-D-deoxyfuranose ribose moiety. The term encompasses double-stranded DNA, single-stranded DNA, DNA having double-stranded and single-stranded regions, isolated DNA, such as partially purified DNA, substantially pure DNA, synthetic DNA, recombinantly produced DNA, and altered or analog DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides.

[0047] As used herein, the terms "connect," "connected," "link," "linked," or "tied" refer to any method known in the art for functionally connecting two or more entities, such as connecting a protein to a DNA molecule or a protein to a protein. For example, a protein can be covalently linked to another protein, such as in a recombinant fusion protein, with or without an intervening sequence or domain. Exemplary covalent bonds can be formed, for example, by SpyCatcher / SpyTag interactions, cysteine-maleimide conjugation, or azide-alkyne click chemistry, among other means known in the art. Additionally, a DNA molecule can be linked to another DNA molecule by hybridization of complementary DNA sequences.

[0048] As used herein, the term "nanopore" generally refers to a hole, channel, or passage formed or otherwise provided in a membrane. The membrane can be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed from a polymeric material. The membrane can be a polymeric material. The nanopore can be configured adjacent to or in proximity to an electrode of a sensing circuit or coupled to a sensing circuit, such as a complementary metal oxide semiconductor (CMOS) or field effect transistor (FET) circuit. In some example embodiments, the nanopore has a characteristic width or diameter on the order of 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins. For example, α-hemolysin monomers oligomerize to form a protein. The membrane includes a trans side (i.e., the side facing the sensing electrode) and a cis side (i.e., the side facing the corresponding electrode).

[0049] The term "nucleic acid molecule" or "nucleic acid" includes RNA, DNA, and cDNA molecules. It is understood that as a result of the degeneracy of the genetic code, many nucleotide sequences encoding a given protein, such as α-hemolysin and / or variants thereof, can be produced. The present disclosure encompasses every possible variant nucleotide sequence encoding a variant α-hemolysin, all of which are possible given the degeneracy of the genetic code.

[0050] As is generally recognized in the art, the term "nucleotide" is used herein to include natural bases (standard) and modified bases well known in the art. Such bases are generally located at the 1' position of the nucleotide sugar portion. Nucleotides generally comprise a base, a sugar, and a phosphate group.

[0051] "Synthetic" as used herein, such as with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or a polypeptide molecule produced by recombinant methods and / or by chemical synthesis methods.

[0052] As used in this article, the protein that uses the well-known method of molecular biology to express by cloned DNA encoding is represented by the recombinant method production of using recombinant DNA method.For example, standard technique can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (for example, electroporation, lipofection).According to the specification sheets of manufacturer, or as commonly used in this area or as described herein, enzyme reaction and purification technique can be carried out.Aforesaid technique and rule can be performed according to conventional method well-known in the art usually, and as described in the various general and more specific references that are quoted and discussed throughout this specification sheets. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference in its entirety for any purpose.

[0053] As used herein, "vector" (or plasmid) refers to a discrete DNA element that is used to introduce a heterologous nucleic acid into a cell to express or replicate it. The vector typically remains episomal, but can be designed to achieve integration of a gene or part thereof into the chromosome of the genome. Vectors that serve as artificial chromosomes, such as bacterial artificial chromosomes, yeast artificial chromosomes, and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles are well known to those skilled in the art.

[0054] "Expression" as used herein generally refers to the process by which nucleic acid is transcribed into mRNA and translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, expression may include processing, such as splicing of mRNA, in the case of selecting an appropriate eukaryotic host cell or organism.

[0055] As used herein, "expression vectors" include vectors capable of expressing DNA operably linked to regulatory sequences (such as promoter regions) that enable expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that, upon introduction into a suitable host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and / or prokaryotic cells, as well as those that remain episomal or integrate into the host cell genome. As used herein, vectors also include "viral vectors" or "viral vectors." Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer them into cells (as a vehicle or shuttle).

[0056] The term "host cell" refers to a cell that contains a vector and supports replication and / or transcription or transcription and translation (expression) of an expression construct. The host cell can be a prokaryotic cell, such as Escherichia coli or Bacillus subtilis, or a eukaryotic cell, such as a yeast, plant, insect, amphibian, or mammalian cell. Generally, the host cell is prokaryotic, for example, Escherichia coli.

[0057] The terms "cellular expression" or "cellular gene expression" generally refer to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell. Thus, gene expression involves the processes of transcription and translation, but may also involve post-transcriptional and post-translational processes that may affect the biological activity of a gene or gene product. These processes include, for example, RNA synthesis, processing, and transport, and polypeptide synthesis, transport, and post-translational modification of polypeptides. In addition, processes that affect protein-protein interactions within the cell may also affect gene expression as defined herein.

[0058] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, the optional step of attaching an analyte sensing complex to a nanopore component monomer means that the analyte sensing complex may or may not be attached.

[0059] As used herein, the term "phospholipid" refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid may comprise a phosphorus-containing group and a saturated or unsaturated alkyl group, which may be optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl.

[0060] As used herein, the term "membrane" refers to a continuous bilayer sheet or layer of lipid molecules in which membrane proteins are embedded. Membrane lipid molecules are generally amphiphilic, and most spontaneously form a bilayer when placed in water. "Phospholipid membrane" refers to any structure composed of phospholipids arranged so that the hydrophobic heads of the lipids point in one direction and the hydrophilic tails point in the opposite direction. Examples of phospholipid membranes include the lipid bilayer of cell membranes.

[0061] As used herein, "identity" or "sequence identity" in the context of sequences refers to the similarity between two nucleic acid sequences or two amino acid sequences, and is expressed in terms of similarity between sequences, otherwise referred to as sequence identity. Sequence identity is often measured as a percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. For example, 80% homology refers to something that is identical to 80% sequence identity as determined by a well-defined algorithm, and thus homologs of a given sequence have greater than 80% sequence identity over the length of the given sequence. Example levels of sequence identity include, for example, 80%, 85%, 90%, 95%, 98% or more sequence identity to a given sequence (e.g., the coding sequence of any of the polypeptides of the invention described herein).

[0062] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman Adv. Appl. Math. 2: 482, 1981; Needleman and Wunsch J. Mol. Biol. 48: 443, 1970; Pearson and Lipman Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp Gene 73: 237-244, 1988; Higgins and Sharp CABIOS 5:151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al. Meth. Mol. Biol. 24, 307-31, 1994. Altschul et al. (J. Mol. Biol. 215:403-410, 1990) presents detailed considerations of sequence alignment methods and homology calculations.

[0063] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J. Mol. Biol. 215:403-410, 1990) is available from a number of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use with sequence analysis programs, including, for example, the suite of BLAST programs, such as BLASTN, BLASTX and TBLASTX, BLASTP, and TBLASTN.

[0064] When evaluating given nucleotide sequence relative to the nucleotide sequence in GenBank DNA sequence and other public databases, BLASTN program is used to carry out sequence search usually.BLASTX program is preferably used for the nucleotide sequence that the amino acid sequence in GenBank protein sequence and other public databases is searched in all reading frames.BLASTN and BLASTX use the default parameters of 11.0 open gap penalty (open gap penalty) and 1.0 extension gap penalty (extended gap penalty) to run, and utilize BLOSUM-62 matrix (referring to, for example, Altschul, SF, et al., Nucleic Acids Res. 25:3389-3402,1997).

[0065] In certain example embodiments, a preferred alignment of selected sequences to determine "% identity" between two or more sequences is performed using, for example, the CLUSTAL-W program in MacVector version 13.0.7, which is run with default parameters, including an open gap penalty of 10.0, an extension gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0066] As used herein, the term "variant" refers to a modified protein that exhibits altered characteristics (eg, altered ionic conductance) compared to a parent protein.

[0067] As used herein, the term "sample" or "test sample" is used in its broadest sense. As used herein, a "biological sample" includes, but is not limited to, any amount of material from a living organism or a pre-living organism (such as from a subject). A biological sample can include a sample of biological tissue or fluid origin obtained in vivo or in vitro. Such a sample can be derived from, but is not limited to, body fluids, organs, tissues, fractions, and cells isolated from a biological subject. A biological sample can also include an extract from a biological sample, such as an extract from a biological fluid (e.g., blood or urine).

[0068] As used herein, "biological fluid" or "biological fluid sample" refers to any physiological fluid (e.g., blood, plasma, sputum, lavage fluid, lens fluid, cerebrospinal fluid, urine, semen, sweat, tears, milk, saliva, synovial fluid, peritoneal fluid, amniotic fluid) and solid tissue that has been at least partially converted to liquid form by one or more known protocols or from which a liquid has been extracted. For example, a liquid tissue extract (such as from a biopsy) can be a biological fluid sample. In certain embodiments, the biological fluid sample is a urine sample collected from a subject. In certain embodiments, the biological fluid sample is a blood sample collected from a subject. As used herein, the terms "blood," "plasma," and "serum" include fractions or processed portions thereof. Similarly, in the case of a sample obtained from a biopsy, swab, smear, etc., "sample" includes processed fractions or portions derived from the biopsy, swab, smear, etc.

[0069] Further, " fluid solution ", " fluid sample " or " fluid " comprises biological fluid, but also can comprise and encompass non-physiological components, for example any analyte that may be present in environmental sample.For example, the sample can come from a river, lake, pond or other reservoir.In certain embodiment embodiments, the fluid sample can be modified.For example, a buffer or preservative can be added to the fluid sample, or the fluid sample can be diluted.In other embodiment embodiments, the fluid sample can be modified by common methods known in the art to increase the concentration of one or more solutes in the solution.In any case, the fluid solution remains the fluid solution as described herein.For example, when the fluid sample is to be tested, the fluid sample can be referred to as a "test sample".

[0070] As used herein, "subject" refers to an animal, including vertebrates. The vertebrate can be a mammal, such as a human. In certain embodiments, the subject can be a human patient. The subject can be a "patient," for example, such as a patient who has or is suspected of having a disease or condition and who may require treatment or diagnosis, or who may require monitoring of the progression of the disease or condition. A patient can also be receiving a therapeutic therapy whose efficacy needs to be monitored. Mammal means any animal classified as a mammal, including, for example, humans, chimpanzees, domestic and farm animals, and zoo, sport or pet animals, such as dogs, cats, cows, rabbits, horses, sheep, pigs, and the like.

[0071] As used herein, the term "wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.

[0072] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.It should be understood that modifications can be made to the procedures described without departing from the spirit of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Figure 1 Schematic diagram of an analyte detection complex according to certain example embodiments.

[0074] Figure 2 A Figure 2 A is a diagram showing three nanopore assemblies, each comprising an analyte detection complex for a different analyte, according to certain example embodiments.

[0075] Figure 2 B Figure 2 B is a diagram showing a method according to certain example embodiments. Figure 2 A three nanopore assembly, but in which each analyte ligand is shown binding its respective analyte.

[0076] Figure 2 C Figure 2 C is a diagram showing a method for Figure 2 The same three nanopore assemblies in A-2B, but showing the nanopore assemblies in a configuration in which each analyte-sensing complex is pulled toward opposite sides of the nanopore assembly.

[0077] Figure 3 Figure 3 Graphs illustrating evaluation of weak binding interactions between an analyte ligand and an analyte, and changes in electrical signals associated with the binding and dissociation of the analyte-ligand pair, according to certain example embodiments.

[0078] Figure 4 Figure 4 Schematic diagram showing evaluation of a strong binding interaction between an analyte ligand and an analyte according to certain example embodiments.

[0079] Figure 5 Figure 5 Schematic diagram showing the evaluation of a very strong interaction between an analyte ligand and an analyte according to certain example embodiments.

[0080] Figure 6 Figure 6 The diagram shows evaluation of a test sample when the target analyte is not present in the test solution, according to certain example embodiments.

[0081] Figure 7 Figure 7 The graph of FIGURE 1 shows an example confidence level distribution for single analyte capture and dissociation for weak, strong, and very strong analyte-ligand interactions according to certain example embodiments.

[0082] Figure 8 Figure 8 Schematic diagram showing identification of specific analyte-ligand interactions on a chip according to certain example embodiments.

[0083] Example implementation plan

[0084] Example embodiments will now be described in detail, in part with reference to the accompanying drawings.Where reference is made to the drawings, like numerals indicate similar (but not necessarily identical) elements throughout the figures.

[0085] Analyte detection complex

[0086] Figure 1 is a diagram of an analyte detection complex according to embodiment 1 of certain examples. Figure 1 , the analyte detection complex 1 includes, for example, an analyte ligand 2, a transmissive element 3, and one or more signaling elements 4a and 4b disposed within or associated with the transmissive element 3. In certain exemplary embodiments, the analyte detection complex 1 also includes an anchor tag 5 located on the distal end of the analyte detection complex.

[0087] The analyte ligand 2 of the analyte detection complex 1 can be any ligand having binding affinity for any analyte described herein. Figure 1 As shown in , for example, analyte ligand 2 can be an antibody, and the analyte is an antigen that has binding affinity for the antibody. As will be appreciated by those skilled in the art, given the present disclosure, any antibody or functional fragment thereof can be used as the analyte ligand. In other example embodiments, analyte ligand 2 of analyte detection complex 1 can be used to detect environmental analytes. In certain example embodiments, analyte ligand 2 of analyte detection complex 1 can be used to identify protein analytes in complex biological fluid samples (e.g., tissue and / or body fluids). In certain example embodiments, the analyte targeted by analyte ligand 2 can be present at low concentrations compared to other components of the biological or environmental sample. In certain example embodiments, analyte ligand 2 can also be used to target subpopulations of macromolecular analytes based on the conformational or functional properties of the analyte. Example analyte ligands 2 include those defined herein, as well as aptamers, antibodies or functional fragments thereof, receptors, and / or peptides known to bind to target analytes. Regarding aptamers, the aptamer can be a nucleic acid aptamer, including DNA, RNA, and / or nucleic acid analogs. In certain example embodiments, the aptamer can be a peptide aptamer, such as a peptide aptamer comprising a variable peptide loop attached at both ends to a scaffold.The aptamer can be selected, for example, to bind a specific target protein analyte.

[0088] As will be understood by those skilled in the art, the analyte and the analyte ligand 2 represent two members of a binding pair, i.e., two different molecules, one of which specifically binds to the second molecule through a chemical and / or physical interaction. In addition to the well-known members of antigen-antibody binding pairs, other binding pairs include, for example, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, peptide sequences and antibodies specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonucleases, S-peptides, and ribonuclease S-proteins), sugars and boronic acids, and similar molecules having an affinity that allows them to bind in a binding assay.

[0089] Further, the analyte-ligand binding pair may include members similar to the original binding member, for example, analyte analogs or binding members prepared by recombinant techniques or molecular engineering. If the analyte ligand is an immunoreactant, it can be, for example, an antibody, antigen, hapten, or a complex thereof. If an antibody is used, it can be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a chimeric antibody, a mixture or fragment thereof, and a mixture of antibodies and other binding members. Details of the preparation of such antibodies, peptides, and nucleotides and their suitability for use as binding members in binding assays are well known in the art.

[0090] As in Figure 1 As shown in FIG, an analyte ligand 2 (such as an antibody) is attached to a transversal element 3. When bound to a nanopore, the transversal element 3 can penetrate the pore of the nanopore. The transversal element 3 can be any structure capable of penetrating the pore of the nanopore assembly. In certain example embodiments, the transversal element 3 can be a single-stranded or double-stranded nucleic acid sequence or other molecular polymer. For example, the transversal element 3 can be an amino acid sequence and can include a carbon spacer. In certain example embodiments, the transversal element 3 has an overall charge of one polarity, and varying the voltage across the nanopore assembly as described herein can cause the transversal element to move in one direction or the other.

[0091] Associated with the transmissive element 3 of the analyte detection complex 1 are one or more signaling elements, such as 1, 2, 3, 4 or 5 signaling elements. Figure 1As shown in , for example, the transverse element 3 can be combined with a pair of signal elements 4a and 4b. When positioned in the pore of a nanopore, one or more signal elements 4a and 4b can be used, for example, to determine the position of the transverse element 3 within the nanopore assembly. The signal elements can be used, for example, to provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signal that is detectable and provides an indication of the position of the transverse element 3 in the pore of a nanopore assembly as described herein. In certain example embodiments, the signal element 4a can be the same as the signal element 4b. In other example embodiments, the individual elements 4a can be different from the signal element 4b. In certain example embodiments, when the total charge of the transverse element 3 is a given charge, the signal element can represent a constriction site of a specific charge, which can be used to determine the position of the transverse element in the pore of the nanopore assembly.

[0092] In certain example embodiments, the signaling element can be an oligonucleotide, peptide, or polymer sequence that is associated with the crossing element 3. In certain example embodiments, for example, when the crossing element 3 is a nucleotide sequence and the signaling element is a specific sequence within the nucleotide sequence of the crossing element 3, the signaling element can be integrated as part of the crossing element 3. For example, the signaling element can be a sub-portion of the crossing element. Additionally or alternatively, the signaling element can be attached to the crossing element 3.

[0093] One or more signaling elements, such as signaling elements 4a and 4b, can be associated with multiple locations on the transversal element 3, thereby enabling detection of various signals and / or signal changes, as described herein, during use. For example, when signaling elements 4a and 4b are different, the electrical signal associated with the nanopore assembly can be different depending on which signaling element (4a or 4b) is located within the pore, as described herein. In certain example embodiments, one or more signaling elements can be located at the proximal end of the transversal element, while in other example embodiments, one or more signaling elements 4 can be located more distally on the analyte detection complex 1. In other example embodiments, one signaling element 4a can be associated with the proximal end of the transversal element 3, while another signaling element 4b can be associated with a more distal portion of the transversal element 3.

[0094] In certain example embodiments, one or more signal elements, such as signal elements 4a and 4b, can be single-stranded nucleic acid sequences, such as a series of repeated nucleic acid residues. For example, the signal element can be a repeated single-stranded oligonucleotide sequence of about 10-100 nucleotides in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides. In certain example embodiments, the signal element can be a 30-50 oligonucleotide sequence, such as a 40-mer oligonucleotide sequence.

[0095] In other embodiment embodiments, one or more signal elements can be double-stranded nucleic acid sequences, for example a series of nucleic acid base pairs repeated.For example, signal element can be the repetition double-stranded oligonucleotide sequence of about 10-100 nucleotide length, such as about 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 base pairs.In certain embodiment embodiments, signal element can be 30-50 oligonucleotide sequence, for example 40 aggressiveness base pair sequence.In certain embodiment embodiments, one or more signal elements can comprise a series of T residues and a series of N3-cyanoethyl-T residues.In certain embodiment embodiments, the signal element that passes through element can comprise Sp2 unit, Sp3 unit, dSp unit, methylphosphonate-T unit etc.

[0096] As in Figure 1 As shown in , the analyte detection complex 1 also includes an anchor tag 5 on the distal end of the analyte detection complex 1. For example, when the analyte detection complex 1 penetrates the nanopore, the anchor tag 5 can be used to prevent the analyte detection complex 1 from migrating through the cis side of the nanopore assembly or being pulled to the cis side of the nanopore assembly as described herein. Therefore, the anchor tag 5 can be any protein, nucleic acid or chemical entity that can be used to anchor the distal end of the analyte detection complex 1 to the trans side of the nanopore assembly. For example, the anchor tag 5 can be biotin-streptavidin, double-stranded DNA or RNA, a DNA or RNA ternary structure, a SpyTag-Catcher, an antibody-antigen.

[0097] Nanopore assembly

[0098] In certain example embodiments, the analyte detection complex 1 described herein is combined with a nanopore to form a nanopore assembly, which, together with the nanopore assembly, interacts with the analyte. To detect the interaction of the analyte detection complex 1 with the analyte, the nanopore assembly comprising the analyte detection complex 1 is embedded in a membrane, and a sensing electrode is positioned adjacent to or near the membrane. For example, the nanopore assembly comprising the analyte detection complex 1 can be formed or otherwise embedded in a membrane adjacent to a sensing electrode disposed on a sensing circuit (such as an integrated circuit). The integrated circuit can be an application-specific integrated circuit (ASIC). In certain example embodiments, the integrated circuit is a field-effect transistor or a complementary metal oxide semiconductor (CMOS). The sensing circuit can be located within a chip or other device comprising the nanopore, or outside of the chip or device, such as in an off-chip configuration. The semiconductor can be any semiconductor, including, but not limited to, Group IV (e.g., silicon) and Group III-V semiconductors (e.g., gallium arsenide). For information on devices and apparatus arrangements that can be used in accordance with the compositions and methods described herein, see, for example, WO 2013 / 123450, the entire contents of which are hereby expressly incorporated herein by reference.

[0099] As will be understood by those skilled in the art, pore-based sensors (e.g., biochips) can be used for electrical interrogation of single molecules. A pore-based sensor can include a nanopore assembly as described herein, formed in a membrane positioned adjacent to or near a sensing electrode. The sensor can include, for example, a counter electrode. The membrane includes a trans side (i.e., the side facing the sensing electrode) and a cis side (i.e., the side facing the counter electrode). Thus, a nanopore assembly disposed in the membrane also includes a trans side (i.e., the side facing the sensing electrode) and a cis side (i.e., the side facing the counter electrode). As described herein, for example, the analyte ligand 2 is located on the cis side of the nanopore assembly, while the anchor tag 5 is located on the trans side of the nanopore assembly.

[0100] The nanopore of a nanopore assembly is typically a multimeric protein embedded in a matrix, such as a membrane. Examples of protein nanopores include, for example, α-hemolysin, voltage-dependent mitochondrial porin (VDAC), OmpF, OmpC, OmpG, MspA, and LamB (maltoporin). See alsoRhee, M. et al., Trends in Biotechnology, 25(4)(2007): 174-181). Other example nanopores include nanomotors packaged with phi 29 DNA, ClyA, FhuA, Aeromonas lysin, and Sp1. In certain example embodiments, the nanopore protein can be a modified protein, such as a modified natural protein or a synthetic protein. For example, in the case of α-hemolysin, the nanopore of the nanopore assembly can be an oligomer of seven α-hemolysin monomers (i.e., a heptameric nanopore assembly). The monomer subunits of the α-hemolysin heptameric nanopore assembly can be identical copies of the same polypeptide, or they can be different polypeptides, as long as the ratio totals seven subunits. The nanopore can be assembled by any method known in the art. For example, the α-hemolysin nanopore assembly can be assembled according to the methods described in WO2014 / 074727, which is hereby incorporated herein in its entirety.

[0101] refer to Figure 2 A, according to certain example embodiments, provides a diagram showing three nanopore assemblies, each nanopore assembly comprising an analyte detection complex 1. As shown, the proximal end of the analyte detection complex 1, comprising the analyte ligand 2, is located on the cis side of the nanopore assembly. In this way, the analyte ligand 2 of the analyte detection complex 1 can be presented to the analyte on the cis side of the nanopore assembly, thereby promoting the binding of the analyte ligand 2 to the analyte described herein. Figure 2 In the embodiment shown, each analyte ligand 2 is specific to a different analyte ligand. In addition, the anchor tag 5 is located on the opposite side of the nanopore assembly ( Figure 2 A). Transmissive element 3, for example, extends through the pore of a nanopore, thereby positioning one or more signaling elements (e.g., 4a or 4b) within the pore of the nanopore assembly. As shown, first signaling element 4a is positioned within the pore of the nanopore assembly, while second signaling element 4b is positioned cis-lateral to the pore. Each nanopore assembly can, for example, be positioned within a single pore of a biochip.

[0102] refer to Figure 2 B, according to certain example embodiments provides a display Figure 2 Schematic representation of the three nanopore assemblies of A, but showing each analyte ligand 2 binding its respective analyte 6. The nanopore assembly is also shown in a configuration where the analyte detection complex is pulled towards the cis side of the nanopore assembly. Figure 2 As in A, each analyte ligand 2 is located on the cis side of the nanopore assembly, so analyte binding occurs on the cis side of the nanopore assembly ( Figure 2 B). And with Figure 2A, the first signal element 4a of the traversing element 3 is still located in the pore of the nanopore assembly, while the second signal transmission element 4b of the traversing element 3 is located on the cis side of the nanopore assembly ( Figure 2 B).

[0103] refer to Figure 2 C, according to certain example embodiments provides a display and Figure 2 A-2B is an illustration of the same three nanopore assemblies, but showing the nanopore assembly in a configuration where each analyte detection complex is pulled toward the opposite side of the nanopore assembly. As shown, the second signal element 4b of traversing element 3 is now located within the pore of the nanopore assembly, while the first signal element 4a of traversing element 3 has moved to the opposite side of the nanopore assembly. Figure 2 In the embodiment shown in C, the binding of the analytes to their respective analyte ligands can prevent the analyte detection complex from moving to the trans side of the nanopore assembly. However, as further described below, if the force pulling the analyte detection complex to the trans side of the nanopore assembly overcomes the binding force of the analyte-ligand interaction, the analyte ligand 3 of the analyte detection complex 1 can dissociate from the analyte. The analyte detection complex 1 can then be transferred to the trans side of the nanopore assembly.

[0104] Methods and systems for evaluating analyte-ligand interactions

[0105] In certain example embodiments, methods and systems are provided for evaluating the binding interaction between a ligand and an analyte of the ligand, including evaluating the binding strength between an analyte ligand and an analyte. For example, a nanopore assembly comprising the analyte detection complex 1 described herein can be integrated into a biochip. The biochip can then be contacted with a fluid sample to be analyzed. If the analyte is present in the fluid solution, the analyte ligand 2 of the analyte detection complex 1 can bind to the analyte, resulting in a discernible electrical signal (i.e., a binding signal) associated with the nanopore assembly. In addition, the binding strength between the analyte ligand 2 and the analyte can be determined based on the electrical signal associated with the pore. If the analyte is not present in the fluid sample, the analyte ligand 2 does not bind to the analyte, in which case the absence of a binding event can be determined from the electrical signal associated with the nanopore assembly. Without wishing to be bound by any particular theory, such methods and systems are Figure 3-8 Shown in.

[0106] refer to Figure 3 , provides a diagram showing the evaluation of weak binding interactions between analyte ligand 2 and analyte 6, and the evaluation of electrical signal changes associated with the binding and dissociation of the analyte-ligand pair, according to certain example embodiments. Figure 3As shown at point "A" of the chip, the nanopore can be positioned as an "open pore" within the membrane of the chip. That is, in certain example embodiments, the pore may initially not include the analyte detection complex 1, in which case a baseline electrical signal can be obtained from the nanopore via an electrode associated with the pore. For example, upon application of a first voltage across the nanopore assembly, in certain example embodiments, the nanopore can capture the analyte detection complex 1, thereby positioning the first signaling element 4a within the pore (see point "B") and forming a nanopore assembly, as described herein.

[0107] In certain example embodiments, an electrical signal may be detected from the nanopore assembly at point "B," which signal indicates penetration of the analyte-sensing complex 1 within the nanopore of the nanopore assembly ( Figure 3 For example, the signal may be a crossing signal corresponding to the presence of the first signal element 4a positioned in the pore of the nanopore ( Figure 3 ).like Figure 3 As shown, for example, application of the first voltage also pulls the analyte detection complex 1 toward the cis side of the nanopore assembly. However, the anchor tag 5 can prevent the analyte detection complex 1 from being pulled to the cis side of the membrane. For example, the size of the anchor tag 5 relative to the size of the pore can prevent the analyte detection complex 1 from transferring to the cis side of the nanopore assembly.

[0108] Once the analyte detection complex 1 is within the nanopore, for example, the chip, and therefore the nanopore assembly disposed within the chip membrane, is contacted with a fluid sample. That is, the nanopore assembly is contacted with a sample to be tested or examined, for example, for the presence of a target analyte 6. For example, to test a fluid solution for the presence of an analyte, the fluid solution can be flowed through a nanopore assembly that is configured to include an analyte detection complex 1 as described herein, wherein the analyte ligand 2 of the analyte detection complex 1 has a binding affinity for the target analyte.

[0109] As the fluid flows through the nanopore assembly, the analyte 6 (when present) has the opportunity to contact the analyte ligand 2 of the analyte detection complex 1 and can thus bind to the analyte ligand 2. However, if the analyte is not present in the fluid solution, no binding of the analyte to the analyte ligand 2 of the analyte detection complex 1 will occur. Figure 3 As shown in the embodiment of FIG, binding of analyte 6 to analyte ligand 2 occurs at point "C." However, because analyte 6 does not block the pore of the nanopore assembly, the electrical signal associated with the nanopore assembly can remain substantially unchanged. For example, first signal element 4a can remain positioned within the pore of the nanopore assembly.

[0110] After the chip is brought into contact with the fluid sample and thus provides any analyte with an opportunity to bind to the analyte ligand 2, a second voltage having a polarity opposite to the first voltage is gradually applied across the membrane. That is, the first voltage is gradually transformed into a second voltage having a polarity opposite to the first voltage. For example, the first voltage may have a negative potential, which is then transformed into a voltage having a positive potential. As in Figure 3 As shown in , for example, localization of the analyte 6 detection complex 1 in the open pore and binding of the analyte ligand 2 to the analyte may occur during a negative cycle, after which the voltage is slowly changed to a second (positive) voltage having a polarity opposite to the first voltage.

[0111] For example, as a voltage having a polarity opposite to the first voltage is gradually applied across the membrane, the analyte ligand 2 and its bound analyte 6 are pulled toward the opposite side of the nanopore assembly ( Figure 3 However, the bound analyte 6 may prevent the analyte-detecting complex 1 from being pulled through the nanopore assembly to the reverse side of the nanopore assembly. Furthermore, a second signaling element 4b (e.g., a forward-side signaling element) may be positioned within the pore of the nanopore assembly.

[0112] As in Figure 3 As shown in FIG, the binding of analyte 6 to analyte ligand 2 and the relocation of analyte detection complex 1 within the pore can generate a binding signal that is different and distinguishable from the transit signal. The binding signal is, for example, a detectable electrical signal associated with the nanopore assembly that corresponds to the presence of analyte 6 bound to analyte ligand 2 ( Figure 3 ). Detection of the binding signal can therefore also provide an indication of the presence of the analyte in the test sample. In certain exemplary embodiments, comparison of the transit signal with the binding signal can provide an indication of the binding of analyte 6 to analyte ligand 2 (and, therefore, the presence of the analyte in the test sample). For example, a change in the electrical signal from the transit signal to the binding signal indicates that analyte 6 is bound to analyte ligand 2.

[0113] In certain example embodiments, the positioning of the second signal element 4b within the pore of the nanopore assembly results in a binding signal. For example, the second signal element 4b can generate a specific electrical signal associated with the second signal element 4b positioned within the nanopore. Thus, detection of the electrical signal associated with the second signal element 4b corresponds to a binding signal. Additionally or alternatively, in certain example embodiments, analyte 6 binding to the analyte ligand 2 can result in a detectable signal change, such as compared to a transit signal, thereby indicating the presence of the analyte in the sample. For example, and without being bound by any particular theory, the presence of analyte 6 at or near the pore opening can block or partially block the pore of the nanopore assembly, thereby affecting the electrical signal generated from the nanopore assembly (and resulting in a detectable binding signal).

[0114] After determining the binding signal, in certain example embodiments, a voltage having a polarity opposite to the first voltage may be further increased to further increase the force pulling the analyte detection complex 1 toward the reverse side of the nanopore assembly. At some point during the voltage increase, the force pulling the analyte detection complex 1 toward the reverse side of the nanopore assembly may become strong enough to pull the analyte ligand 2 away from the analyte 6. In this regard, as shown in FIG. Figure 3 As shown at point "E" in FIG, the analyte ligand 2 and the analyte 6 can dissociate, and the analyte detection complex 1 moves to the opposite side of the nanopore assembly. As a result, any signaling element located within the pore can be completely moved out of the pore, and the nanopore assembly transitions to an open nanopore state. Further, an electrical signal can be obtained by an electrode associated with the nanopore, which electrical signal corresponds to a dissociation signal. In other words, the dissociation signal corresponds to an electrical signal obtained from the nanopore assembly at or about the time when the analyte ligand 2 dissociates from the analyte 6. As shown in FIG. Figure 3 As shown in , the interaction between the analyte and the analyte ligand 2 is a weak interaction because, as described herein, the analyte dissociates from the analyte ligand 2 relatively early as the voltage increases.

[0115] In certain example embodiments, once the analyte ligand 2 of the analyte detection complex 1 dissociates from the analyte 6 and the analyte detection complex 1 moves to the opposite side of the nanopore, the voltage can be reversed again and the pore can be reused ( Figure 3 That is, after the dissociation event described herein, a voltage having a polarity opposite to the second voltage can be applied across the membrane. For example, the voltage can be the same or similar in magnitude and polarity to the first voltage described herein. Thus, the pore can then capture the Figure 3 The analyte detection complex 1 described at points "A" and "B" of FIG. Thereafter, the process of points "C" to "F" can be repeated. In certain example embodiments, a given nanopore assembly including the analyte detection complex 1 can be reused multiple times during the analysis of a given sample.

[0116] refer to Figure 4 , according to certain example embodiments, a diagram showing an evaluation of a strong binding interaction between an analyte ligand 2 and an analyte 6 is provided. Figure 4 As shown at point "A" in FIG. , the nanopore can be provided as an "open pore" within the membrane of the chip. Figure 3 In the example shown, in certain example embodiments, the nanopore can capture the analyte detection complex 1, thereby positioning the first signal element 4a within the pore (see point "B"). A cross-over signal can then be detected from the nanopore assembly at point "B", indicating the presence of the analyte detection complex 1 within the nanopore of the nanopore assembly ( Figure 4For example, the signal may correspond to the presence of a first signaling element 4a positioned in the pore of the nanopore assembly ( Figure 4 ). Further, like Figure 3 Similarly, the anchor tag 5 can prevent the analyte detection complex 1 from being pulled to the cis side of the nanopore assembly ( Figure 4 ).

[0117] Once the analyte detection complex 1 is within the nanopore, the chip is contacted with a fluid sample as described herein, thereby promoting the binding of the analyte ligand 2 to its corresponding analyte 6. Figure 4 As shown in FIG, binding of the analyte to the analyte ligand 2 occurs at point "C". However, because the analyte 6 does not block the pore of the nanopore assembly, for example, the electrical signal associated with the nanopore assembly can remain substantially unchanged ( Figure 4 ). For example, the first signaling element 4a may remain positioned in the pore of the nanopore assembly, while the second signaling element 4b may remain on the opposite side of the nanopore assembly.

[0118] After the chip is contacted with the fluid sample and thus provides the analyte with an opportunity to bind to the analyte ligand 2, a second voltage having a polarity opposite to the first voltage may be gradually applied across the nanopore assembly. For example, the second voltage is gradually applied across the nanopore assembly. Figure 2 As in the weak binding example, for example, the positioning of the analyte detection complex 1 in the open pore and the binding of the analyte ligand 2 to the analyte may occur during a negative cycle, after which the voltage is slowly changed to a second (positive) voltage having a polarity opposite to the first voltage ( Figure 4 ).

[0119] As described herein, as a voltage of opposite polarity to the first voltage is gradually applied across the membrane, the analyte ligand 2 and its bound analyte are pulled toward the opposite side of the nanopore assembly ( Figure 4 Further, the second signal element 4b (eg, the positive side signal element) can be positioned within the pore of the nanopore and retained therein, thereby providing a binding signal. Figure 3 As with the exemplary weak binding embodiment shown, detection of the binding signal provides an indication of the presence of the analyte in the sample being tested (see Figure 4 And in certain example embodiments, the presence of bound analyte can additionally or alternatively provide a binding signal, as described herein.

[0120] like Figure 4 As shown at point "E" of FIG. , further increasing the second voltage can result in dissociation of the analyte ligand 2 from the analyte, which is associated with a discernible dissociation signal. Figure 3 Compared to point "E" in Figure 4The stronger binding shown in results in a greater force required to separate analyte ligand 2 from the analyte. Figure 4 As shown, the analyte remains bound to Analyte Ligand 2 for a longer period of time (compared to Figure 3 Compared to the weak binding shown). Thus, Figure 4 The dissociation signal associated with the nanopore assembly shown (strong binding at point "E") is different from Figure 3 The dissociation signal is shown (weak binding at point "E"). After the analyte ligand 2 dissociates from the analyte, the analyte-detection complex 1 can move to the opposite side of the membrane and the nanopore can be reused as described herein (point "F", Figure 4 ).

[0121] refer to Figure 5 , according to certain example embodiments, provides a diagram illustrating the evaluation of a very strong interaction between analyte ligand 2 and analyte 6. Figure 5 As shown, the nanopore assembly passes through the reference Figure 3 and 4 The process proceeds from point A to point D. For example, analyte 6 binds to analyte ligand 2 at point "C," and as a second voltage having a polarity opposite to the first voltage is gradually applied, analyte-detection complex 1 is pulled toward the opposite side of the nanopore at point "D." For example, a dissociation signal may be obtained at point "D."

[0122] But with reference Figure 3 and 4 Unlike the analyte-ligand interaction described above, the binding between analyte 6 and analyte ligand 2 is so strong that increasing the second voltage cannot overcome the binding force between the analyte and analyte ligand 2 ( Figure 5 , at point "E"). Therefore, since there is no dissociation between the analyte and the analyte ligand 2, no dissociation signal is obtained ( Figure 5 ). In this way, the signaling element 4b can remain in the pore throughout the positive cycle (the signaling element 4a is outside the pore, point "D"), thereby providing an indication that the analyte is very strongly bound to the analyte ligand 2 ( Figure 5 ). In other words, determination of a binding signal as described herein, followed by absence of a dissociation signal as described herein, can provide an indication that the analyte remains bound to the analyte ligand 2 despite the second voltage increase. In such example embodiments, the nanopore is not reused. Figure 5 As shown, for example, even if a voltage having a polarity opposite to that of the second voltage is applied across the nanopore assembly ( Figure 5 At point "F"), the analyte remains bound to Analyte Ligand 2.

[0123] refer to Figure 6, according to certain example embodiments, provides a diagram showing the evaluation of a test sample when the target analyte is not present in the test solution. Figure 6 As shown, the nanopore assembly travels through point AB, as in reference Figure 3-5 For example, by applying a first voltage as described herein and detecting a crossing signal, the analyte detection complex 1 can be positioned at point "B" in the pore of the nanopore assembly ( Figure 6 As shown, the signal element 4a is located inside the hole, while the signal element 4b is located outside the hole ( Figure 6 However, because no analyte is present in the test sample, no binding occurs between the analyte and the analyte ligand 2 at point "C." Furthermore, as the polarity of the voltage is changed as described herein, the analyte detection complex 1 is pulled out of the nanopore assembly at point "D." Figure 6 ), i.e., very early in the application of the second voltage. For example, because there is no analyte-ligand binding, the analyte does not prevent the analyte-detection complex 1 from transferring back to the opposite side of the nanopore (as opposed to Figure 3-5 Thus, no binding signal is determined. Similarly, as the voltage with a polarity opposite to the first voltage is further increased to point "E", the nanopore remains open, and the dissociation voltage ( Figure 6 ). In contrast, open channel signals can be detected in both "positive" and "negative" states.

[0124] In certain example embodiments, reusing a nanopore can be used to increase the confidence level of the nanopore's analyte-ligand binding assessment. That is, in examples where the analyte dissociates from the analyte ligand 2, the same nanopore can be reused multiple times as described herein to assess (and then reassess) the interaction of the analyte with the analyte ligand 2. In this way, reusing the nanopore can provide multiple data points for each nanopore assembly, thereby providing additional information about the analyte-ligand interaction.

[0125] Additionally or alternatively, in certain example embodiments, multiple nanopore assemblies for the same analyte can be used on a chip to further increase the confidence of the analyte-ligand binding assessment. For example, each such nanopore assembly can be used to assess the analyte-ligand binding interaction, and when dissociation occurs, the multiple nanopores can also be reused as described herein, thereby further increasing the confidence of the analyte-ligand binding assessment (through the multiple nanopores and nanopore reuse). Thus, by increasing the number of nanopore assemblies for a given analyte and by reusing a given nanopore assembly as described herein, the confidence of the analyte-ligand binding assessment can be greatly increased.

[0126] In certain example embodiments, subsets of different nanopore assemblies can be formed on a single chip, with each individual subset targeting the same target analyte. Thus, in such embodiments, a single chip can be used to assess binding interactions between different analytes and their respective ligands on the chip as described herein. Further, for each subset of nanopore assemblies, the confidence level of the analyte-ligand assessment can be increased as described herein, for example, by increasing the number of nanopore assemblies in the subset and / or reusing each nanopore assembly as described herein.

[0127] As will be appreciated by those skilled in the art, a variety of methods can be used to distinguish between different populations of nanopores on a chip. For example, different nanopore types, such as pores with smaller or larger pore sizes, can be used and readily distinguished based on techniques known in the art. For example, using this configuration, nanopores with larger openings can provide a larger current signal than pores with smaller openings, thereby allowing differentiation between pores on the same chip. Different nanopores can then be associated with the analytes they are configured to detect, thereby allowing identification of different analytes on the same chip. Other methods of differentiation include the level of blockade of the analyte-detecting complex 1 as a whole and / or across the element, and electrical signals associated with pores in the absence of analyte, including current-voltage curves of the pores. In certain example embodiments, control analytes can be used to distinguish between different nanopore assemblies. That is, a known analyte can be displayed to identify populations of nanopore assemblies that bind a specific analyte. Using such methods, for example, nanopore assemblies targeting analyte AA can be distinguished from nanopore assemblies targeting analytes BB or CC.

[0128] refer to Figure 7 According to certain example embodiments, a diagram is provided showing example confidence level distributions for single analyte capture and dissociation for weak, strong, and very strong analyte-ligand interactions. In such example embodiments, the relative binding strengths between different analyte-ligand pairs on the same chip can be evaluated and compared. For example, for multiple subsets of nanopore assemblies (where each subset is directed to the same analyte, but where different subsets are directed to different analytes), the voltage level applied throughout a given binding-dissociation cycle can be plotted against the probability of analyte binding. Peaks, for example, correspond to the dissociation of an analyte-ligand binding pair. For weak interactions, such as Figure 3 For those shown in , dissociation requires lower voltages than for stronger binding interactions ( Figure 7 ). For strong interactions, such as Figure 4 For those shown in , dissociation requires higher voltages ( Figure 7 ). And for very strong interactions, such as Figure 5 Those shown in , despite the higher voltage, do not dissociate ( Figure 7 ). The different voltages can then be compared, for example, to provide an indication of the relative binding strengths of different analyte-ligand pairs.

[0129] In certain example embodiments, the methods and systems described herein can be used to identify the analyte detected. For example, when detecting an analyte as described herein, such as via a binding signal, the specific identity of the analyte can be determined based on the known identity of the analyte ligand. If, for example, the analyte ligand 2 is a specific antibody, such as a monoclonal antibody or a functional fragment thereof, then the detection of the antigen via the methods and systems described herein can be used to identify the specific antigen found in the fluid solution. If the analyte ligand 2 is directed to a specific disease marker, such as a protein marker, then the methods and systems described herein can be used to identify that a specific marker is present in a sample. For example, when analyzing a fluid sample from a subject for the presence of a specific analyte, such an embodiment is useful.

[0130] In certain example embodiments, the methods and systems described herein can be used on a single chip to detect and identify multiple known analytes on the same chip. Such embodiments are useful, for example, for analyzing the presence of multiple known analytes in a test sample. As will be appreciated by those skilled in the art, current chip technology allows for the deposition of hundreds of thousands of nanopores (or more) on a single chip. Thus, by using the methods and compositions described herein, thousands of different nanopore assemblies can be used on the same chip to test for thousands of different analytes in a fluid sample.

[0131] For example, multiple subsets of nanopore assemblies can be assembled as described herein, wherein each subset is arranged to detect a different known analyte. For example, each subset of nanopore assemblies can include the same analyte ligand 2 and therefore target the same known analyte, while different subsets target different analytes. In order to distinguish different subsets of nanopore assemblies, for example, each subset of nanopore assemblies can include subset-specific signal transmission elements. For example, one subset can have a specific signal element 4b that is different from another subset of nanopore assemblies having a different signal element 4b. In certain example embodiments, different subsets can be distinguished based on the inclusion of additional signal elements, such as a third signal element. In other example embodiments, one subset of nanopore assemblies can include an analyte detection complex that has three signal elements bound thereto, while other subsets can have four signal elements bound thereto. As will be appreciated by those skilled in the art, different subsets of nanopore assemblies can be distinguished in many ways.

[0132] Once the different subsets of nanopore components are assembled on the chip, the chip can be contacted with a test sample as described herein, for example, with a fluid sample from a subject. As described herein, if any known analyte is present in the test sample, the binding of the analyte to the analyte ligand can be assessed by switching the polarity of the voltage and determining the binding signal. The binding of the analyte to the analyte ligand 2 can then be determined based on the binding signal. In other words, the binding signal provides an indication that the analyte is present in the test sample. In certain example embodiments, the binding strength of different analyte-ligand pairs can also be assessed by continuing to increase the second voltage as described herein. Therefore, when analyzing multiple analytes on the same chip, not only analyte-ligand pairs are identified, but also those with the strongest binding can be identified.

[0133] Similarly, in certain example embodiments, a single chip can be used to discover new analyte-ligand pairs. Such embodiments have many useful applications, for example, in drug discovery and diagnostic reagent development. For example, different subsets of nanopore assemblies can be formed on a chip, each subset comprising a different analyte ligand for an unknown ligand. Furthermore, nanopore assemblies can be differentiated as described herein. For example, as described herein, a nanopore assembly comprising analyte ligand X can be distinguished from a nanopore assembly comprising analyte ligand Y or analyte ligand Z. The nanopore assembly can then be contacted with a test sample comprising several different candidate analytes for the ligands. Any binding of the candidate analytes to a particular ligand can then be determined as described herein. For example, some analytes may bind only to ligand X (and not to other ligands). Furthermore, among analytes that bind to ligand X, those with the strongest analyte-ligand binding can also be identified by increasing a second voltage as described herein.

[0134] refer to Figure 8 , according to certain example embodiments, provides a diagram illustrating the identification of specific analyte-ligand interactions on a chip. As shown, a plurality of different nanopore assemblies are formed on a chip at a given first voltage (e.g., a negative polarity voltage) (left figure). Based on signal data from the nanopore (in an open state) or from the nanopore assembly, the different nanopore assemblies can be distinguished. As shown, different subsets of the same nanopore can be formed on a chip, such as Figure 8 As shown (left). After the nanopore assembly is brought into contact with the test sample, a second voltage (e.g., a positive voltage) having a polarity opposite to that of the first voltage is applied ( Figure 8 (right). With the application of a second voltage, any analyte-ligand binding pair can be identified as described herein. Figure 8 As shown, for example, signal analyte-ligand interactions can be identified.

[0135] In other example embodiments, the methods and systems described herein can be used to determine the dissociation constant between an analyte-ligand pair. For example, the dissociation voltage of the analyte-ligand pair can be obtained based on the dissociation signal. The dissociation voltage corresponds to the voltage at which analyte-ligand dissociation occurs, which is consistent with the detection of the dissociation signal.

[0136] In certain example embodiments, to determine the dissociation constant, the dissociation voltage of an analyte-ligand pair can be compared to a predetermined reference dissociation voltage, which then allows identification of the dissociation constant of the analyte-ligand pair. The reference dissociation voltage corresponds, for example, to the voltage at which the reference analyte-ligand pair dissociates when the methods described herein are performed on a known reference analyte-ligand pair. If the dissociation constant of the reference analyte-ligand pair is known, the dissociation constant can be assigned to the analyte-ligand pair to be tested. For example, the dissociation voltage for the analyte-ligand pair being tested can be matched to the reference dissociation voltage, the matched dissociation voltage having a relevant dissociation constant that can be assigned to the analyte-ligand pair being tested.

[0137] In certain example embodiments, a reference dissociation voltage can be obtained from a curve of the dissociation voltage of a reference analyte-ligand pair and its known dissociation constant. For example, as described herein, a nanopore assembly having analyte ligands for different reference analytes can be formed on a chip. In certain example embodiments, a nanopore assembly having analyte ligands for the analyte to be tested can also be formed on the same chip. Thereafter, the chip is contacted with the control analyte, and in certain example embodiments, the analyte to be tested (i.e., the test analyte) can also be applied to the chip. For example, in embodiments where the test analyte is tested together with the control analyte on the same chip, the control analyte and the test analyte can be mixed before contacting the chip with the mixture.

[0138] After contacting the chip with the mixture, the dissociation voltage of the reference analyte can be determined as described herein, and a curve can be generated by plotting the dissociation voltage against the known dissociation constant of the reference analyte-ligand pair. By then matching the dissociation voltage of the test analyte-ligand pair to the voltage on the curve (i.e., the reference dissociation voltage), the dissociation constant of the test analyte-ligand pair can be determined. In certain example embodiments, multiple cycles of binding and dissociation can be performed as described herein to increase the confidence level in the dissociation voltage determination for the test analyte-ligand pair and any reference analyte-ligand pair.

[0139] In addition to detecting analyte binding and determining analyte-ligand binding strength, the methods and systems described herein can also be used to determine the concentration of one or more analytes in a fluid solution applied to a chip. That is, analyte-ligand binding interactions can be assessed and identified as described herein, thereby allowing the concentration of the analyte in the solution to be determined. For example, multiple nanopore assemblies (each associated with an analyte detection complex for a specific analyte) can be formed on a chip as described herein. Similarly, nanopore assemblies for control analytes can be formed on the chip. Thereafter, the chip containing the nanopore assembly can be contacted with one or more test analytes and a predetermined concentration of a control analyte as described herein, thereby allowing the analytes to bind to their corresponding analyte ligands 2. A second voltage of opposite polarity to the first voltage is then applied across the nanopore assembly until a binding signal is obtained, as described herein.

[0140] By counting the number of binding signals associated with the test analyte-ligand pair on the chip, a binding count for the analyte-ligand pair can be determined. Thus, the binding count corresponds to the total number of analyte-ligand bindings that occurred when the second voltage was applied across the nanopore assembly. In certain example embodiments, the confidence level of the binding count can be increased by cycling the test analyte-ligand pair between bound and unbound states (i.e., reusing the nanopore) as described herein. For example, the binding count can correspond to the average or median of analyte-ligand binding over multiple association and dissociation cycles, as described herein.

[0141] In addition to determining the binding counts for the test analyte-ligand pair, a reference count for the control analyte-ligand binding pair can also be simultaneously determined. The reference count corresponds, for example, to the total number of control analyte-ligand bindings that occur when a second voltage is applied across the nanopore assembly. And as with the test analyte-ligand pair, the confidence level of the reference count can be increased by cycling the control analyte-ligand pair between the bound and unbound states as described herein. For example, as described herein, the reference count can correspond to the average or median of the control analyte-ligand bindings over multiple binding and dissociation cycles.

[0142] To determine the concentration of a test analyte in solution, for example, the determined binding counts can be compared to the determined reference counts. As an example, if a control analyte is known to be present at a concentration of 10µM after addition to the chip, and the nanopore assembly for the control analyte binds an average of 1000 captures per cycle, then for a 10µM sample, the reference count would be 1000. For example, if the average binding count for the test analyte during the same set of cycles is also 1000, then the concentration of the test analyte can be inferred to be 10µM. However, if the average binding count for the test analyte is 2000, twice that of the control analyte, then the concentration of the test analyte would be 10µM. Alternatively, if the average binding count for the test analyte is 500, half that of the control analyte, then the concentration of the test analyte would be 5µM.

[0143] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated exemplary embodiments are merely preferred embodiments of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. An analyte detection complex, comprising an analyte ligand, a crossing element, a first signal element and an anchor tag, wherein the first signal element comprises an oligonucleotide sequence having a length in the range of 10-100 nucleotides and includes a motif selected from a series of T residues, a series of N3-cyanoethyl-T residues, an Sp2 unit, an Sp3 unit, a dSp unit, and a methylphosphonate-T unit; the analyte detection complex further comprises a second signal element, wherein the second signal element comprises an oligonucleotide sequence having a length in the range of 10-100 nucleotides and includes a motif selected from a series of T residues, a series of N3-cyanoethyl-T residues, an Sp2 unit, an Sp3 unit, a dSp unit, and a methylphosphonate-T unit, wherein the analyte ligand is located at the proximal end of the analyte detection complex and the crossing element, the anchor tag is located at the distal end of the analyte detection complex, and wherein the signal element is located within the crossing element or is associated with the crossing element.

2. The analyte detection complex of claim 1, wherein the analyte ligand is an antibody or a functional fragment thereof.

3. The analyte detection complex of claim 1, wherein the anchor tag comprises a biotin tag.

4. The analyte detection complex of claim 1, wherein the signaling element comprises an oligonucleotide sequence of about 40 nucleotide pairs.

5. The analyte detection complex of claim 4, wherein the oligonucleotide sequence comprises a series of T residues or a series of N3-cyanoethyl-T residues.

6. The analyte detection complex of claim 1, wherein the second signaling element comprises an oligonucleotide sequence of about 40 nucleotide pairs.

7. The analyte detection complex of claim 6, wherein the oligonucleotide sequence comprises a series of T residues or a series of N3-cyanoethyl-T residues.

8. A nanopore assembly comprising the analyte detection complex of any one of claims 1-7.

9. The nanopore assembly of claim 8, wherein the nanopore assembly is a heptameric α-hemolysin nanopore assembly.

10. A method for evaluating the binding strength between an analyte and an analyte ligand, the method comprising: providing a chip comprising a nanopore assembly according to claim 8 in the presence of a first voltage, wherein the nanopore assembly is disposed within a membrane, and wherein the sensing electrode is located adjacent to or near the membrane; contacting the chip with a fluid solution comprising the analyte, wherein the analyte comprises a binding affinity for an analyte ligand of an analyte detection complex; applying a gradually increasing second voltage across the membrane, wherein the second voltage has a polarity opposite to the first voltage; determining a binding signal with the sensing electrode in response to applying a gradually increasing second voltage across the membrane, wherein the binding signal provides an indication of binding of the analyte to the analyte ligand, wherein a second signaling element positioned within the pore of the nanopore assembly generates the binding signal; determining a dissociation signal by means of the sensing electrode as the second voltage further increases, wherein the dissociation signal provides an indication of the strength of binding between the analyte and the analyte ligand; The method further comprises determining a transit signal with the sensing electrode, wherein the transit signal provides an indication that a transit element is located within a pore of the nanopore assembly, wherein the transit signal corresponds to the presence of a first signal element positioned in the pore of the nanopore; and The method further comprises comparing the crossing signal to the binding signal, wherein the comparison provides an indication of binding of the analyte to the analyte ligand.

11. The method of claim 10, wherein a first voltage across the membrane positions the analyte ligand on the cis side of the membrane.

12. The method of claim 10, further comprising determining a dissociation voltage associated with dissociation of the analyte from the analyte ligand from the dissociation signal.

13. The method of claim 12, further comprising comparing the determined dissociation voltage to a reference dissociation voltage.

14. The method of claim 13, further comprising determining a dissociation constant for the analyte and analyte-ligand binding pair from a comparison of the determined dissociation voltage to the reference dissociation voltage.

15. A method of determining the concentration of an analyte in a fluid solution, comprising: providing a chip comprising a plurality of nanopore assemblies according to claim 8, wherein the nanopore assemblies are disposed within a membrane and wherein at least a first subset of the nanopore assemblies comprises a first analyte ligand, in the presence of a first voltage; positioning a plurality of sensing electrodes adjacent to or near the membrane; contacting the chip with a fluid solution comprising a first analyte, wherein the first analyte comprises a binding affinity for the first analyte ligand; determining a binding count with the aid of the plurality of sensing electrodes and a computer processor, wherein the binding count provides an indication of the number of binding interactions between the first analyte ligand and the first analyte; comparing the determined bound counts to a reference count; determining a concentration of an analyte in the fluid solution based on a comparison of the bound counts to the reference counts; Wherein determining the binding count comprises: determining, with the plurality of sensing electrodes and for each nanopore assembly of a first subset of nanopore assemblies, a transit signal, wherein the transit signal provides an indication that a transit element is located within a nanopore of the nanopore assembly, wherein the transit signal corresponds to the presence of a first signal element positioned in a pore of the nanopore; applying a gradually increasing second voltage across the membrane, wherein the second voltage has a polarity opposite to the first voltage; determining, in response to applying a gradually increasing second voltage across the membrane, a binding signal with the plurality of sensing electrodes and for each nanopore assembly of the first subset of nanopore assemblies, wherein a second signaling element positioned within the pore of the nanopore assembly generates the binding signal; for each nanopore assembly of the first subset of nanopore assemblies, comparing the determined transit signal to the determined binding signal, wherein the comparison provides an indication of binding of the first analyte to the first analyte ligand; and A total number of indications of binding of the first analyte to the first analyte ligand is determined from a comparison of each determined crossing signal to the determined binding signal, wherein the total number of indications corresponds to the binding count.

16. The method of claim 15, wherein the plurality of nanopore assemblies further comprises a second subset of nanopore assemblies, wherein each nanopore assembly of the second subset comprises a second analyte ligand comprising a binding affinity to a control analyte.

17. The method of claim 16, further comprising determining the reference count, wherein determining the reference count comprises contacting the fluid solution with a predetermined amount of the control analyte to provide a predetermined concentration of the control analyte in the fluid solution.

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