Nanopore protein conjugates for detection and analysis of analytes
阅读说明：本技术 用于分析物的检测和分析的纳米孔蛋白缀合物 (Nanopore protein conjugates for detection and analysis of analytes ) 是由 M·R·安布罗索 K·M·S·巴雅吉 T·K·克雷格 于 2019-02-13 设计创作，主要内容包括：提供了用于检测目标分析物的方法、组合物和系统。还提供了用于确定流体溶液中一种或多种目标分析物的浓度的方法、组合物和系统。所述组合物包括其中纳米孔蛋白单体与捕获标签连接的纳米孔缀合物。与纳米孔蛋白缀合物相连的是针对特定分析物的分析物配体。当在包括纳米孔缀合物的纳米孔组件上施加电压时,纳米孔以给定的捕获速率捕获捕获标签。然而,在有分析物配体的分析物存在下,捕获标签的捕获率改变,从而允许通过纳米孔组件检测分析物。此外,基于与分析物和分析物配体之间的结合相关的捕获率,可以使用结合/解离动力学确定分析物的浓度。(Methods, compositions, and systems for detecting target analytes are provided. Methods, compositions, and systems for determining the concentration of one or more target analytes in a fluid solution are also provided. The compositions include a nanopore conjugate in which a nanopore protein monomer is linked to a capture tag. Attached to the nanopore protein conjugate is an analyte ligand for a particular analyte. When a voltage is applied across a nanopore assembly comprising a nanopore conjugate, the nanopore captures a capture tag at a given capture rate. However, in the presence of analyte ligand, the capture rate of the capture tag is altered, thereby allowing detection of analyte through the nanopore assembly. Furthermore, the concentration of the analyte may be determined using binding/dissociation kinetics based on the capture rate associated with the binding between the analyte and the analyte ligand.)
1. A nanopore protein conjugate comprising a nanopore protein monomer and a capture tag.
2. The nanopore protein conjugate of claim 1, wherein the capture tag comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to the sequence set forth in SEQ ID NO: 8 (capture tag sequence).
3. The nanopore protein conjugate of claim 1, wherein the nanopore protein monomer of the nanopore protein conjugate comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to a sequence set forth in SEQ ID No. 16 (α -HL) or SEQ ID No. 6 (OMPG).
4. The nanopore protein conjugate of claim 1, wherein the nanopore protein monomer of the nanopore protein conjugate comprises an alpha-hemolysin monomer, and wherein the capture tag comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to the sequence set forth in SEQ ID No. 8 (capture tag sequence).
5. The nanopore protein conjugate of claim 1, wherein the nanopore protein conjugate comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to a sequence set forth in SEQ ID No. 4 or SEQ ID No. 7.
6. An analyte detection complex, wherein the analyte detection complex comprises the nanopore protein conjugate of any one of claims 1-5 and an analyte ligand attached to the nanopore protein conjugate.
7. The analyte detection complex of claim 6, wherein the analyte ligand is a nanobody.
8. The analyte detection complex of claim 6 or 7, wherein the analyte ligand is linked to the nanopore protein conjugate by an isopeptide bond.
9. The analyte detection complex of claim 8, wherein the isopeptide bond is a SpyTag/SpyCatcher bond.
10. A nanopore assembly comprising at least one analyte detection complex of any of claims 6-9.
11. The nanopore assembly of claim 10, wherein the nanopore assembly is a heptameric nanopore assembly, and wherein each monomer of the heptameric nanopore assembly comprises an alpha-hemolysin monomer.
12. A method for detecting an analyte in a fluid solution, the method comprising:
providing a chip comprising a nanopore assembly according to claim 10 or 11, wherein the nanopore assembly is disposed within a membrane;
positioning a sensing electrode adjacent or near the membrane;
determining, with the aid of a computer processor and the sensing electrode, a first capture rate of capture tags of the nanopore assembly;
contacting the nanopore assembly with an analyte, wherein the analyte has a binding affinity for an analyte ligand of the analyte detection complex; and the combination of (a) and (b),
determining, with the aid of a computer processor and a sensing electrode, a second capture rate of the capture tag bound to the nanopore assembly, wherein the second capture rate provides an indication that the analyte is bound to the analyte ligand of the analyte detection complex and is therefore present in the fluid solution.
13. The method of claim 12, wherein the second capture rate is greater than or less than the first capture rate.
14. The method of claim 12 or 13, further comprising identifying the analyte based on the identity of the analyte ligand and based on an indication that the analyte binds to the analyte ligand.
15. A method for determining an analyte concentration in a fluid solution, the method comprising:
providing a chip comprising a nanopore assembly according to claim 10 or 11, wherein the nanopore assembly is disposed within a membrane;
positioning a sensing electrode adjacent or near the membrane;
determining, with the aid of a computer processor and the sensing electrode, a first capture rate of capture tags of the nanopore assembly;
contacting the nanopore assembly with an analyte, wherein the analyte has a binding affinity for an analyte ligand of the analyte detection complex;
identifying, with the aid of a computer processor and a sensing electrode, a first transition from the first capture rate to a second capture rate of a capture tag of the analyte detection complex;
identifying, with the aid of the computer processor and the sensing electrode, a second transition from the second capture rate to near the first capture rate; and the combination of (a) and (b),
determining a concentration of an analyte in the fluid solution based at least in part on the identification of the first transition and the second transition.
16. The method of claim 15, wherein the second capture rate is greater than or less than the first capture rate.
17. The method of claim 15 or 16, wherein determining the concentration of an analyte in the fluid solution further comprises measuring a time interval between the first transition and the second transition.
18. The method of any one of claims 15-17, wherein determining the concentration of an analyte in the fluid solution further comprises determining a rate of binding between the analyte and an analyte ligand of an analyte detection complex of the nanopore assembly.
19. A system for determining an analyte concentration in a fluid solution, the system comprising:
a chip comprising a nanopore assembly, wherein the nanopore assembly is disposed within a membrane of the chip, and wherein at least one nanopore protein monomer of the nanopore assembly comprises a capture tag and an analyte ligand;
a sensing electrode positioned adjacent or near the membrane, wherein the sensing electrode is configured to detect a signal from the nanopore assembly; and the combination of (a) and (b),
a computer processor, wherein the computer processor and sensing electrode are configured to identify a first transition and a second transition associated with a nanopore assembly, the first transition corresponding to binding of the analyte to the analyte ligand and the second transition corresponding to dissociation of the analyte from the analyte ligand.
20. The system of claim 19, wherein the concentration of the analyte in the fluid solution is determined based at least in part on the identified first transition and the identified second transition.
21. The system of claim 19 or 20, wherein the nanopore protein monomer is an alpha-hemolysin monomer.
22. The system of claim 19 or 20, wherein the nanopore protein monomer comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to a sequence set forth in SEQ ID No. 4 (α -HL alone) or 7 (OMPG alone).
23. The system of any one of claims 19-22, wherein the capture tag comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to the sequence set forth in SEQ ID No. 8 (capture tag sequence).
Technical Field
The present disclosure relates generally to methods, compositions, and systems for detecting analytes of interest, and more particularly to the use of nanopore protein conjugates for identifying analytes in fluid solutions and determining analyte concentrations in solutions.
Background
Biologically active components, such as small molecules, proteins, antigens, immunoglobulins, and nucleic acids, are involved in a number of biological processes and functions. Thus, any disturbance in the levels of such components can lead to disease or accelerate the disease process. For this reason, much effort has been expended in developing reliable methods for rapidly detecting and identifying bioactive components for use in patient diagnosis and therapy. For example, detecting proteins or small molecules in a blood or urine sample can be used to assess the metabolic state of a patient. Similarly, detection of antigens in blood or urine samples can be used to identify pathogens to which a patient has been exposed, thereby facilitating appropriate treatment. And with the recent progress in the identification of fetal cell-free DNA, the ability to diagnose certain genetic disorders prenatally from maternal blood samples by detecting cell-free DNA is now possible. 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 compare the component to a reference value to facilitate further assessment of the health of the patient.
However, while many detection and identification methods are available, many are expensive and can be quite time consuming. For example, many diagnostic tests may take several days to complete and require significant laboratory resources. And in some cases, delay in diagnosis may negatively impact patient care, for example, in analyzing markers associated with myocardial infarction. Furthermore, the complexity of many diagnostic tests aimed at identifying biologically active components lends itself to error, thereby reducing accuracy. Also, many detection and identification methods can only analyze one or a few bioactive components (and not at a determinable concentration) at a time.
There is a need for additional methods, compositions, and systems that can rapidly detect and identify biologically active components, particularly in an efficient and cost-effective manner. There is also a need for methods, compositions, and systems that can simultaneously assay multiple bioactive components, thereby reducing costs. In addition, methods, compositions, and systems are needed to determine the concentration of bioactive components in a fluid solution.
Disclosure of Invention
In certain example aspects, a nanopore protein conjugate is provided that includes a nanopore protein monomer and a capture tag. For example, a nanopore protein conjugate may comprise an amino acid sequence having at least 60%, 65%, 70%, 80%, 90%, or 95% or more sequence identity to the sequence set forth in SEQ ID NO. 4 or SEQ ID NO. 7.
In certain example aspects, an analyte detection complex comprising a nanopore protein conjugate is provided. For example, the analyte detection complex includes an analyte ligand linked to a nanopore protein conjugate, such as via an isopeptide bond.
In certain example aspects, a nanopore assembly is provided that includes at least one analyte detection complex. For example, the nanopore assembly may be a heptameric nanopore assembly comprising an analyte detection complex. In certain example aspects, the nanopore monomer of the conjugate is an α -hemolysin monomer, and each of the other six monomers of the heptamer is an α -hemolysin monomer.
In certain example aspects, a method for detecting an analyte in a fluid solution is provided. The method includes providing a chip comprising a nanopore assembly described herein. For example, the nanopore assembly includes an analyte detection complex that includes a nanopore monomer, a capture tag, and an analyte ligand. Further, a nanopore assembly is disposed within the membrane of the chip. The sensing electrode is then placed adjacent or near the membrane. A first capture rate at which the tags are captured is determined with the aid of a computer processor and a sensing electrode. The nanopore assembly is then contacted with an analyte having a binding affinity for the analyte ligand of the analyte detection complex. A second capture rate for capturing the tags is then determined using the computer processor and the sensing electrode. For example, the second capture rate provides an indication that the analyte is bound to the analyte ligand of the analyte detection complex, thus indicating that the analyte is present in the fluid solution. The second capture rate may be less than or greater than the first capture rate, for example. Furthermore, the identity of the analyte may be determined based on the identity of the analyte ligand-and based on an indication that the analyte binds to the analyte ligand.
In other example aspects, a method for determining a concentration of an analyte in a fluid solution is provided. The method includes providing a chip having a nanopore assembly as described herein disposed within a membrane. The nanopore assembly includes, for example, an analyte detection complex comprising a nanopore monomer, a capture tag, and an analyte ligand. The sensing electrode is located adjacent or near the membrane. Thereafter, a first capture rate of the capture tag of the nanopore assembly is determined with the aid of the computer processor and the sensing electrode. The nanopore assembly is then contacted with an analyte having a binding affinity for the analyte ligand of the analyte detection complex. A first transition of the capture tag of the analyte detection complex from the first capture rate to the second capture rate is identified with the aid of the computer processor and the sensing electrode. Then, with the aid of the computer processor and the sensing electrode, a second transition from the second capture rate to a capture rate that approximates the first capture rate is identified. The concentration of the analyte in the fluid solution is then determined based at least in part on the identification of the first transition and the second transition. For example, the time interval between transition rates can be used to determine the concentration of the analyte.
In other example aspects, a system for determining an analyte concentration in a fluid solution is provided. The system may also be used to detect analytes in a solution. The system includes, for example, a chip having a nanopore assembly disposed within a membrane of the chip. The nanopore assembly, for example, comprises at least one nanopore protein monomer with a capture tag and an analyte ligand. The system also includes a sensing electrode located adjacent or near the membrane. The sensing electrode is configured to detect a signal from the nanopore assembly, for example. The system also includes a computer processor, which together with the sensing electrode is configured to identify a first transition and a second transition associated with the nanopore assembly. The first transition corresponds, for example, to binding of the analyte to the analyte ligand. The second transition corresponds, for example, to dissociation of the analyte from the analyte ligand.
In certain example aspects of the system, the concentration of the analyte in the fluid solution is determined based at least in part on the identified first transition and the identified second transition. In certain example aspects of the system, the nanopore protein monomer is an alpha-hemolysin monomer or an OmpG monomer. In certain example aspects of the system, the nanopore protein monomer comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to a sequence set forth in SEQ ID No. 4 or SEQ ID No. 7. In certain exemplary aspects of the system, the capture tag comprises an amino acid sequence having at least 80%, 90%, 95%, 98% or more sequence identity to the sequence set forth in SEQ ID NO. 8.
These and other aspects, objects, features and advantages of the example embodiments will become apparent to those of ordinary skill in the art upon consideration of the following detailed description of the example embodiments.
Drawings
Fig. 1 is a diagram showing a nanopore protein conjugate, according to certain example embodiments.
Fig. 2 is a diagram showing an analyte detection complex according to certain example embodiments.
Figure 3 is a diagram showing a nanopore assembly including an analyte detection complex according to certain example embodiments.
Fig. 4A-4C are graphs showing different pore states associated with different capture rates, according to some example embodiments. More specifically, figure 4A shows a nanopore assembly including a nanopore protein conjugate, but not an analyte ligand (and therefore not an analyte detection complex). Such wells correlate with a baseline capture rate for capture of the tag (solid versus dashed, showing capture). Figure 4B shows the same nanopore assembly, but with an analyte ligand (e.g., nanobody) attached. Thus, the nanopore assembly of fig. 4B includes an analyte detection complex. The nanopore assembly of fig. 4B is associated with a first capture rate of the capture tag (solid line versus dashed line, showing capture). Fig. 4C shows the same well as fig. 4B, but now with the analyte (e.g., antigen of the nanobody) attached. Such a well is associated with a second capture rate for capturing the tag (solid line versus dashed line, capture shown).
Figures 5A-5C are graphs showing various capture rates according to certain example embodiments, as those capture rates correspond to binding states of nanopore assemblies. More specifically, fig. 5A is a graph showing a measurement of a baseline capture rate. Fig. 5B is a graph showing a measurement of a first capture rate, where an analyte ligand binds to a nanopore protein conjugate of a nanopore assembly, thereby forming an analyte detection complex. In fig. 5B, no analyte is present. Fig. 5C shows capture rate measurements for a first capture rate (no analyte binding) and a second capture rate (analyte binding). Also shown is a first transition from the first capture rate to the second capture rate and a second transition from the second capture rate back to the first capture rate.
Detailed Description
The embodiments described herein can be understood more readily by reference to the following detailed description, examples and claims, and their previous and following description. Before the present systems, devices, compositions, and/or methods are disclosed and described, it is to be understood that the embodiments described herein are not limited to the specific systems, devices, and/or compositions disclosed unless otherwise specified, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
Further, the following description is provided as an enabling teaching of various embodiments in their best, currently known aspect. One skilled in the relevant art will recognize that many changes can be made to the aspects described, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present invention can be obtained by selecting some of the features of the various embodiments without utilizing other features. Thus, those who work in the art will recognize that many modifications and adaptations to the various embodiments described herein are possible and may even be desirable in certain circumstances and are a part of the present disclosure. Accordingly, the following description is provided as illustrative of the principles of the embodiments described herein and not in limitation thereof.
SUMMARY
As disclosed herein, methods, compositions, and systems for detecting target analytes are provided. Methods, compositions, and systems for determining the concentration of one or more target analytes in a fluid solution are also provided. The composition includes a nanopore conjugate in which a nanopore protein monomer is linked to a capture tag. Attached to the nanopore protein conjugate is an analyte ligand that can be directed to a particular analyte. As described herein, the nanopore captures the capture tag at a given capture rate when a voltage is applied across a nanopore assembly comprising a nanopore conjugate. However, in the presence of analyte for the analyte ligand, the capture rate of the capture tag is altered, allowing detection of the analyte through the nanopore assembly. Furthermore, the concentration of the analyte may be determined in certain embodiments based on the capture rate associated with the binding between the analyte and the analyte ligand.
More particularly, the nanopore protein monomer of the nanopore conjugates described herein can be any type of biological nanopore protein monomer. For example, the nanopore protein monomer may be an alpha-hemolysin (alpha-HL) monomer, an OmpG monomer, or other protein nanopore monomer. For example, when the nanopore protein monomer is alpha-hemolysin, the resulting nanopore protein conjugate is an alpha-hemolysin/capture tag protein conjugate.
By forming conjugates using alpha-hemolysin nanopore monomers, for example, the alpha-hemolysin monomer of a conjugate can oligomerize with other alpha-HL monomers, forming a heptameric nanopore assembly. The heptameric component may, for example, have one alpha-hemolysin/capture tag conjugate and six alpha-hemolysin monomers, e.g., six wild-type alpha-hemolysin monomers. In such embodiments, the capture tag of the protein conjugate is configured to interact with the heptameric nanopore assembly in the presence of a voltage. In certain embodiments, during synthesis of the α -hemolysin monomer, the capture tag is fused to the α -hemolysin via a linker sequence, thereby forming a nanopore protein conjugate. A capture tag is, for example, any molecule that a nanopore can capture and release resulting in a detectable capture rate.
In addition to including a capture tag, the nanopore protein conjugate is also linked to an analyte ligand. The analyte ligand may be, for example, any ligand that binds the analyte. In certain embodiments, the analyte ligand is a nanobody, such as an antibody or fragment thereof that binds an antigen or fragment thereof in the presence of the antigen. Analyte ligands may be bound to the nanopore protein conjugate directly or indirectly (e.g., through a peptide linker sequence). For example, the nanopore protein conjugate may include a region for attaching an analyte ligand to the nanopore assembly. In certain embodiments, the nanopore protein conjugate may comprise a SpyTag sequence. In such embodiments, the analyte ligand may be linked to a SpyCatcher sequence. Thus, the analyte ligand may be linked to the nanopore protein conjugate through a SpyTag/SpyCatcher linkage. In certain embodiments, the capture tag described herein can be linked to an analyte ligand.
When assembled into a membrane of a chip, a nanopore protein assembly comprising a nanopore protein conjugate described herein and an analyte ligand may be used to identify an analyte in a fluid solution and/or determine the concentration of the analyte. Without wishing to be bound by any particular theory, when the chip is contacted with a fluid solution containing an analyte, it is believed that the proximity of the analyte-ligand binding pair to the capture tag alters the interaction of the capture tag with the well, thereby affecting the capture rate of the capture tag. Electrodes in proximity to the membrane may then detect the change in capture rate, thereby providing an indication of the binding of analyte to the analyte ligand and thus indicating the presence of ligand in the fluid solution. Furthermore, using the binding/dissociation kinetics, the concentration of the analyte can be determined based on the capture rate of the capture tag. That is, the change in capture rate can be used to determine the K of the analyteaAnd/or KdAnd, together with other known variables, KaAnd/or KdCan be used to determine the concentration of an analyte in a fluid solution.
In other embodiments, different nanopore protein assemblies for different analytes may be used on a single chip to detect and/or determine the concentration of different analytes on the same chip. In such embodiments, different nanopore protein assemblies may be configured to generate different signals that may be detected by the sensing electrode. For example, various parameters (e.g., the size of the open pore channel of the pore, the placement of different capture tags, and/or other capture tags on the reverse side of the membrane) can be used to alter the baseline signal produced by a given set of pores. For example, with this configuration, a well with a larger opening may provide a stronger signal than a well with a smaller opening, allowing the wells on the same chip to be distinguished. Different wells can then be associated with the analytes they are configured to detect, thereby allowing different analytes to be identified on the same chip. In addition, the concentration of each detected analyte can be determined as described herein using the binding/dissociation kinetics.
Summary of the terms
The invention will now be described in detail by reference only, using the following definitions and examples. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, the preferred methods and materials are described. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.
The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Ranges or values may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value of the range and/or to the other particular value of the range. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. In certain example embodiments, the term "about" is understood to be within the normal tolerance in the art for a given measurement, such as within 2 standard deviations of the mean, for example. In certain example embodiments, "about" may be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value, depending on the measurement. Unless otherwise apparent from the context, all numbers provided herein may be modified by the term "about". Furthermore, terms used herein, such as "embodiment," "exemplary," or "example," are not meant to indicate a preference, but rather to explain that the aspect discussed thereafter is merely one example of the aspect presented.
The term "antibody" as used herein broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof, which retains the essential epitope binding characteristics of an Ig molecule. Such mutant, variant or derivative antibody entities are known in the art. A functional fragment of an antibody, for example, includes a portion 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", e.g., single domain antibody fragments.
The term "amino acid" as used herein is an organic compound containing an amino group and a carboxylic acid group. A peptide or polypeptide contains two or more amino acids. For purposes herein, amino acids include 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).
"polypeptide" as used herein means 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 natural or artificial proteins, protein fragments and polypeptide analogs of the protein sequence. Polypeptides may be monomeric or polymeric, and may include a number of modifications. Typically, the peptide or polypeptide has a length of greater than or equal to 2 amino acids, and typically has a length of less than or equal to 40 amino acids.
As used herein, "alpha-hemolysin," "alpha-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 transmembrane channels formed by seven monomeric proteins. In certain example embodiments, the α -hemolysin is a "modified α -hemolysin," meaning that the α -hemolysin originates from another (i.e., parent) α -hemolysin and contains one or more amino acid alterations (e.g., amino acid substitutions, deletions, or insertions) as 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 by 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 alpha-hemolysin has at least one altered phenotype compared to the parent alpha-hemolysin. In certain example embodiments, the α -hemolysin originates 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 removing, adding and/or manipulating the coding sequence, or the amino acid sequence of the expressed protein has been modified in accordance with the invention described herein, respectively.
The term "analyte" or "target analyte" as used herein broadly refers to any target compound, molecule or other substance to be detected, identified or characterized. For example, the term "analyte" or "target analyte" includes any target physiological molecule or agent that is a particular substance or component being detected and/or measured. In certain example embodiments, the analyte is a target physiological analyte. Additionally or alternatively, the analyte may be a chemical substance having a physiological effect, such as a drug or a pharmacological agent. Additionally or alternatively, the analyte or target analyte may be an environmental factor or other chemical agent or entity. The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from a biological material. For example, the agent may be a cytotoxic agent.
In certain example embodiments, the term "analyte" or "analyte of interest" includes toxins, organic compounds, proteins, peptides, microorganisms, amino acids, carbohydrates, nucleic acids, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), lipids, viral particles, and metabolites of or antibodies to any of the foregoing. For example, the analyte may include ferritin; creatinine kinase MIB (CK-MIB); digoxin; phenytoin; phenobarbital (phenobarbitol); carbamazepine; vancomycin; gentamicin; theophylline; valproic acid; quinidine (quinidine); luteinizing Hormone (LH); follicle Stimulating Hormone (FSH); estradiol, progesterone, IgE antibody; vitamin B2 microglobulin; glycated hemoglobin (gly. Hb); cortisol; digitoxin, N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, such as rubella-IgG and rubella-IgM; antibodies against toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; a salicylate; acetaminophen; hepatitis b virus surface antigen (HBsAg); antibodies against hepatitis b core antigen, such as anti-hepatitis b core antigen IgG and IgM (anti-HBC);
Other example analytes or analytes of interest include folate, folate RBC, iron, soluble transferrin receptor, transferrin, vitamin B12, lactate dehydrogenase, osteocalcin, N-MID osteocalcin, P1NP, phosphorus, PTH (1-84), B-CrossLaps, vitamin D, cardiac apolipoprotein A1, apolipoprotein B, Cholesterol, CK-MB (mass) STAT, CRPs hs, cystatin C, D-dimer, cardiac digoxigenin, digoxin, GDF-154, direct HDL Cholesterol (HDL Cholesterol direct), homocysteine, hydroxybutyrate dehydrogenase, direct LDL Cholesterol (LDL direct), lipoprotein (a), myoglobin STAT, NT-proBNP, STAT, 1 troponin I, 1 troponin I STAT, troponin T hs STAT, coagulation ATIII, D-dimer, abuse drug test amphetamine (Ecstasy), benzodiazepines (serum), cannabinoids, ***e, ethanol, methadone metabolite (EDDP), mequinone, 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 confirmation (confirrmatory), TSH, T-uptake, Muellian resistant hormone, fertility EA-S, estradiol, FSH, 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, direct bilirubin, total bilirubin, cholinesterase acetyl, 3 cholinesterase butyryl, gamma glutamyltransferase, glutamate dehydrogenase, HBeAg, HBsAg, lactate dehydrogenase, infectious disease anti-HAV, anti-HAV IgM, anti-HBc IgM, anti-HBe, HBeAg, anti-HBsAg, HBsAg confirmation, HBsAg quantification, anti-HCV, Chagas 4, CMV IgG affinity, IgM, HIV-com PT, HIV-CMV, HIV-Ag-IgG confirmation, HSV-1 IgG, HSV-2 IgG, HTLV-I/II, rubella IgG, rubella IgM, syphilis, Toxo IgG avidity, Toxo IgM, TPLA (syphilis), anti-CCP, ASLO, C3C, C4, ceruloplasmin, CRP (latex), haptoglobin, IgA, IgE, IgG, immunoglobulin A CSF, immunoglobulin M CSF, interleukin 6, kappa light chain free6, 2,3, lambda light chain free6, 2,3, prepurin, procalcitonin, rheumatoid factor, a1-Acid glycoprotein, a 1-antitrypsin, bicarbonate (CO2), calcium, chloride, fructosamine, glucose, HbA1C (hemolysis), HbA1C (whole blood), insulin, lactate, direct LDL sterol, magnesium, potassium, sodium, cholesterol, Triglycerides (Triglycerides), Triglycerides (triglyceride), Glycerol (total protein), total protein (triglyceride), and triglyceride (triglyceride), Total vitamin D, acid phosphatase, AFP, CA 125, CA 15-3, CA19-9, CA 72-4, calcitonin, Cyfra 21-1, hCG + beta, HE4, kappa light chain free6, 2,3, lambda light chain free6, 2,3, NSE, proGRP, PSA free, total PSA, SCC, S-100, thyroglobulin (TG II), thyroglobulin confirmation, b 2-microglobulin, albumin (BCG), albumin (BCP), immunological albumin, creatinine (enzymatic), creatinine (Jaffe), cystatin C, potassium, PTH (1-84), total protein, urine/CSF, urea/BUN, uric acid, a 1-microglobulin, b 2-microglobulin, acetaminophen (p-acetamidophenol), amikacin, carbamazepine, cyclosporin, and the like, Digitoxin, digoxin, everolimus, gentamicin, lidocaine, lithium, ISE mycophenolic acid, NAPA, phenobarbital, phenytoin, primidone, procainamide, quinidine, salicylate, sirolimus, tacrolimus, theophylline, tobramycin, valproic acid, vancomycin, anti-Mueller hormone, AFP, b-Crosslaps, DHEA-S, estradiol, FSH, free betｃA hCG, hCG + betｃA, hCG STAT, HE4, LH, N-MID osteocalcin, PAPP-A, PlGF, sFIt-1, P1NP, progesterone, prolactin, SHBG, testosterone, CMV IgG avidity, CMV IgM, rubellｃA, IgM, Toxo IgG and/or Toxo IgM.
The term "ligand" or "analyte ligand" as used herein broadly refers to any compound, molecule, group of molecules, 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, the ligand forms a complex with the analyte for biological purposes. As will be understood by those skilled in the art, the association between a ligand and its binding partner (e.g., an analyte) varies with charge, hydrophobicity, and molecular structure. Binding may occur through a variety of intermolecular forces, such as ionic bonds, hydrogen bonds, and van der waals forces. In certain embodiments, the ligand or analyte ligand is an antibody or functional fragment thereof having binding affinity for an antigen.
The term "DNA" as used herein denotes a molecule comprising at least one deoxyribonucleotide residue, as commonly understood in the art. A "deoxyribonucleotide" is a nucleotide that has no hydroxyl group but a hydrogen at the 2' position of the β -D-deoxyribofuranosyl moiety. The term encompasses double-stranded DNA, single-stranded DNA, DNA having both double-stranded and single-stranded regions, isolated DNA, e.g., partially purified DNA, substantially pure DNA, synthetic DNA, recombinantly produced DNA, and altered or similar DNA, as distinguished from naturally occurring DNA by the addition, deletion, substitution, and/or modification of one or more nucleotides.
The term "linked" as used herein denotes any method known in the art for functionally linking two or more entities, such as linking a protein to a DNA molecule or linking a protein to a protein. For example, one protein may be linked to another protein by a covalent bond, e.g., in a recombinant fusion protein, with or without intervening sequences or domains. Exemplary covalent bonds may be formed, for example, as follows: by SpyCatcher/SpyTag interaction, cysteine-maleimide conjugation, or azide-alkyne click chemistry, among other means known in the art. In addition, one DNA molecule can be linked to another by hybridization of complementary DNA sequences.
The term "nanopore" as used herein generally refers to a hole, channel, or channel formed or otherwise provided in a membrane. The membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed from a polymeric material. The membrane may be a polymeric material. The nanopore may be configured adjacent or proximate to a sensing circuit or an electrode coupled to the 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 proteins. The membrane comprises a trans side (i.e. the side facing the sensing electrode) and a cis side (i.e. the side facing the corresponding electrode).
The term "nucleic acid molecule" or "nucleic acid" includes RNA, DNA and cDNA molecules. It will be appreciated that as a result of the degeneracy of the genetic code, a number of nucleotide sequences encoding a given protein, such as α -hemolysin and/or variants thereof, may be produced. The present disclosure encompasses every possible variant nucleotide sequence encoding a variant alpha-hemolysin, all of which are possible given the degeneracy of the genetic code.
As is recognized in the art, the term "nucleotide" is used herein to include both natural bases (standards) and modified bases well known in the art. Such bases are typically located at the 1' position of the sugar portion of the nucleotide. Nucleotides typically comprise a base, a sugar and a phosphate group.
"synthetic" as used herein, for example 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 polypeptide molecule produced by recombinant methods and/or by chemical synthetic methods.
As used herein, production by recombinant methods using recombinant DNA methods means the use of well-known methods of molecular biology to express the protein encoded by the cloned DNA. For example, standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions, or as is commonly used in the art or as described herein. The foregoing techniques and protocols may generally be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al Molecular Cloning: A Laboratory Manual (2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
As used herein, "vector" (or plasmid) refers to a discrete DNA element used to introduce a heterologous nucleic acid into a cell for its expression or replication. Vectors are typically maintained episomally, but can be designed to achieve integration of a gene or portion thereof into the chromosome of the genome. Vectors that are artificial chromosomes, such as bacterial artificial chromosomes, yeast artificial chromosomes, and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles is well known to those skilled in the art.
As used herein, "expression" generally refers to the process of transcribing a nucleic acid into mRNA and translating into a peptide, polypeptide, or protein. If the nucleic acid is derived from genomic DNA, expression may include processing, such as splicing of mRNA, in the case of selection of an appropriate eukaryotic host cell or organism.
As used herein, "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences (such as promoter regions) capable of effecting expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and optionally may 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 means a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which 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 those that integrate into the genome of the host cell. Vectors as used herein also include "viral vectors" or "viral vectors". Viral vectors are engineered viruses that are operably linked to a foreign gene to transfer (as a vector or shuttle) the foreign gene into a cell.
The term "host cell" refers to a cell that comprises a vector and supports the replication and/or transcription and translation (expression) of an expression construct. The host cell may be a prokaryotic cell, such as E.coli or Bacillus subtilis, or a eukaryotic cell, such as a yeast, plant, insect, amphibian, or mammalian cell. Generally, the host cell is prokaryotic, e.g., E.coli.
The term "cellular expression" or "cellular gene expression" generally refers to the cellular process by which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in a cell. Thus, gene expression involves 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, as well as polypeptide synthesis, transport and post-translational modification of polypeptides. In addition, processes that affect protein-protein interactions within a cell may also affect gene expression as defined herein.
The term "optional" or "optionally" as used herein means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the optional step of attaching the analyte detection complexes to the nanopore assembly monomer means that the analyte detection complexes may or may not be attached.
The term "phospholipid" as used herein denotes a hydrophobic molecule comprising at least one phosphorus group. For example, the phospholipid may comprise a phosphorus-containing group and a saturated or unsaturated alkyl group, optionally substituted with: OH, COOH, oxo, amine or substituted or unsubstituted aryl.
The term "membrane" as used herein means 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 bilayers when placed in water. "phospholipid membrane" means any structure composed of phospholipids arranged such that the hydrophobic head of the lipid points in one direction and the hydrophilic tail points in the opposite direction. Examples of phospholipid membranes include the lipid bilayer of the cell membrane.
As used herein, "identity" or "sequence identity" refers in the context of sequences to the similarity between two nucleic acid sequences or two amino acid sequences and in the manner of similarity between sequences, otherwise referred to as sequence identity. Sequence identity is often measured in terms of percent identity (or similarity or homology); the higher the percentage, the more similar the two sequences. For example, 80% homology refers to something identical to 80% sequence identity as determined by a well-defined algorithm, and thus homologues of a given sequence have greater than 80% sequence identity over the length of the given sequence. Exemplary 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).
Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in the following documents: 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-; and Pearson et al, meth. mol.Bio.24, 307-31, 1994. Altschul et al (J. mol. biol. 215: 403. ang. 410, 1990) propose detailed considerations for sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al J. mol. biol. 215: 403-.
In evaluating a given nucleic acid sequence against nucleic acid sequences in GenBank DNA sequences and other public databases, sequence searches are typically performed using the BLASTN program. The BLASTX program is preferably used to search amino acid sequences in GenBank protein sequences and other public databases for nucleic acid sequences that have been translated in all reading frames. BLASTN and BLASTX were run using default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix. (see, e.g., Altschul, S. F., et al, Nucleic Acids Res. 25:3389-3402, 1997).
In certain example embodiments, a preferred alignment of selected sequences is performed using, for example, the CLUSTAL-W program in MacVector 13.0.7 version to determine "% identity" between two or more sequences, which is run with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.
The term "variant" as used herein denotes a modified protein that exhibits altered characteristics (e.g., altered ionic conductance) as compared to the parent protein.
The term "sample" as used herein is used in its broadest sense. As used herein, a "biological sample" includes, but is not limited to, any amount of a substance from an organism or a prior organism (such as from a subject). The biological sample may comprise a sample of biological tissue or fluid origin obtained in vivo or in vitro. Such samples may be from, but are not limited to, bodily fluids, organs, tissues, fractions, and cells isolated from a biological subject. The biological sample may also include an extract from the biological sample, such as an extract from a biological fluid (e.g., blood or urine).
As used herein, "biological fluid" or "biological fluid sample" means any physiological fluid (e.g., blood, plasma, sputum, eluate, ocular lens fluid, cerebrospinal fluid, urine, semen, sweat, tears, milk, saliva, synovial fluid, peritoneal fluid, amniotic fluid) and solid tissue that has been converted to a liquid form or for which a liquid has been extracted, at least in part, by one or more known protocols. For example, the liquid tissue extract (such as from a biopsy) may 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. The terms "blood", "plasma" and "serum" as used herein include fractions or processed portions thereof. Similarly, where a sample is obtained from a biopsy, swab, smear, or the like, the "sample" includes a processed fraction or portion derived from the biopsy, swab, smear, or the like.
Further, "fluid solution," "fluid sample," or "fluid" includes biological fluids, but may also include and encompass non-physiological components, such as any analyte that may be present in an environmental sample. For example, the sample may be from a river, lake, pond, or other reservoir. In certain example embodiments, the fluid sample may be modified. For example, a buffer or preservative may be added to the fluid sample, or the fluid sample may be diluted. In other example embodiments, the fluid sample may be modified by ordinary methods known in the art to increase the concentration of one or more solutes in the solution. Regardless, the fluid solution remains a fluid solution as described herein.
As used herein, "subject" means an animal, including vertebrates. The vertebrate may be a mammal, such as a human. In certain embodiments, the subject may be a human patient. A subject may be a "patient," e.g., a patient having or suspected of having a disease or disorder, and may require treatment or diagnosis, or may require monitoring of the progression of the disease or disorder. The patient may also receive a treatment regimen requiring monitoring of efficacy. Mammal means any animal classified as a mammal, including, for example, humans, chimpanzees, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cows, rabbits, horses, sheep, pigs, and the like.
The term "wild-type" as used herein refers to a gene or gene product that has the characteristics of the gene or gene product when isolated from a naturally occurring source.
As used herein, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, sequence variants are described by using the following nomenclature: original amino acids: position: a substituted amino acid. According to this nomenclature, for example, a substitution of threonine at position 17 with arginine is shown as Thr17Arg or T17R. Multiple mutations are separated by plus signs, for example: thr17Arg + Glu34Ser or T17R + E34S, representing mutations at positions 17 and 34, replacing threonine and glutamic acid with arginine and serine, respectively.