Systems and methods for assessing target molecule

Abstract

To provide systems and methods for analyzing or identifying a target molecule.SOLUTION: For example, a system of the present disclosure may comprise (i) a sensing electrode, (ii) a binding unit coupled to the sensing electrode and configured to bind at least a portion of the target molecule, and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of a surface of the sensing electrode. The sensor may be configured to detect one or more signals indicative of an impedance or impedance change in the sensor when the at least the portion of the target molecule is bound by the binding unit. The one or more signals may be usable to analyze or identify the target molecule.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 881,254, filed July 31, 2019, which is incorporated by reference in its entirety into this specification. [Background technology]

[0002] background

[0002] Nucleic acid sequencing methods can be used to provide sequence information of a nucleic acid sample. Such sequence information can be useful in diagnosing or treating a condition (e.g., a disease) of a subject (e.g., an individual, a patient, etc.). For example, a subject's nucleic acid sequence information can be used to identify, diagnose, or develop a treatment for one or more genetic diseases. In another example, nucleic acid sequence information of one or more pathogens can lead to a treatment for one or more infectious diseases.

[0003]

[0003] The detection of one or more rare sequence variants (e.g., mutations) may be useful in healthcare. The detection of rare sequence variants may be important for one or more pathological mutations and for their early detection. The detection of one or more cancer-associated mutations (e.g., point mutations) in clinical samples may improve the identification of one or more minimal residual disease during chemotherapy or the detection of tumor cells in relapsed patients. In addition, such detection of one or more mutations may be important for the evaluation of exposure to environmental mutagens, the monitoring of endogenous DNA repair, or the study of the accumulation of one or more somatic mutations in the elderly. The detection of one or more rare sequence variants may enhance prenatal diagnosis and allow the characterization of fetal cells present in maternal blood.

[0004]

[0004] Alternatively or in addition, detection of small molecules and / or polypeptides (e.g., growth factors, enzymes, etc.) from biological samples (e.g., blood, urine, tissue biopsies, etc.) may be used to identify or monitor one or more health conditions, such as cancer. Summary of the Invention [Means for solving the problem]

[0005] overview

[0005] One aspect of the present disclosure provides a system for analyzing or identifying a target molecule, the system including a sensor including (i) a sensing electrode, (ii) a binding unit coupled to the sensing electrode and configured to bind to at least a portion of the target molecule, and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of a surface of the sensing electrode, the sensor configured to detect one or more signals indicative of an impedance or an impedance change of the sensor when at least a portion of the target molecule is bound by the binding unit, the one or more signals being usable to analyze or identify the target molecule.

[0006]

[0006] In some embodiments, the one or more signals indicate one or more of the following: (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, and (iii) the electrical inductance of the sensor or a change therein.

[0007] In some embodiments of any one of the subject systems, the one or more signals is a current or a voltage. In some embodiments of any one of the subject systems, the one or more signals is not a tunneling current.

[0008] In some embodiments of any one of the subject systems, the average cross-sectional dimension of the sensing electrode is no more than 20 times the average size of the target molecules. In some embodiments, the average cross-sectional dimension of the sensing electrode is no more than 2 times the average size of the target molecules.

[0009] In some embodiments of any one of the subject systems, the average cross-sectional dimension of the sensing electrode is smaller than the average size of the target molecules.

[0010] In some embodiments of any one of the subject systems, the binding unit is coupled to the sensing electrode via a conductive material.

[0011] In some embodiments of any one of the subject systems, the dielectric material is a self-assembled monolayer.

[0012] In some embodiments of any one of the subject systems, the target molecule includes a tag, the tag configured to induce a change in one or more signals.

[0013] In some embodiments of any one of the subject systems, the sensor further includes a reference electrode in electrical communication with the sensing electrode, and the one or more signals are indicative of an impedance or an impedance change between the sensing electrode and the reference electrode. In some embodiments, the sensing electrode and the reference electrode are configured to provide a first electric field along a first direction, and the system further includes an additional electric field generator configured to apply a second electric field along a second direction different from the first direction. In some embodiments, the second direction is substantially orthogonal to the first direction.

[0014] In some embodiments of any one of the subject systems, the sensor is further configured to determine a residence time of at least a portion of the target molecule on the binding unit.

[0015] In some embodiments of any one of the subject systems, the binding unit comprises one or more members selected from the group consisting of a small molecule, an enzyme, an antibody, a functional fragment thereof, and a functional variant thereof.

[0016]

[0016] In some embodiments of any one of the subject systems, the target molecule comprises one or more members selected from the group consisting of small molecules, nucleotides, polynucleotides, amino acids, peptides, polypeptides, and variants thereof.

[0017]

[0017] Another aspect of the present disclosure provides a kit for analyzing or identifying a target molecule, the kit comprising (i) any one of the subject systems including a sensor, and (ii) instructions for providing a sample to be analyzed by the sensor, the sample containing or suspected of having the target molecule.

[0018]

[0018] Different aspects of the present disclosure provide methods for analyzing or identifying a target molecule, the methods including: (a) providing a sensor including (i) a sensing electrode, (ii) a binding unit coupled to the sensing electrode and configured to bind to at least a portion of the target molecule, and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of a surface of the sensing electrode; (b) detecting one or more signals indicative of an impedance or impedance change of the sensor when at least a portion of the target molecule is bound by the binding unit; and (c) analyzing or identifying the target molecule using the one or more signals.

[0019]

[0019] In some embodiments, the one or more signals indicate one or more of the following: (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, and (ii) the electrical inductance of the sensor or a change therein.

[0020] In some embodiments of any one of the subject methods, the one or more signals is a current or a voltage. In some embodiments of any one of the subject methods, the one or more signals is not a tunneling current.

[0021] In some embodiments of any one of the subject methods, the average cross-sectional dimension of the sensing electrode is no more than 20 times the average size of the target molecules. In some embodiments, the average cross-sectional dimension of the sensing electrode is no more than 2 times the average size of the target molecules.

[0022] In some embodiments of any one of the subject methods, the average cross-sectional dimension of the sensing electrode is smaller than the average size of the target molecules.

[0023] In some embodiments of any one of the subject methods, the binding unit is coupled to the sensing electrode via a conductive material.

[0024] In some embodiments of any one of the subject methods, the dielectric material is a self-assembled monolayer.

[0025] In some embodiments of any one of the subject methods, the target molecule includes a tag, the tag configured to induce a change in one or more signals.

[0026] In some embodiments of any one of the subject methods, the sensor further includes a reference electrode in electrical communication with the sensing electrode, and the one or more signals are indicative of an impedance or an impedance change between the sensing electrode and the reference electrode. In some embodiments, the sensing electrode and the reference electrode provide a first electric field along a first direction, and the method further includes applying a second electric field along a second direction different from the first direction using an additional electric field generator. In some embodiments, the second direction is substantially orthogonal to the first direction.

[0027] In some embodiments of any one of the subject methods, the method further comprises determining a residence time of at least a portion of the target molecule on the binding unit.

[0028] In some embodiments of any one of the subject methods, the binding unit comprises one or more members selected from the group consisting of a small molecule, an enzyme, an antibody, a functional fragment thereof, and a functional variant thereof.

[0029]

[0029] In some embodiments of any one of the subject methods, the target molecule comprises one or more members selected from the group consisting of small molecules, nucleotides, polynucleotides, amino acids, peptides, polypeptides, and variants thereof.

[0030]

[0030] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which shows and describes merely exemplary embodiments of the present disclosure. As will be appreciated, the present disclosure is capable of other different embodiments, and its several details can be modified in various obvious respects, all without departing from the present disclosure. Thus, the drawings and description should be regarded as illustrative in nature, and not restrictive.

[0031] Incorporation by Reference

[0031] All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, it is intended that the specification take precedence and / or supersede any such conflicting material.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the invention are set forth with particularity in the appended claims. A further understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG.") in which: [Brief description of the drawings]

[0033] [Figure 1A]

[0033] An example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 1B]

[0033] An example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 1C]

[0033] An example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 1D]

[0033] An example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 1E]

[0033] An example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 2A]

[0034] 1 illustrates generally another example of a sensor for analyzing or identifying a target molecule. [Figure 2B]

[0034] Another example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 2C]

[0034] Another example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 2D]

[0034] Another example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Figure 2E]

[0034] Another example of a sensor for analyzing or identifying a target molecule is illustrated diagrammatically. [Diagram 3]

[0035] 1 illustrates a schematic diagram of an example sensor with modified channel height. [Figure 4A]

[0036] 1 illustrates a schematic diagram of an example of a sensor having an additional electric field generator. [Figure 4B]

[0036] An example of a sensor having an additional electric field generator is illustrated diagrammatically. [Figure 5A]

[0037] 1 illustrates a schematic diagram of an example of a sensor configured to detect the redox potential of a target molecule. [Figure 5B]

[0037] An example of a sensor configured to detect the redox potential of a target molecule is illustrated generally. [Figure 6]

[0038] 1 illustrates a computer system that is programmed or otherwise configured to implement the methods provided herein. [Figure 7]

[0039] 1 illustrates an exemplary process for analyzing or identifying a target molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description

[0040] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

[0035]

[0041] As used in this specification and claims, the singular forms "a," " "An" and "the" can include plural references unless the context clearly dictates otherwise. For example, the term "transmembrane receptor" can include a plurality of transmembrane receptors.

[0036]

[0042] The term "about" or "approximately" as used herein may refer to being within an acceptable error range for a particular value as determined by one of ordinary skill in the art, and the acceptable error will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within one standard deviation or more than one standard deviation, according to the conventions of the art. Alternatively, "about" may mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% from a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold. When a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise specified.

[0037]

[0043] The term "dielectric material" as used herein refers to an electrically insulating material that can be polarized by the action of an applied electric field. When a dielectric material is placed in an electric field, charge may not flow through the dielectric material. In some cases, in such an electric field, the dielectric material may exhibit dielectric polarization, where positive charges may move to positions along the electric field and negative charges may move in the opposite direction, creating an internal electric field that may partially cancel the external electric field inside the dielectric material. Examples of dielectric materials may include, but are not limited to, polyester, polyethylene, polypropylene, fabrics (such as nylon), paper, laminates, glass, self-assembled monolayers (SAMs), and the like.

[0038]

[0044] The terms "self-assembled monolayer" and "SAM" as used interchangeably herein refer to an ordered or relatively ordered collection (e.g., one or more layers) of molecules adsorbed on a surface (e.g., the surface of an electrode). The molecules in a SAM may be oriented approximately parallel to one another. The molecules in a SAM may be approximately perpendicular to the surface. Each of the molecules may include a functional group configured to adhere to the surface and a portion configured to interact (e.g., by hydrophobic interactions) with one or more neighboring molecules in the SAM to form a relatively ordered array. The functional group may be configured to covalently or non-covalently bind to the surface. In one example, the functional group may be a thiol that covalently couples to a metal surface, such as a gold surface. In some cases, a SAM may include a single molecular population or a mixed molecular population. The mixed molecular population may exhibit (expose) multiple additional functionalities (e.g., biological functionalities, binding moieties, etc.). In some embodiments, SAMs can be used in sensors (e.g., biosensors, chemical sensors, etc.) to detect molecules (e.g., small molecules, biological molecules, etc.) in a sample, such as a biological sample. Examples of SAMs include, but are not limited to, 3-mercaptopropyltrimethoxysilane (3MPT), 3-aminopropyltrimethoxysilane (APTES), p-aminophenyltrimethoxysilane (APTS), and 4-[2-(triethoxysilyl)ethyl]pyridine. Optionally, one or more conductive materials can be mixed in the SAM and in contact with the surface.

[0039]

[0045] The term "conductive material" as used herein may refer to any material capable of conducting electric current, metals (e.g., tungsten, titanium, tantalum, aluminum, copper) and non-metals (e.g., conductive small molecules or conductive polymers). Examples include polyaniline, polypyrrole, polythiophene (e.g., poly(3,4-ethylenedioxythiophene)), polyfuran, polyphenylene (e.g., poly(p-phenylenevinylene)), functional variants thereof, and combinations thereof. In some cases, the degree of polymerization of the conductive polymer may be in the range of 10 to 100,000 monomer units.

[0040]

[0046] The term "binding unit" as used herein generally refers to a molecule that has the ability to interact with (e.g., bind to) one or more target molecules. Such binding can be covalent and / or non-covalent (e.g., hydrogen bonds, hydrophobic interactions, etc.). The interaction between the binding unit and one or more target molecules can be reversible or irreversible. Alternatively or in addition, the binding unit can be configured to bind to a tag (or probe) coupled to the target molecule. The target molecule can be a biomolecule. The target molecule can be a cell or one or more components of a cell or derivatives thereof.

[0041]

[0047] The term "biomolecule" as used herein may refer to any molecule found in a biological system, its derivatives or its functional variants. Biological molecules may be naturally occurring or the result of an external perturbation of the system (e.g., disease, poisoning, genetic engineering, etc.), and may be synthetic analogs and derivatives thereof. Non-limiting examples of biological molecules may include amino acids (naturally occurring or synthetic), peptides, polypeptides, glycosylated and non-glycosylated proteins (e.g., polyclonal and monoclonal antibodies, receptors, interferons, enzymes, etc.), nucleosides, nucleotides, oligonucleotides (e.g., DNA, RNA, PNA oligos), polynucleotides (e.g., DNA, cDNA, RNA, etc.), carbohydrates, hormones, haptens, steroids, toxins, etc. Biological molecules may be isolated from natural sources or they may be synthetic.

[0042]

[0048] The term "cell" as used herein generally refers to a biological cell or cell derivative. A cell may be the basic structural, functional and / or biological unit of an organism. A cell may be derived from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, cereals, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, mosses), algae cells, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Natto, and the like). Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), These include algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cells are not derived from a natural organism (e.g., the cells can be synthetically produced cells, sometimes called artificial cells).

[0043]

[0049] The terms "nucleotide," "nucleobase," and "base," as used interchangeably herein, generally refer to a base-sugar-phosphate combination. A nucleotide can include synthetic nucleotides. A nucleotide can include synthetic nucleotide analogs. A nucleotide can be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and deoxyribonucleic acid (GTP). The nucleotides may include ribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, etc., or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules that include them. The term nucleotide as used herein generally refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. The nucleotides may be unlabeled or detectably labeled. Labeling may also be performed with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), cascade blue, Oregon green, Texas red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, available from Perkin Elmer, Foster City, Calif. Amersham, Arlington FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, and FluoroLink available from Heights, Ill. Cy3-dUTP and FluoroLink Cy5-dUTP; fluorescein-15-dATP, fluorescein-12- dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP and fluorescein-15-2'-dATP; and chromosome markers available from Molecular Probes, Eugene, Oreg. Nucleotides include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modification. Chemically modified single nucleotides can be biotin-dNTPs. Some non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0044]

[0050] The naturally occurring nucleotides guanine, cytosine, adenine, thymine and uracil may be abbreviated as G, C, A, T and U, respectively. Nucleotides may include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be specific for A, C, G, T or U, or one or more of the complementary A, C, G, T or U, or any other subunit that is complementary to a purine (i.e., A or G or variants thereof) or a pyrimidine (i.e., C, T or U or variants thereof). Subunits The nucleic acid sequence may allow for the resolution of individual nucleobases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA or their uracil counterparts).

[0045]

[0051] The terms "polynucleotide", "oligonucleotide", "oligomer" and "nucleic acid" as used interchangeably herein generally refer to a polymeric form of nucleotides (either deoxyribonucleotides or ribonucleotides) or analogs thereof of any length, in either single-stranded, double-stranded or multiple stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars or nucleobases). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein coupled to a sugar), thiol-containing nucleotides, biotin-coupled nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queousine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, one or more loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozymes, complementary DNA (cDNA, such as double-stranded cDNA (dd-cDNA) or single-stranded cDNA (ss-cDNA)), circulating tumor DNA (ctDNA), damaged DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes (e.g., fluorescent in situ hybridization (FISH) probes), and primers. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0046]

[0052] The term "gene" as used herein generally refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence involved in encoding an RNA transcript. The term "gene" as used herein in relation to genomic DNA can include intervening non-coding regions as well as regulatory regions, and can include the 5' and 3' ends. In some instances, the term encompasses the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will contain an "open reading frame" that encodes a polypeptide. In some instances, a "gene" includes only coding sequences (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. A gene may not encode a polypeptide, such as, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. The term "gene" not only includes the transcribed sequence, but can also include non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may be an "endogenous gene" or a native gene in its natural location in the genome of an organism. A non-native gene can be a "naturally occurring gene" or non-native gene. A non-native gene can be a gene that is not normally found in the host organism, but that is introduced into the host organism by gene transfer (e.g., a transgene). A non-native gene can be a naturally occurring nucleic acid or polypeptide sequence that contains mutations, insertions and / or deletions (e.g., a non-native sequence).

[0047]

[0053] The term "mutation" as used herein generally refers to a change in nucleotide sequence in a normally conserved nucleic acid sequence, resulting in the formation of a mutant as distinguished from a normal (unmodified) or wild-type sequence. The position (e.g., relative position to a gene or sample polynucleotide) and sequence of the mutation may not be determined prior to sequencing. Alternatively, the position (e.g., relative position to a gene or sample polynucleotide) and sequence of the mutation may be determined prior to sequencing, in which case sequencing may be performed to detect the presence or absence of a mutation in the sample polynucleotide. Mutations may include base pair substitutions (e.g., single nucleotide substitutions) and frameshift mutations. Frameshift mutations may require the insertion or deletion of one to several nucleotide pairs.

[0048]

[0054] The term "probe" as used herein generally refers to a nucleotide or polynucleotide tagged with a marker (e.g., a fluorescent marker) useful for detecting or identifying its corresponding target nucleotide or polynucleotide by hybridization with the corresponding target sequence in a hybridization reaction. The terms "nucleotide probe", "nucleotide tag" and "tagged nucleotide" as used interchangeably herein generally refer to a probe having a single nucleotide. The terms "polynucleotide probe", "polynucleotide tag" and "tagged polynucleotide" as used interchangeably herein generally refer to a probe having a polynucleotide. A polynucleotide probe may be tagged with at least one marker (e.g., one marker for each nucleotide of the polynucleotide probe). A probe may be hybridizable with one or more target nucleotides or polynucleotides. A polynucleotide probe may be fully complementary to one or more target polynucleotides in a sample or may contain one or more nucleotides that are not complementary (i.e., mismatches) to one or more nucleotides of one or more target polynucleotides in a sample.

[0049]

[0055] In some embodiments, the marker may be a redox species. The term "redox species" as used herein generally refers to a molecule or compound or a portion thereof (e.g., a molecular portion or functional group portion of a molecule or compound) that can be oxidized and / or reduced (i.e., "redox") upon or under electrical stimulation (e.g., upon or under application of an electric potential) or can undergo a faradaic reaction. In some instances, a redox species may include one or more molecular portions that accept and / or donate one or more electrons depending on its redox state. In some cases, a redox species may form part (e.g., a molecular portion) of a small molecule, compound, polymer molecule, or may exist as a separate molecule or compound. Examples of redox species include imidazolium, pyrrolidinium, tetraalkylammonium, [OTf]-, [FAP]-, [PF6]-, [BF4]-, [DCA]-, [NTf2]-, [FSI]-, [B(CN)4]-ferrocene (Fc), derivatives thereof, functional variants thereof, and combinations thereof. Examples of Fc derivatives include methylferrocene, dimethylferrocene, ethylferrocene, propylferrocene, n-butylferrocene, t-butylferrocene, and 1,1-dicarboxylate ferrocene.

[0050]

[0056] The same nucleotide may be tagged with the same marker. Alternatively, the same nucleotide may be tagged with different markers. For example, A first nucleotide A may be tagged with a first marker, and a second nucleotide A may be tagged with a second marker, where the first marker and the second marker are different. If the sensor can separately detect and distinguish the first marker and the second marker from each other, the use of multiple markers at the same nucleotide may aid in solving the sequence of identical nucleotides provided sequentially.

[0051]

[0057] The terms "complement," "multiple complements," "complementary," and "complementarity," as used interchangeably herein, generally refer to a sequence that is completely complementary to and can hybridize to a given sequence. A sequence that hybridizes to a given nucleic acid is said to be the "complement" or "reverse complement" of the given molecule if its base sequence over a given region has the ability to bind complementarily to the base sequence of its binding partner, for example, such that AT, AU, GC, and GU base pairs are formed. In general, a first sequence that can hybridize to a second sequence can specifically or selectively hybridize to the second sequence, i.e., hybridization to a second sequence or set of second sequences during a hybridization reaction is favored (e.g., more thermodynamically stable under a given set of conditions, such as stringent conditions commonly used in the art) compared to hybridization to a non-target sequence. Typically, hybridizable sequences share some degree of sequence complementarity over all or a portion of their respective lengths, such as from 25% to 100% complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence complementarity. Each length may comprise a region of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides.

[0052] For purposes of assessing percent complementarity, sequence identity may be determined using any of a variety of methods, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner, optionally with default settings, available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html), the BLAST algorithm (see, e.g., the EMBOSS Needle aligner, available at blast.ncbi.nlm.nih.gov / Blast.cgi), and the BLAST algorithm (see, e.g., the EMBOSS Needle aligner, available at blast.ncbi.nlm.nih.gov / Blast.cgi). Refer to the available BLAST alignment tools (optionally with default settings). Alignment can be measured by any suitable alignment algorithm, including the EMBOSS Water aligner (optionally with default settings), available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, or the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner, optionally with default settings, available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html). Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including default parameters.

[0053]

[0058] Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex where each base in the duplex binds to a complementary base according to Watson-Crick pairing rules. Substantial / sufficient complementarity can mean that the sequence of one strand is not entirely and / or completely complementary to the sequence of the opposite strand, but that sufficient binding occurs between the bases of the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by predicting the Tm of the hybridized strands using their sequences and standard mathematical calculations, or by empirically determining the Tm using routine methods.

[0054]

[0059] The term "hybridization" as used herein generally refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonding between the bases of the nucleotide residues, whether by Watson-Crick base pairing, Hoogsteen type bonding, or any other method based on base complementarity. The hybridization reaction may occur in a sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR or the enzymatic cleavage of a polynucleotide by an endonuclease. A second sequence that is complementary to a first sequence may be referred to as the "complement" of the first sequence. The term "hybridizable" as applied to polynucleotides generally refers to the ability of the polynucleotide to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues in a hybridization reaction.

[0055]

[0060] The term "target polynucleotide" as used herein generally refers to a nucleic acid molecule or polynucleotide in a population of nucleic acid molecules having a target sequence for which it is desired to determine the presence, amount and / or nucleotide sequence or one or more changes thereof. The term "target sequence" generally refers to a nucleic acid sequence on a single strand of nucleic acid. The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, ctDNA, RNA including mRNA, miRNA, rRNA, etc. The target sequence may be a target sequence from a sample or may be a secondary target, such as the product of an amplification reaction. The target polynucleotide may be a portion of a gene (or a fragment thereof) that contains one or more mutations.

[0056]

[0061] The term "target site" as used herein generally refers to a polynucleotide sequence that includes a target polynucleotide (or target nucleotide). The target polynucleotide (or target nucleotide) of a target site can be one or more sequence variants. Examples of one or more sequence variants can include single nucleotide changes, insertion or deletion of one or more nucleotides (e.g., consecutive or non-consecutive nucleotides), copy number variation (CNV) that includes one or more repeats of one or more nucleotides (e.g., CNVs with an average size of at least 1, 5, 10, 50, 100, 150, 200 kilobases (kb) or more; CNVs with an average size of up to 200, 150, 100, 50, 10, 5, 1 kb or less) and microsatellite instability (MSI). A target site may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000 nucleotides or more. A target site may contain up to 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides. A target site may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000 nucleotides or more relative to the target polynucleotide. A target site may contain up to 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide relative to the target polynucleotide. In some examples, a target site may be a target polynucleotide.

[0057]

[0062] The term "stringent conditions" as used herein generally refers to one or more hybridization conditions under which the nucleic acid having complementarity with target sequence mainly hybridizes with target sequence and does not substantially hybridize with non-target sequence.Stringent conditions can be sequence-dependent and can vary according to a number of factors.In some cases, the longer the sequence, the higher the temperature at which the sequence can specifically hybridize with its target sequence.

[0058]

[0063] As used herein, the term "recognition moiety" generally refers to a nucleic acid sequence, i.e., a nucleic acid sequence that is capable of interacting with a "recognition sequence" or "recognition site," such as a desired (or target) nucleic acid sequence. A recognition moiety refers to a molecule (e.g., a small molecule, a polynucleotide, a protein, variations thereof, or combinations thereof) having a specific biological activity, including, but not limited to, one or more of the following activities: a nuclease (e.g., a double-stranded nuclease), a nickase, a transcriptional activator, a transcriptional repressor, a nucleic acid methylase, a nucleic acid demethylase, and a recombinase. A recognition moiety may include a domain (e.g., a moiety that includes a domain) that has the ability to bind (e.g., hybridize) with a recognition sequence. Such a domain may include one or more amino acids, one or more nucleotides, variations thereof, or combinations thereof. Alternatively or in addition, a recognition moiety may associate with (e.g., bind to) a secondary molecule that includes such a domain. In some examples, a recognition moiety may include a nucleic acid molecule that has the ability to hybridize with a recognition sequence. In some examples, a recognition moiety may include a moiety that exhibits a specific biological activity, including, but not limited to, one or more of the following activities: a nuclease (e.g., a double-stranded nuclease), a nickase, a transcriptional activator, a transcriptional repressor, a nucleic acid methylase, a nucleic acid demethylase, and a recombinase. The recognition sequence may contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides. The recognition sequence may contain up to 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less nucleotides.

[0059]

[0064] The recognition moiety may be used to isolate a desired molecule comprising a desired nucleic acid sequence from a plurality of molecules (e.g., a plurality of nucleic acid molecules). The recognition moiety may be used to enrich a desired molecule comprising a desired nucleic acid in a composition or reaction mixture. In some examples, the recognition moiety may be captured by a capture system (e.g., magnetic beads) using one or more interactions (e.g., avidin-biotin binding, magnetic binding, etc.). In some examples, the recognition moiety may include biotin, which may form a complex with streptavidin magnetic beads for isolation or enrichment.

[0060]

[0065] Exemplary recognition moieties can include CRISPR-associated (Cas) systems (e.g., Cas proteins, including catalytically active or inactive Cas polypeptides); zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); Cas RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); variants thereof; and combinations thereof. The recognition moiety can include a polynucleotide (e.g., a sequence at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length) that can be captured by a capture system using one or more interactions, such as, for example, a polynucleotide sequence that can be captured by one or more avidin-functionalized magnetic beads tagged with biotin. At least a portion of the polynucleotide can share complementarity with a recognition sequence of a target nucleic acid molecule.

[0061]

[0066] The term "nickase" as used herein generally refers to a molecule (e.g., an enzyme) that cleaves one strand of a double-stranded nucleic acid molecule (i.e., "nicks" the double-stranded molecule). A nickase can be a nuclease that cleaves only a single DNA strand, either due to its natural function or because it has been engineered (e.g., modified by mutation and / or deletion of one or more nucleotides) to cleave only a single DNA strand. A nickase can be a nicking enzyme (e.g., a restriction endonuclease, a nicking endonuclease, etc.). A nickase can bind to a nicking site of a double-stranded nucleic acid molecule to create a nick (or gap) in one strand of the double-stranded nucleic acid molecule. A nick can occur within the nicking site. Alternatively, a nick can occur adjacent to the nicking site. Optionally, a nickase can bind to a nickase binding site adjacent to the nicking site. A nick can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, The nick may be up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide in length. Exemplary nickases include those of the Cas system (e.g., Cas nickases such as Cas9n), N.Alw I, Nb.BbvCl, Nt.BbvCl, Nb.BsmI, Nt.BsmAI, Nt.BspQl, Nb.BsrDI, Nt.BstNBI, Nb.BstsCI, Nt.CviPII, Nb.Bpu I OI, Nt.Bpu I OI, and Nt,Bst9I, variations thereof, and combinations thereof. In some examples, a nucleic acid molecule (e.g., a double-stranded nucleic acid molecule or a self-complementary single-stranded nucleic acid molecule) can include at least one nicking site that already contains at least one nick.

[0062]

[0067] The terms "CRISPR-associated system", "Cas system" and "Cas complex" as used interchangeably herein generally refer to a bicomponent ribonucleoprotein complex that includes a guide RNA (gRNA) and a Cas polypeptide or protein (e.g., a Cas endonuclease, catalytic or non-catalytic derivatives thereof, etc.) or other protein with endonuclease activity. The term "CRISPR" refers to clustered regularly interspaced short palindromic repeats and its associated systems. At least a portion of the gRNA may have complementarity to at least a portion of the target region. The target region may include a "protospacer" and a "protospacer adjacent motif" (PAM), and both domains may be required for the nuclease activity (e.g., cleavage) of the Cas polypeptide. The protospacer may also be referred to as a target site (or genomic target site). The gRNA may be paired (or hybridized) with the opposite strand (binding site) of the protospacer to guide the Cas polypeptide to the target region. A PAM site generally refers to a short sequence that is recognized by a Cas polypeptide and may in some cases be necessary for nuclease (or nickase) activity. The sequence and number of nucleotides in a PAM site may vary depending on the type of Cas enzyme.

[0063]

[0068] The Cas polypeptide may contain nuclease (or nickase) activity, and the gRNA may interact with the Cas polypeptide to direct the nuclease (or nickase) activity of the Cas polypeptide to a desired target region. Alternatively, the Cas polypeptide may be non-catalytic and may not contain nuclease activity. Non-catalytic Cas polypeptides are sometimes referred to as dead Cas or inactive Cas (dCas).

[0064]

[0069] Cas proteins may include proteins of CRISPR-associated type I, type II or type III systems or proteins derived therefrom that may have RNA-guided polynucleotide binding activity or nuclease activity. Examples of suitable Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (i.e., Csnl and Csxl2), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or Ca sC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, homologs thereof, and modified versions thereof (e.g., catalytic or non-catalytic). In some cases, the Cas protein can include a protein of a CRISPR-associated Type V or Type VI system or a protein derived therefrom, such as Cpf1 (or Cas12a), C2c1 (or Cas12b), C2c2, homologs thereof, and modified versions thereof (e.g., catalytic or non-catalytic).

[0065]

[0070] Although specific examples herein refer to Cas proteins, Other proteins with cascade enzyme activity can be used. Such other proteins do not have to be Cas proteins, but can be configured for use with, for example, a gRNA.

[0066]

[0071] Cas polypeptides or proteins can be engineered to convert nuclease activity to nickase activity. For example, Cas9 from S. pyogenes Substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain can convert Cas9 from a nuclease that cleaves both strands to a Cas9n nickase that cleaves only one strand. This Cas9n nickase mutant can introduce a single-strand break into DNA targeted by gRNA instead of the double-strand break created by wild-type Cas polypeptide. Other examples of mutations that turn Cas9 into a nickase include H840A, N854A, and N863A.

[0067]

[0072] The term "guide RNA (gRNA)" as used herein generally refers to an RNA molecule that can bind to a Cas polypeptide and serve to target the Cas polypeptide to a specific location within a target nucleic acid region (e.g., DNA or gene). The degree of complementarity between the gRNA and the specific location within the target nucleic acid region can be at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. A guide RNA can include a CRISPR RNA (crRNA) segment and a transactivating crRNA (tracrRNA) segment. The terms "crRNA" and "crRNA segment" as used interchangeably herein generally refer to an RNA molecule or a portion thereof that includes a polynucleotide targeting guide sequence, a stem sequence, and optionally a 5'-overhang sequence. The terms "tracrRNA" and "tracrRNA segment" as used interchangeably herein generally refer to an RNA molecule or a portion thereof that includes a protein-binding segment (e.g., a protein-binding segment capable of interacting with a CRISPR-associated protein such as Cas9). In some cases, the guide RNA may be a single guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located on the same RNA molecule. The gRNA may include one or more peptide nucleic acids.

[0068]

[0073] The crRNA may comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 RNA bases or more. The crRNA may comprise up to 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 RNA bases or less. The target nucleic acid sequence of the gRNA of the Cas system may comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 DNA bases or more. The target nucleic acid sequence of the gRNA of the Cas system may comprise up to 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 DNA bases or less. The crRNA sequence can be selected to target any target sequence. The target sequence can be a sequence within the genome of the cell. The target sequence can include those that are unique to the target genome.

[0069]

[0074] The term "polymerase" as used herein generally refers to an enzyme (e.g., natural or synthetic) capable of catalyzing a polymerization reaction. Examples of polymerases can include nucleic acid polymerases (e.g., DNA polymerases or RNA polymerases), transcriptases, and ligases. A polymerase can be a polymerizing enzyme. The term "DNA polymerase" generally refers to an enzyme capable of catalyzing a DNA polymerization reaction.

[0070]

[0075] As used herein, the term "linked polymerase" generally refers to a Refers to a polymerase, such as a DNA polymerase, that is coupled (e.g., fused) to a linker. The linker may have the ability to couple (e.g., bind or conjugate to) another entity (e.g., a nanopore, such as a protein nanopore or a solid-state nanopore).

[0071]

[0076] The terms "sequence variant" and "sequencing variant" as used interchangeably herein generally refer to any change in a sequence compared to one or more reference sequences. Typically, for a given population of individuals to which a reference sequence is provided, sequence variants occur less frequently than the reference sequence. For example, a particular bacterial genus may have a consensus reference sequence for the 16S rRNA gene, but an individual species within the genus may have one or more sequence variants within the gene or a portion of the gene that are useful in identifying the species in the bacterial population. As a further example, sequences of multiple individuals of the same species or multiple sequencing reads of the same individual may produce a consensus sequence when optimally aligned, and sequence variants of the consensus may be used to identify mutants that represent dangerous contaminations in the population. In general, a "consensus sequence" refers to a nucleotide sequence that reflects the most common base choice at each position in a sequence when a set of related nucleic acids is subjected to intensive mathematical and / or sequence analysis, such as optimal sequence alignment according to any of a variety of sequence alignment algorithms. The reference sequence may be a single reference sequence, such as a predetermined genomic sequence of a single individual. The reference sequence may be a consensus sequence formed by aligning multiple sequences, such as predetermined genomic sequences of multiple individuals serving as a reference population, or multiple sequencing reads of polynucleotides from the same individual. The reference sequence may be a consensus sequence formed by optimally aligning sequences from the sample under analysis, such that sequence variants represent changes compared to corresponding sequences in the same sample. The sequence variants may occur at low frequencies in a population (also referred to as "rare" sequence variants). For example, the sequence variants may occur at frequencies less than 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.1%, 0.075%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.001%, or less. Sequence variants may occur at a frequency of less than 0.1%.

[0072]

[0077] A sequence variant can be any change relative to a reference sequence. A sequence change can consist of a single nucleotide or multiple nucleotide changes, insertions or deletions, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. When a sequence variant contains two or more nucleotide differences, the different nucleotides can be contiguous or discontinuous with each other. Examples of types of sequence variants include single nucleotide polymorphisms (SNPs), deletion / insertion polymorphisms (DIPs), copy number variants (CNVs), short tandem repeats (STRs), simple sequence repeats (SSRs), highly variable repeats (VNTRs), amplified fragment length polymorphisms (AFLPs), retrotransposon-based insertion polymorphisms, sequence-specific amplified polymorphisms, and epigenetic mark differences (e.g., methylation differences) that can be detected as sequence variants.

[0073]

[0078] The term "sequencing" as used herein generally refers to a procedure that determines the order in which nucleotides appear in a target nucleotide sequence. Sequencing methods can include high-throughput sequencing, such as next-generation sequencing (NGS). Sequencing can be whole genome sequencing or targeted sequencing. Sequencing can be single molecule sequencing or massively parallel sequencing. Next-generation sequencing methods can be useful for obtaining millions of sequences in a single run. In some examples, sequencing can be performed using one or more nanopore sequencing methods, such as sequencing by synthesis, sequencing by ligation, or sequencing by cleavage.

[0074]

[0079] The term "nanopore" as used herein generally refers to a pore, channel, or passageway 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 of a polymeric material, such as a protein nanopore. The membrane may be a solid membrane (e.g., a silicon substrate). The nanopore may be disposed adjacent to or in close proximity to a sensing circuit or an electrode coupled to the sensing circuit, such as, for example, a complementary metal oxide semiconductor (CMOS) or field effect transistor (FET) circuit. The nanopore may be part of the sensing circuit. The nanopore may have a characteristic width or diameter, for example, on the order of about 0.1 nanometer (nm) to 1000 nm. The nanopore may be a biological nanopore, a solid-state nanopore, a hybrid biological-solid-state nanopore, variations thereof, or combinations thereof. Examples of biological nanopores include, but are not limited to, OmpG and OmpG pores from E. coli, sp., Salmonella sp., Shigella sp., and Pseudomonas sp. and alpha-hemolysin from S. aureus sp., smegma MspA from M. smegmatis sp., functional variants thereof or combinations thereof The sequencing may include forward sequencing and / or reverse sequencing. Examples of solid-state nanopores include, but are not limited to, silicon nitride, silicon oxide, graphene, molybdenum sulfide, functional variants thereof, or combinations thereof. Solid-state nanopores may be fabricated by high energy beam fabrication methods, imprinting (e.g., nanoimprinting), laser ablation, chemical etching, plasma etching (e.g., oxygen plasma etching), and the like.

[0075]

[0080] The term "nanopore sequencing complex" as used herein generally refers to a nanopore linked or coupled to an enzyme, e.g., a polymerase, which is then associated with a polymer, e.g., a polynucleotide template. The nanopore sequencing complex may be located in a membrane, e.g., a lipid bilayer, where it serves to identify polymer components, e.g., nucleotides or amino acids.

[0076]

[0081] The terms "nanopore sequencing" and "nanopore-based sequencing," as used interchangeably herein, generally refer to methods of determining the sequence of a polynucleotide using a nanopore. In some cases, the sequence of the polynucleotide may be determined in a template-dependent manner. In some cases, the methods, systems, or compositions disclosed herein may not be limited to any particular nanopore sequencing method, system, or device.

[0077]

[0082] The term "barcode" as used herein generally refers to a predetermined nucleic acid sequence that allows identification of some feature of the polynucleotide with which the barcode is associated (e.g., a polynucleotide that includes at least a portion of the barcode or has complementarity with at least a portion of the barcode). In some examples, the feature of the polynucleotide identified may be the original sample from which the polynucleotide originates. The barcode may be at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length or more. The barcode may be up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides in length. The barcode associated with a polynucleotide from a first sample may be different (e.g., different sequence and / or different length) from the barcode associated with a polynucleotide from a second sample that is different from the first sample. In that case, identifying the barcode of each polynucleotide can facilitate identifying one or more sample sources of the polynucleotides. Thus, different samples with different barcodes can be analyzed (e.g., sequenced) together (e.g., in a batch) and separated during analysis, at least in part, based on the barcode. In some examples, the barcodes can indicate mutations, insertions, or deletions of one or more nucleotides in the barcode sequence. can be accurately identified even after deletion (e.g., mutation, insertion or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides). Multiple polynucleotides from the same sample may have the same barcode. Alternatively, multiple polynucleotides from the same sample may have different barcodes. A first barcode may differ from a second barcode by at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide positions. Multiple barcodes may appear in a pool of samples, with each sample containing polynucleotides containing one or more barcodes that are different from the barcodes contained in polynucleotides from other samples in the pool. Samples of polynucleotides containing one or more barcodes may be pooled based on the barcode sequence to which it is spliced, such as all four nucleotide bases A, G, C and T appearing approximately evenly at one or more positions along each barcode in the pool (such as 1, 2, 3, 4, 5, 6, 7, 8 or more positions or all positions of the barcode). In some examples, the disclosed method may include identifying the original sample from which the target polynucleotide originates based on the barcode sequence to which the target polynucleotide is spliced. The barcode may include a nucleic acid sequence that, when spliced ​​to the target polynucleotide, may serve as an identifier of the original sample from which the target polynucleotide originates. In one example, an oligonucleotide primer (e.g., an amplification primer) may include one or more barcodes. In another example, a nucleic acid molecule may be coupled (e.g., ligated) to an adaptor nucleic acid (e.g., for circularization), and the adaptor nucleic acid may include one or more barcodes.

[0078]

[0083] The term "sample" as used herein generally refers to any sample that may contain one or more components (e.g., nucleic acid molecules) for processing or analysis. The sample may be a biological sample. The sample may be a cell sample or a tissue sample. The sample may be an acellular sample, such as blood (e.g., whole blood), plasma, serum, sweat, saliva, or urine. The sample may be obtained in vivo or cultured in vitro.

[0079]

[0084] The term "subject" as used herein generally refers to the individual or entity from which the sample is derived, for example, a vertebrate (e.g., a mammal such as a human) or an invertebrate. A mammal can be a mouse, a monkey, a human, an agricultural animal (e.g., a cow, a goat, a pig, or a chicken), or a pet (e.g., a cat or a dog). A subject can be a plant. A subject can be a patient. A subject can be asymptomatic with respect to a disease (e.g., cancer). Alternatively, a subject can be symptomatic with respect to a disease.

[0080]

[0085] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first number in a series of two or more numbers, the term "at least," "greater than," or "greater than or equal to" applies to each and every number in the series. For example, 1, 2, or 3 or more is equal to 1 or more, 2 or more, or 3 or more.

[0081]

[0086] Whenever the first number in a series of two or more numerical values ​​is preceded by the terms "not greater than," "less than," or "equal to or less than," the terms "not greater than," "less than," or "equal to or less than" apply to each of the numerical values ​​in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0082]

[0087] I. Systems and methods for analysis and detection of target molecules

[0088] In certain embodiments, the present disclosure provides a system for analyzing or identifying a target molecule. The system may include a sensor configured to detect one or more signals indicative of an impedance or impedance change of the sensor when at least a portion of the target molecule is bound by or in proximity to at least a portion of the sensor. The one or more signals may be usable to analyze or identify the target molecule.

[0083]

[0089] The system may include at least one of the sensors disclosed herein. The system may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more sensors. The system may include up to 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less sensors.

[0084]

[0090] The detected signal indicative of the impedance or impedance change of the sensor induced by the target molecule can be a single measurement, or alternatively, the detected signal can be the median or average of multiple measurements.

[0085]

[0091] Upon detection of one or more signals indicative of the impedance or impedance change of the sensor, at least a portion of the target molecule may bind to the binding moiety of the sensor. The binding moiety may be configured to bind to at least a portion of the target molecule (e.g., nucleotides, amino acids, small molecules, ions, etc.). The sensors disclosed herein may include at least one binding moiety. The sensors may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more binding moieties. The sensors may include up to 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less sensors.

[0086]

[0092] 1A-1E illustrate schematic examples of systems including sensors of the present disclosure, for example, to evaluate (e.g., analyze, identify, quantify, rank, etc.) one or more target molecules. With reference to FIG. 1A, the sensor may be immersed in a liquid 105 (e.g., water or a buffer, such as saline, high electrolyte buffer, low electrolyte buffer, etc.). The sensor may include a sensing electrode 110 in electrical communication with a reference electrode 115. The sensor may be configured to detect or measure one or more LCR (inductance (L), capacitance (C), and resistance (R)) properties of a volume 112 of the liquid 105, the volume 112 being in the vicinity or adjacent to the sensing electrode 110. The target molecules (labeled 102-1, 120-2, and 120-3) may be present or suspended in the liquid. The target molecules may or may not be coupled to a probe (e.g., a fluorescent tag or a redox species). Referring to FIG. 1B, when at least a portion of any of the target molecules (e.g., target molecule 120-1) is adjacent to or enters volume 112 of liquid 105, one or more signals indicative of the impedance or impedance change of volume 112 can be detected by a sensor.

[0087]

[0093] In some cases, impedance or impedance change can be detected by measuring the current (e.g., change in current) while applying a constant voltage (e.g., a sinusoidal voltage perturbation). The impedance value (Z) can be measured by dividing the value of the applied voltage (V) by the value of the measured current (I).

[0088]

[0094] For example, the detected impedance or impedance change between the sensing electrode 110 and the reference electrode 115 can be at least about 1 microohm, 2 microohms, 3 microohms, 4 microohms, 5 microohms, 6 microohms, 7 microohms, 8 microohms, 9 microohms, 10 microohms, 20 microohms, 30 microohms, 40 microohms, 50 microohms, 60 microohms, 70 microohms, 80 microohms, 90 microohms, 10 ... Ohms, 200 microohms, 300 microohms, 400 microohms, 500 microohms, 600 microohms, 700 microohms, 800 microohms, 900 microohms, 1 milliohm, 2 milliohms, 3 milliohms, 4 milliohms, 5 milliohms, 6 milliohms, 7 milliohms, 8 milliohms, 9 milliohms, 10 milliohms, 20 milliohms, 30 milliohms ohms, 40mOhms, 50mOhms, 60mOhms, 70mOhms, 80mOhms, 90mOhms, 100mOhms, 200mOhms, 300mOhms, 400mOhms, 500mOhms, 600mOhms, 700mOhms, 800mOhms, 900mOhms, 1 Ohm, 2 Ohms, 3 Ohms, 4 Ohms, 5 Ohms, 6 Ohms, 7 Ohms, 8 Ohms, 9 Ohms, 10 ohms, 20 ohms, 30 ohms, 40 ohms, 50 ohms, 60 ohms, 70 ohms, 80 ohms, 90 ohms, 100 ohms, 200 ohms, 300 ohms, 400 ohms, 500 ohms, 600 ohms, 700 ohms, 800 ohms, 900 ohms, 1k ohms, 2k ohms, 3k ohms, 4k ohms, 5k ohms, 6k ohms, 7k ohms, 8k ohms, 9k ohms The impedance may be 10 kOhms, 20 kOhms, 30 kOhms, 40 kOhms, 50 kOhms, 60 kOhms, 70 kOhms, 80 kOhms, 90 kOhms, 100 kOhms, 200 kOhms, 300 kOhms, 400 kOhms, 500 kOhms, 600 kOhms, 700 kOhms, 800 kOhms, 900 kOhms, 1,000 kOhms or more.For example, the detected impedance or impedance change between the sensing electrode 110 and the reference electrode 115 may be up to about 1,000 kilohms, 900 kilohms, 800 kilohms, 700 kilohms, 600 kilohms, 500 kilohms, 400 kilohms, 300 kilohms, 200 kilohms, 100 kilohms, 90 kilohms, 80 kilohms, 70 kilohms, 60 kilohms, 50 kilohms, 40 kilohms, 30 kilohms, 20 kilohms, 10 kilohms, 9 kilohms, 8 kilohms, 7 kilohms, 8 kilohms, 9 kilohms, 10 ... Lo ohms, 6 kOhms, 5 kOhms, 4 kOhms, 3 kOhms, 2 kOhms, 1 kOhm, 900 Ohms, 800 Ohms, 700 Ohms, 600 Ohms, 500 Ohms, 400 Ohms, 300 Ohms, 200 Ohms, 100 Ohms, 90 Ohms, 80 Ohms, 70 Ohms, 60 Ohms, 50 Ohms, 40 Ohms, 30 Ohms, 20 Ohms, 10 Ohms, 9 Ohms, 8 Ohms, 7 Ohms, 6 Ohms, 5 Ohms, 4 Ohms, 3 Ohms, 2 Ohms, 1 Ohm, 900 MilliOhms, 800 MilliOhms, 700 MilliOhms , 600 milliohms, 500 milliohms, 400 milliohms, 300 milliohms, 200 milliohms, 100 milliohms, 90 milliohms, 80 milliohms, 70 milliohms, 60 milliohms, 50 milliohms, 40 milliohms, 30 milliohms, 20 milliohms, 10 milliohms, 9 milliohms, 8 milliohms, 7 milliohms, 6 milliohms, 5 milliohms, 4 milliohms, 3 milliohms, 2 milliohms, 1 milliohm, 900 microohms, 800 microohms, 700 microohms, 600 microohms, 5 The impedance may be 00 microohms, 400 microohms, 300 microohms, 200 microohms, 100 microohms, 90 microohms, 80 microohms, 70 microohms, 60 microohms, 50 microohms, 40 microohms, 30 microohms, 20 microohms, 10 microohms, 9 microohms, 8 microohms, 7 microohms, 6 microohms, 5 microohms, 4 microohms, 3 microohms, 2 microohms, 1 microohm or less.

[0089]

[0095] For example, the detected impedance or impedance change between the sensing electrode 110 and the reference electrode 115 may be greater than or equal to at least about 1 nanosecond, 2 nanoseconds, 3 nanoseconds, 4 nanoseconds, 5 nanoseconds, 6 nanoseconds, 7 nanoseconds, 8 nanoseconds, 9 nanoseconds, 10 nanoseconds, 20 nanoseconds, 30 nanoseconds, 40 nanoseconds, 50 nanoseconds, 60 nanoseconds, 70 nanoseconds, 80 nanoseconds, 90 nanoseconds, 100 nanoseconds, 200 nanoseconds, 300 nanoseconds, 400 nanoseconds, 500 nanoseconds, 600 nanoseconds, 700 nanoseconds, 800 nanoseconds, 900 nanoseconds, 1 microsecond, 2 microseconds, 3 microseconds, 4 microseconds, 5 microseconds, 6 microseconds, 7 microseconds, 8 microseconds, 9 microseconds, 10 microseconds, 20 microseconds, 30 microseconds, 40 microseconds, 50 microseconds, 60 microseconds, 70 microseconds, 80 microseconds, 90 microseconds, 100 microseconds, 200 microseconds, 300 microseconds, 400 microseconds, 500 microseconds, 600 microseconds, 700 microseconds, 800 microseconds, 900 microseconds, 1 millisecond, 2 milliseconds, 3 milliseconds, 4 milliseconds, 5 milliseconds, 6 milliseconds, 7 milliseconds, 8 milliseconds, 9 milliseconds, 10 milliseconds The measurements may be taken over a period of time (e.g., a single measurement, multiple measurements to average multiple measurements) of 1, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more seconds.For example, the detected impedance or impedance change between the sensing electrode 110 and the reference electrode 115 may be greater than or equal to about 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, 900 milliseconds, 800 milliseconds, 700 milliseconds, 600 milliseconds, 500 milliseconds, 400 milliseconds, 300 milliseconds, 200 milliseconds, 100 milliseconds, 90 milliseconds, 80 milliseconds, 70 milliseconds, 60 milliseconds, 50 milliseconds, 40 milliseconds, 30 milliseconds, 20 milliseconds, 10 milliseconds, 9 milliseconds, 8 milliseconds, 7 milliseconds, 6 milliseconds, 5 milliseconds, 4 milliseconds, 3 milliseconds, 2 milliseconds, 1 millisecond, 900 microseconds, 800 microseconds, 700 microseconds, 600 microseconds, 500 microseconds, 400 microseconds, 300 microseconds, 200 microseconds, 100 microseconds, 90 microseconds, 80 microseconds, , 70 microseconds, 60 microseconds, 50 microseconds, 40 microseconds, 30 microseconds, 20 microseconds, 10 microseconds, 9 microseconds, 8 microseconds, 7 microseconds, 6 microseconds, 5 microseconds, 4 microseconds, 3 microseconds, 2 microseconds, 1 microsecond, 900 nanoseconds, 800 nanoseconds, 700 nanoseconds, 600 nanoseconds, 500 nanoseconds, 400 nanoseconds, 300 nanoseconds, 200 nanoseconds, 100 nanoseconds, 90 nanoseconds, 80 nanoseconds, 70 nanoseconds, 60 nanoseconds, 50 nanoseconds, 40 nanoseconds, 30 nanoseconds, 2 nanoseconds, 1 nanosecond or less (e.g., a single measurement, multiple measurements to average multiple measurements).

[0090]

[0096] Referring to FIG. 1A and FIG. 1B, an electric field may be applied between the sensing electrode and the reference electrode. Optionally, a function generator may be operatively coupled to the sensor, which may apply a voltage (e.g., a small amplitude sinusoidal voltage), which may generate a current in the circuit of the sensor. The current, as well as the amplitude and phase (of the alternating current (i.e., ac) component) may then be measured. Such values ​​may be converted into one or more signals indicative of the impedance or impedance change of the sensor. The measurements may be repeated at a series of different voltages (or frequencies), thus accumulating an impedance spectrum of the sensor. Optionally, an LCR meter may be operatively coupled to the sensor, which may measure one or more of the LCR signals or signatures of the sensor when a target molecule comes into contact (e.g., directly or indirectly) with or in the vicinity of the sensing electrode.

[0091]

[0097] Referring to FIG. 1C, the sensing electrode 110 of the sensor may be disposed adjacent to a substantially flat and planar surface 130. Referring to FIG. 1D, the sensing electrode 110 of the sensor may be disposed within a well 135, for example, at a bottom portion of the well 135. The well 135 may be a closed-bottom well. Optionally, at least a portion of the surface of the well (e.g., the portion of the surface of the well that is not covered by the sensing electrode) may be covered with a dielectric material (e.g., an insulating material). Alternatively, the surface of the well may not be covered with a dielectric material. The well may facilitate localizing or capturing the target molecule within the well for a longer period of time and / or with a higher binding affinity, thereby improving the accuracy and sensitivity of the sensor. Optionally, (i) a first target molecule may be captured in the well and a first signal indicative of the impedance or impedance change of the sensor may be measured, and (ii) Subsequent target molecules may be captured in the well and additional signals indicative of the impedance or impedance change of the sensor may be measured. During (ii), the first target molecule may remain captured in the well. For example, the target molecule may bind to a substrate disposed in the well. Alternatively, the first target molecule may not be captured in the well during (ii). For example, the first target molecule may exit the well prior to (ii). Referring to FIG. 1E, the sensing electrode 110 of the sensor may be disposed under a nanopore (e.g., a protein nanopore or a solid-state nanopore). In this example, the sensing electrode 110 may be disposed under a protein nanopore 140 embedded in a lipid bilayer 145. The lipid bilayer 145 may or may not have a capacitance. The nanopore 140 may be configured to function as a charge and / or size filter, thus controlling what passes through the nanopore 140 into the well 135 and induces a change in the LCR characteristic of the sensor. The size of the nanopore 145 may be adjustable. The overall charge of the nanopore 145 may be adjustable. In one example, applying a positive electrical signal to the nanopore 145 may help filter out positively charged molecules and allow only negatively charged molecules to enter and interact with the sensing electrode 110. In some cases, a sensor as shown in FIG. 1E may detect one or more signals indicative of impedance or impedance change within the sensor after (i) a target molecule (or a tag originally coupled to the target molecule) passes through the nanopore 140 and (ii) the passed target molecule (or tag) couples (e.g., directly or indirectly) to the sensing electrode 110. Such one or more signals may not be detected while the target molecule (or tag) is within the nanopore 140. In one example, upon detection of one or more signals, at least a portion of the target molecule (or tag) may be coupled to the sensing electrode (e.g., via a linker such as a conductive material). In another example, upon detection of one or more signals, at least a portion of the target molecule (or tag) may be in proximity to a conductive material.

[0092]

[0098] In another aspect, the present disclosure provides a system for analyzing or identifying a target molecule. The system may include a sensor including a sensing electrode and a reference electrode in electrical communication with each other. The sensor may include a dielectric material coupled to the sensing electrode and covering a first portion of a surface of the sensing electrode. The sensor may include a conductive material coupled to the sensing electrode and covering a second portion of a surface of the sensing electrode. The sensor may include a binding unit coupled to the conductive material, the binding unit configured to bind to the target molecule. The sensor may be configured to detect one or more signals indicative of an impedance or impedance change of the sensor when at least a portion of the target molecule is bound to the binding unit. The one or more signals may be usable to analyze or identify the target molecule. The conductive material may be a bond (e.g., a chemical bond) or may include a linking unit (e.g., a nanorod, a peptide, a small molecule, etc.) of any desired dimension (e.g., length, cross-sectional diameter or area, volume, etc.). In an alternative aspect, the binding unit may be directly coupled to the sensing electrode. In yet another embodiment, the binding unit can be coupled to at least a portion of the dielectric material that is coupled to the sensing electrode.

[0093]

[0099] The one or more signals may be indicative of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, or (ii) the electrical inductance of the sensor or a change therein. The one or more signals may be indicative of at least two of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, and (ii) the electrical inductance of the sensor or a change therein. The one or more signals may be indicative of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, or (ii) the electrical inductance of the sensor and a change therein.

[0094]

[0100] The one or more signals may be a current or a voltage. The one or more signals may be a current and a voltage. The one or more signals do not have to be tunneling currents.

[0095]

[0101] The first portion of the sensing electrode that is covered by the dielectric material can be at least 50 percent (%) of the surface of the sensing electrode. In some cases, the first portion of the sensing electrode can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the surface of the sensing electrode. In some cases, the first portion of the sensing electrode can be up to 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or less of the surface of the sensing electrode.

[0096]

[0102] The second portion of the sensing electrode may be up to 50% of the surface of the sensing electrode. In some cases, the second portion of the sensing electrode may be up to 50%, 45%, 0%, 35%, 0%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the surface of the sensing electrode. In some cases, the second portion of the sensing electrode may be at least 1%, 2%, 3%, 4%, 5%, %, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the surface of the sensing electrode.

[0097]

[0103] The average cross-sectional dimension of the sensing electrode (or the surface area coupled to the dielectric and conductive materials) can be 100 times or less than the average size of the target molecules. In some cases, the average cross-sectional dimension of the sensing electrode can be up to 100 times, 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 25 times, 20 times, 15 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times, 1 times, 0.5 times, or 0.1 times the average size of the target molecules. In some cases, the average cross-sectional dimension of the sensing electrode can be at least 0.1 times, 0.5 times, 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times the average size of the target molecules. In some cases, the average cross-sectional dimension of the sensing electrode can be at least 0.1 nanometers (nm), 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1,000 nm, 5,000 nm, 10,000 nm or more less than the average size of the target molecules. In some cases, the average cross-sectional dimension of the sensing electrode can be at most 10,000 nm, 5,000 nm, 1,000 nm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0.1 nm or less less than the average size of the target molecules.

[0098]

[0104] Alternatively, the average cross-sectional dimension of the sensing electrode can be smaller than the average size of the target molecules. In some cases, the average cross-sectional dimension of the sensing electrode can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% smaller than the average size of the target molecules. In some cases, the average cross-sectional dimension of the sensing electrode can be up to 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% smaller than the average size of the target molecules.

[0099]

[0105] The area of ​​the second portion of the surface of the sensing electrode is 500 square angstroms (Å 2 ), 100 Å2 , 50 Å 2 ,10Å 2 , 9 Å 2 , 8 Å 2 , 7 Å 2 , 6 Å 2 , 5 Å 2 , 4 Å 2 , 3 Å 2 , 2 Å 2 , 1 Å 2 Optionally, the cross-sectional dimension or diameter of the second portion of the surface of the sensing electrode can be approximately equal to the diameter of an atom of the conductive material (e.g., twice the van der Waals radius).

[0100]

[0106] Dielectric materials may be solid layers (e.g., solid metal or semiconductor materials) or self-assembled monolayers. The SAM may be a supramolecular membrane.

[0101]

[0107] The conductive material can be a single molecule (e.g., a single conductive polymer chain). Optionally, one or more features of an atomic force microscopy (AFM) (e.g., a piezoelectric cantilever probe of an AFM) for coupling a single molecule to a specific (or random) location within the surface of the sensing electrode. Alternatively, the conductive material can be a plurality of molecules (e.g., a plurality of identical and / or different conductive polymer chains). The conductive material can include at least 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000 or more molecules. The conductive material can include up to 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, 50, 10, 5 or 1 molecule.

[0102]

[0108] In some cases, a substrate can be bound to a binding unit (e.g., a nuclease or variant thereof) of a sensor of the present disclosure, and the binding unit or an additional element (e.g., an additional enzyme) can be configured to cleave at least a portion of the substrate. The sensor can be configured to (i) detect a difference in one or more signals indicative of an impedance change of the sensor following such cleavage, and (ii) identify what has been cleaved based on an analysis of the one or more signals.

[0103]

[0109] The binding unit may be an enzyme, an antibody, an aptamer, a non-biological material (e.g., a synthetic polymer), a functional fragment thereof, a functional variant thereof, or a combination thereof. The enzyme may be selected from the group consisting of a polymerase, a nuclease (e.g., a double-stranded nuclease), a nickase, a transcription activator, a transcription repressor, a nucleic acid methylase, a nucleic acid demethylase, and a recombinase. The antibody may be a whole antibody or an antigen-binding fragment thereof, such as an scFv, a Fab fragment, a VHH domain, or a VH domain of a heavy chain-only antibody. The antibody may be monospecific or multispecific (e.g., bispecific, trispecific, etc.). The antibody may be monovalent or multivalent (e.g., bivalent, trivalent, etc.).

[0104]

[0110] The binding unit can be directly coupled to the conductive material, for example, covalently or non-covalently attached to the conductive material. Alternatively, the binding unit can be indirectly coupled to the sensing electrode, for example, via a linker that binds the conductive material on one side and the binding unit on the other side. In such a case, the linker can also be a conductive linker, so as to minimize interference with the sensing ability of the sensor. Alternatively, the linker can be non-conductive.

[0105]

[0111] The target molecule may be selected from the group consisting of a small molecule, a nucleotide, a polynucleotide, an amino acid, a peptide, a polypeptide, variants thereof, and combinations thereof.

[0106]

[0112] A target molecule may include one or more tags. A target molecule may include at least 1, 2, 3, 4, 5 or more tags. A target molecule may include up to 5, 4, 3, 2 or 1 tags. The tags may be configured to induce a change in one or more signals of a sensor. In some cases, multiple types of tags may be used for multiple target molecules, and one or more signals of each of the target molecules having their respective tags may be substantially distinguishable from the others. In some cases, one or more features or characteristics of the target molecule (e.g., size, shape, charge, vibration, movement in a fluid, etc.) may further affect one or more signals of a sensor, thereby making one or more signals of each of the target molecules having their respective tags more distinguishable from the others. In some cases, the tag of the present disclosure may be an impedance tag. The impedance tag may be configured to induce a change in the impedance detected of a sensor, for example, between a sensing electrode and a reference electrode. Examples of impedance tags include, but are not limited to, organic compounds, organometallic compounds, nanoparticles, metals, functional variants thereof, and the like. The combination of these can be mentioned.

[0107]

[0113] Alternatively, the target molecule may not include any tag. In such a case, one or more features or characteristics of the target molecule (e.g., size, shape, charge, vibration, movement in fluid, etc.) may further affect one or more signals of the sensor, thereby making one or more signals of each of the target molecules with its respective tag more distinguishable from others. In an example, the binding unit may be a protein in a biological sample such as blood, plasma, or urine. The binding unit may be an enzyme (e.g., a polymerase configured to perform RCA) that binds to a circular nucleic acid for rolling circle amplification (RCA). The target moiety may be a nucleic acid base without any fluorescent tag or redox species tag, but the sensitivity of the sensor of the present disclosure may have the ability to analyze one or more signals that indicate the impedance or impedance change of the sensor when a nucleotide is bound to the circular nucleic acid by a polymerase.

[0108]

[0114] The size, shape and / or resolution of one or more components of a sensor of the present disclosure may be defined or limited by the resolution of a technique, such as photolithography, etching, nanoimprinting, size of a bionanopore, etc.

[0109]

[0115] 2A-2E illustrate schematic examples of systems including sensors of the present disclosure, for example, to evaluate (e.g., analyze, identify, quantify, rank, etc.) one or more target molecules. With reference to FIG. 2A, the sensor may be immersed in a liquid 105 (e.g., water or a buffer solution, such as saline, high electrolyte buffer, low electrolyte buffer, etc.). The sensor may include a sensing electrode 110 in electrical communication with a reference electrode 115. The sensor may include a dielectric material (e.g., a SAM) 205 coupled to at least a top surface of the sensing electrode 110. The SAM 205 may cover most of the top surface of the sensing electrode 110. In addition, a conductive material 210 (e.g., one or more conductive polymer chains) may be coupled to the top surface of the sensing electrode 110. The conductive material 210 may be embedded in a "sea" of the SAM 205. A binding moiety 215 may also be coupled to the conductive material 210 on the opposite (or end) side of the sensing electrode 110. The sensor may be configured to detect or measure one or more LCR properties or changes therein within a volume 217 of liquid 105, which is in the vicinity or adjacent to a binding unit 215. In this example, the binding unit 215 may be an enzyme (e.g., a polymerase), which may be configured to add one or more nucleotides to a polynucleotide substrate 220.

[0110]

[0116] The thickness of the dielectric material (e.g., SAM 205 as shown in FIG. 2A) coupled to at least a portion of the surface of the sensing electrode is at least about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, m, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1,000 μm or more. The thickness of the dielectric material coupled to at least a portion of the surface of the sensing electrode may be at most about ,000 μm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, It may be 30nm, 20nm, 10nm, 9nm, 8nm, 7nm, 6nm, 5nm, 4nm, 3nm, 2nm, 1nm, 0.5nm, 0.1nm or less.

[0111]

[0117] With reference to FIG. 2B, the target molecule 230 may be three nucleotide probes (labeled 1, 2, and 3) suspended in the liquid 105. With reference to FIG. 2C, a binding unit 215 (e.g., a polymerase) coupled to the sensing electrode 110 and embedded in a SAM (not shown) may interact with the target nucleotide probe 230-1 and couple the target nucleotide probe to the nucleotide substrate 220. The sensor may measure one or more signals indicative of the impedance or impedance change of the sensor when (i) the nucleotide probe 230-1 containing the tag binds to the binding unit 215 and / or (ii) the binding unit 215 or an additional enzyme operably coupled to the binding unit 215 cleaves the tag from the nucleotide probe 230-1. In some cases, the impedance or impedance change as measured may be a bulk impedance or impedance change. In some cases, the impedance or impedance change detected is not in the Debye layer (or bilayer) of the electrode, for example the sensor electrode 110 as shown in FIG. 2C. In some examples, the impedance or impedance change detected may be outside of the Debye layer of the electrode. In some examples, the impedance or impedance change detected is not in the Debye layer (or bilayer) of the dielectric material (e.g., SAM 205 as shown in FIG. 2A) that is coupled to the sensing electrode. In some examples, the impedance or impedance change detected may be outside of the Debye layer of the dielectric material.

[0112]

[0118] Referring to FIG. 2D, the target molecule 230 may be four nucleotide probes (labeled 240-1, 240-2, 240-3, and 240-4) suspended in the liquid 105, where the nucleotide probes have the same nucleotide base (e.g., adenine, i.e., A), but different tags, as depicted by the different shaped tags. At a given position (i.e., t=N) along the length of the nucleotide substrate 220, a binding unit 215 (e.g., a polymerase) coupled to the sensing electrode 110 and embedded in a SAM (not shown) may interact with the target nucleotide probe 240-4 (e.g., by random selection based on the free movement of the nucleotide probe in the liquid 105) to couple the target nucleotide probe 240-4 to the nucleotide substrate 220. The sensor may then detect one or more signals (e.g., a capacitance vs. resistance plot) indicative of the impedance or impedance change of the sensor due to the addition of the nucleotide probe 240-4 to the substrate 220. Similarly, for the next position of the nucleotide substrate 220 (i.e., t=N+1), when the substrate again recruits the same nucleobase as before, the binding unit 215 may interact (e.g., by random selection) with another target nucleotide probe 240-1 to couple the target nucleotide probe 240-1 to the nucleotide substrate 220. The sensor may then detect one or more signals (e.g., a capacitance versus resistance plot) indicative of the impedance or impedance change of the sensor due to the addition of the nucleotide probe 240-1 to the substrate 220.

[0113]

[0119] In some cases, the sensor may be configured to detect one or more signals indicative of, e.g., an impedance or impedance change between the sensing electrode 110 and the reference electrode 115, when at least a portion of the target molecule (and / or a tag coupled to the target molecule) is bound (e.g., directly or indirectly via the binding unit 215 and the conductive material 210) to at least a portion of the sensor, e.g., the sensing electrode 110. Alternatively, the sensor may detect, e.g., an impedance or impedance change between the sensing electrode 110 and the reference electrode 115, when at least a portion of the target molecule (and / or a tag coupled to the target molecule) is not bound to, but is in proximity to, at least a portion of the sensor, e.g., the sensing electrode 110. The sensor may be configured to detect one or more signals indicative of a change in impedance.

[0114]

[0120] The sensors of the present disclosure may have a distance between (i) at least a portion of the target molecule (and / or a tag coupled to the target molecule) and (ii) the sensing electrode that is at least about 0.1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 90 For example, at 0 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1,000 μm or more, it may be configured to detect even more signals indicative of impedance or impedance changes between the sensing electrode and the reference electrode. Sensors as disclosed herein may have a distance between (i) at least a portion of a target molecule (and / or a tag coupled to a target molecule) and (ii) a sensing electrode that is at most about 1,000 μm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, For example, the sensing electrode may be configured to detect a signal indicative of an impedance or impedance change between the sensing electrode and the reference electrode when the impedance is less than or equal to 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0.1 nm or less.

[0115]

[0121] The sensors of the present disclosure may be further configured to detect a signal indicative of impedance or impedance change, for example, between a sensing electrode and a reference electrode, when a target molecule (and / or a tag coupled to the target molecule) is within a predetermined space (e.g., a predetermined volume 217 as shown, for example, in FIG. 2A) that is near or adjacent to the sensing electrode. The predetermined space may be at least about 0.1 nm 2 , 0.5 nm 2 , 1nm, 2nm 2 , 3 nm 2 , 4nm 2 , 5nm 2 , 6nm 2 , 7nm 2 , 8nm 2 , 9nm 2 , 10 nm 2 , 20nm 2 , 30nm 2 , 40nm 2 , 50 nm 2 , 60nm 2 , 70nm 2 , 80nm 2 , 90nm 2 , 100 nm 2 , 200 nm 2 , 300 nm 2 , 400nm 2 , 500 nm 2 , 600nm 2 , 700nm 2 , 800nm 2 , 900nm 2 , 1 μm 2 , 2 μm 2 , 3 μm 2 , 4 μm 2 , 5 μm 2 , 6 μm 2 , 7 μm 2 , 8 μm 2 , 9 μm 2 , 10μm 2 , 20μm 2 , 30μm 2 , 40μm 2 , 50μm 2 , 60μm 2 , 70μm 2 , 80μm 2 , 90μm2 , 100μm 2 , 200μm 2 , 300μm 2 , 400μm 2 , 500μm 2 , 600μm 2 , 700μm 2 , 800μm 2 , 900μm 2 , 1,000μm 2 The predetermined space may be characterized by having a volume of at least about 1,000 μm 2 , 900μm 2 , 800μm 2 , 700μm 2 , 600μm 2 , 500μm 2 , 400μm 2 , 300μm 2 , 200μm 2 , 100μm 2 , 90μm 2 , 80μm 2 , 70μm 2 , 60μm 2 , 50μm 2 , 40μm 2 , 30μm 2 , 20μm 2 , 10μm 2 , 9 μm 2 , 8 μm 2 , 7 μm 2 , 6 μm 2 , 5 μm 2 , 4 μm 2 , 3 μm 2 , 2 μm 2 , 1 μm 2 , 900nm 2 , 800nm 2 , 700nm 2 , 600nm 2 , 500 nm 2 , 400nm 2 , 300 nm 2 , 200 nm 2 , 100 nm 2 , 90nm 2 , 80nm 2 , 70nm 2 , 60nm 2 , 50 nm2 , 40nm 2 , 30nm 2 , 20nm 2 , 10 nm 2 , 9nm 2 , 8nm 2 , 7nm 2 , 6nm 2 , 5nm 2 , 4nm 2 , 3 nm 2 , 2 nm 2 , 1 nm 2 , 0.5 nm 2 , 0.1 nm 2 or less.

[0116]

[0122] A sensor of the present disclosure may not require at least a portion of a target molecule (and / or a tag coupled to the target molecule) to enter and / or pass through a pore (e.g., a nanopore, such as a protein nanopore or a solid-state nanopore) in order to detect one or more signals indicative of an impedance or an impedance change. For example, as shown, for example, in Figures 2A-2D, a sensor may not include a nanopore or be operably coupled to one. Instead, at least a portion of a target molecule (and / or a tag coupled to the target molecule) may enter and / or pass through a pore (e.g., a nanopore, such as a protein nanopore or a solid-state nanopore) of the sensor in order for the sensor to detect one or more signals indicative of an impedance or an impedance change. For example, as shown, for example, in Figure IE, a sensor may include a nanopore.

[0117]

[0123] FIG. 2E shows an example of a capacitance vs. resistance plot obtained by the sensor after each of nucleotide probes 240-4 and 240-1 is added to substrate 220 by binding unit 215. A measurement of the impedance signal of liquid 105 may be shown as a background or control. As shown by the plot in FIG. 2E, although coupled to the same nucleotide base (e.g., adenine), different tags result in different capacitance vs. resistance characteristics, which may enable the sensor to resolve two or more sequential additions of the same nucleobase to substrate 220. In one example, a sensor of the present disclosure may be capable of resolving the difference between (i) a first time (t) indicating the addition of an Nth adenine (e.g., adenine 20) and (ii) a second time indicating the subsequent addition of an (N+1)th adenine (e.g., adenine 21).

[0118]

[0124] 3, the height 310 of the channel 305 holding the fluid 105 may be designed to restrict mobility (or diffusivity) of target molecules along an axis parallel to the height 310 of the channel 305. By limiting the effective operational area of ​​the sensor (e.g., the dimensions of the binding units, conductive material, or sensing electrodes), any mobility of target molecules along an axis perpendicular to the effective operational area of ​​the sensor (e.g., an axis parallel to the height 310) may negatively affect one or more signal measurements. Thus, by reducing the height 310 of the channel 305, the system becomes substantially two-dimensional, which may limit any effect due to perpendicular movement of target molecules relative to the sensing electrodes 110. The channel height 310 can be up to 300 micrometers (μm), 250 μm, 200 μm, 150 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 25 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm or less. The channel height 310 can be at least 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm or more.

[0119]

[0125] With reference to FIG. 3 , in some cases, the height 310 of the channel 305 may be a predetermined height (e.g., during manufacturing of the sensor). Alternatively, the height 310 of the channel 305 may be adjustable. In some cases, it may be desirable to adjust the height 310 of the channel 305 depending on the size of the target molecule or the average size of a population of target molecules. In such cases, one or more switches (e.g., mechanical switches, microfluidic switches) may be disposed on or adjacent to at least a portion of the sensor such that the height 310 can be changed in real time. Examples of switches may include, but are not limited to, thermally or piezoelectrically driven actuators, electrically driven actuators, etc.

[0120]

[0126] In some embodiments, the sensing electrode and the reference electrode are aligned along a first direction. In addition, the system may further include an additional electric field generator (e.g., an additional set of electrodes in a different circuit) configured to apply a second electric field in a second direction different (e.g., substantially perpendicular) from the first direction of the first electric field. The difference between the first and second directions may be at least about 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 6 degrees, 7 degrees, 8 degrees, 9 degrees, 10 degrees, 15 degrees, 20 degrees, 30 degrees, 40 degrees, 50 degrees, 60 degrees, 70 degrees, 80 degrees, 90 degrees, 100 degrees, 110 degrees, 120 degrees, 130 degrees, 140 degrees, 150 degrees, 160 degrees, 170 degrees or more. The difference between the first and second directions can be at most about 180 degrees, 170 degrees, 160 degrees, 150 degrees, 140 degrees, 130 degrees, 120 degrees, 110 degrees, 100 degrees, 90 degrees, 80 degrees, 70 degrees, 60 degrees, 50 degrees, 40 degrees, 30 degrees, 20 degrees, 15 degrees, 10 degrees, 9 degrees, 8 degrees, 7 degrees, 6 degrees, 5 degrees, 4 degrees, 3 degrees, 2 degrees, 1 degree or less. In some examples, the difference between the first and second directions can be about 90 degrees (i.e., the first and second directions can be substantially perpendicular to each other). Figure 4A illustrates a system including a sensor, for example, for evaluating (e.g., analyzing, identifying, quantifying, ranking, etc.) one or more target molecules, where the sensor does not include any additional electric field generator. When the polynucleotide substrate 220 is large, it folds when it binds to the binding unit 215. When the polynucleotide substrate 220 folds, one or more locations on the substrate 220 may be inaccessible to a binding unit (e.g., a polymerase enzyme) for adding a target nucleotide, thus negatively impacting the sensitivity and / or accuracy of the sensor. In contrast, Figure 4B illustrates a different system including a sensor and an additional electric field generator configured to apply an additional electric field 405 between an additional pair of electrodes 410 and 415, for example, to evaluate (e.g., analyze, identify, quantify, rank, etc.) one or more target molecules. As illustrated, the direction of the electric field 405 may be substantially perpendicular to the surface of the sensing electrode 110.The applied electric field 405 may unfold and / or stretch the substrate 220 along the direction of the electric field 405, thereby reducing folding and overlapping of the substrate 220 by the binding units 215, the conductive material (not shown) and / or the surface of the sensing electrode 110.

[0121]

[0127] The system may include at least 1, 2, 3, 4, 5 or more additional electric field generators. The system may include up to 5, 4, 3, 2 or 1 additional electric field generators. When multiple additional electric field generators are included, the multiple additional electric field generators may apply multiple electric fields along the same or different directions. Alternatively, the system may not include any additional electric field generators.

[0122]

[0128] In some embodiments, the target molecule may include one or more redox moieties (e.g., as a tag), and the sensor of the present disclosure may be configured to measure the redox potential (e.g., reduction potential or oxidation potential) of the redox moiety. In some examples, the sensor may be configured to measure the oxidation potential of an oxidizable functional group of the target molecule. The buffer solution may negatively affect the activity of the binding unit (e.g., the enzymatic activity of the binding unit), and therefore, for some binding units, it may be desirable to reduce the salt concentration in the solution. However, in some cases, reducing the salt concentration in the buffer may increase the resistance of the system, thereby reducing the sensitivity or accuracy of the sensor. In such cases, it may be advantageous to utilize tags with one or more redox moieties to measure the redox potential of the target molecule that includes such tags, which may improve accuracy.

[0123]

[0129] 5A illustrates generally one example of a system including a sensor for measuring the oxidation potential of one or more target molecules, e.g., to evaluate (e.g., analyze, identify, quantify, rank, etc.) the one or more target molecules. The sensor may include one or more components of the system illustrated in FIG. 2A, such as sensing electrode 110, reference electrode 115, liquid 105 (e.g., a salt solution), binding unit 215 (e.g., a polymerase), and polynucleotide substrate 220. Here, a target molecule attached to substrate 220 by binding unit 215 is labeled 510. The redox moieties of the target molecules are synthetic nucleotides A, T, C and G, as indicated at 510-1, 510-2, 510-3 and 510-4. The redox moieties of the target molecules can be ferrocene (Fc) or a series of Fc derivatives, namely Fc-x, Fc-y and Fc-dm. Fc and its derivatives have different oxidation potentials, such that when each target molecule containing its respective redox tag is attached to substrate 220 by binding unit 215, upon application of different voltages (e.g., differing by a few millivolts), it may be possible to resolve which target molecule has been attached to substrate 220, for example, by detecting electron transfer between (i) the redox moiety of the target molecule and (ii) a sensing electrode when the target molecule attached to the redox moiety is attached to the binding unit and attached to substrate 220.

[0124]

[0130] 5B illustrates a schematic of a time (t, x-axis) vs. current (I, y-axis) plot and a current (indicated by the square peaks) plot at various voltages (indicated V0, V1, V2, V3 and V4 along the y-axis) obtained from the sensor of FIG. 5A. In FIG. 5B, multiple time vs. current plots at various voltages are overlaid to show the different current signatures generated by different target molecule-redox tag complexes as provided herein. The redox tags of the four target molecules 510-1, 510-2, 510-3 and 510-4 may respond to different voltages, and thus the respective measured current signatures may be used to determine the sequence 520 of the region of interest of the target substrate 220.

[0125]

[0131] In some embodiments, the sensor may be configured to determine the residence time of the target molecule on the binding unit. Optionally, different target molecules or different tags coupled to the target molecules may exhibit different residence times with respect to the binding unit of the sensor, which may be a unique or additional signature for analyzing or identifying the target molecule.

[0126]

[0132] The sensor of the present disclosure can be an electrical circuit (e.g., a CMOS or FET circuit). The electrical circuit can be coupled to a voltage source. A constant voltage can be applied to the electrical circuit and the change in current can be measured. Alternatively, the change in voltage required to maintain a constant current can be measured. The sensor can be in an electrolyte (e.g., 0.5 M potassium acetate and 10 mM KCl). Alternatively, the sensor may not be in an electrolyte. In some examples, the sensor can be in an aqueous solution or in a gas.

[0127]

[0133] The one or more signals may be a current or a voltage measured from the sensing circuit. The one or more signals may be a current and a voltage measured from the sensing circuit. The signal may be a tunneling current. Alternatively, the signal may not be a tunneling current. The current may be a faradaic current. Alternatively, the current may not be a faradaic current. The current may be at least 1 picoamp (pA), 10 pA, 100 pA, 1 nanoamp (nA), 10 nA, 100 nA, 1 microamp (mA), 10 mA, 100 mA or more. The current may be up to 100 mA, 10 mA, 1 mA, 100 nA, 10 nA, 1 nA, 100 pA, 10 pA, 1 pA or less. The current may be at least in the picoampere (pA) range, tens of pA range, hundreds of pA range, nanoampere (nA) range, tens of nA range, hundreds of nA range, microampere (mA) range, tens of mA range or more. The current may be up to tens of mA range, mA range, hundreds of nA range, tens of nA range, nA range, hundreds of pA range, tens of pA range, pA range or less. The voltage may be at least 0.1 millivolt (mV), 0.5 mV, 1 mV, 5 mV, 10 mV, 50 mV, 100 mV, 500 mV or more. The voltage may be up to 500 mV, 100 mV, 50 mV, 10 mV, 5 mV, 1 mV, 0.5 mV, 0.1 mV or less. The voltage may be at least in the millivolt (mV) range, tens of mV range, hundreds of mV range or more. The voltage can be up to hundreds of mV range, tens of mV range, mV range or even less.

[0128]

[0134] In some embodiments, the sensors of the present disclosure are provided on a chip or biochip. The sensor array may be provided as an array, such as an array of sensors that are present. The array of sensors may have any suitable number of any of the sensors disclosed herein. The array may include about 10, about 20, about 50, about 100, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 10000, about 15000, about 20000, about 40000, about 60000, about 80000, about 100000, about 200000, about 400000, about 600000, about 800000, about 1000000 or more sensors.

[0129]

[0135] In another aspect, the present disclosure provides a method of analyzing or identifying a target molecule. The method may include using a sensor to detect one or more signals indicative of an impedance or impedance change of the sensor when at least a portion of the target molecule is bound by at least a portion of the sensor. The method may further include analyzing or identifying the target molecule using the one or more signals. The method may utilize any of the systems of the presently disclosed subject matter as illustrated in Figures 1 to 5.

[0130]

[0136] In another aspect, the present disclosure provides a method for analyzing or identifying a target molecule. The method may include providing a sensor including a sensing electrode and a reference electrode in electrical communication with each other. The sensor may further include a dielectric material coupled to the sensing electrode and covering a first portion of a surface of the sensing electrode. The sensor may further include a conductive material coupled to the sensing electrode and covering a second portion of a surface of the sensing electrode. The sensor may further include a binding unit coupled to the conductive material, the binding unit configured to bind to the target molecule. The method may further include detecting one or more signals indicative of an impedance or impedance change of the sensor when at least a portion of the target molecule is bound by the binding moiety. The method may further include analyzing or identifying the target molecule using the one or more signals. The method may utilize any of the systems of the presently disclosed subject matter as illustrated in Figures 1 to 5.

[0131]

[0137] 7 shows an exemplary process 701 of a method for analyzing or identifying a target molecule. The method may include providing a sensor (process 710). The sensor may include a binding unit. The method may include providing a target molecule to the sensor (process 720). The method may include detecting one or more signals indicative of an impedance or impedance change of the sensor when at least a portion of the target molecule is bound by the binding unit (process 730). The method may include analyzing or identifying the target molecule using the one or more signals.

[0132]

[0138] The one or more signals may be indicative of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, or (ii) the electrical inductance of the sensor or a change therein. The one or more signals may be indicative of at least two of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, or (ii) the electrical inductance of the sensor or a change therein. The one or more signals may be indicative of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, and (ii) the electrical inductance of the sensor or a change therein.

[0133]

[0139] The one or more signals may be a current or a voltage. The one or more signals may be a current and a voltage. The one or more signals do not have to be a tunneling current.

[0134]

[0140] In some embodiments, the sensing electrode and the reference electrode may provide a first electric field. Additionally, the method may further include providing an additional electric field generator. The method may further include using the additional electric field generator to generate a second electric field in a second direction substantially perpendicular to the first direction of the first electric field. It may further include applying an electric field.

[0135]

[0141] In some embodiments, the method may further include determining a residence time of the target molecule on the binding unit. The method may further include analyzing or identifying the target molecule using the residence time instead of and / or in addition to one or more signals.

[0136]

[0142] II. Sample

[0143] The sample for analysis can include a plurality of polynucleotides. The polynucleotides can be single-stranded DNA, double-stranded DNA, or a combination thereof. The polynucleotides can include genomic DNA, genomic cDNA, cell-free DNA, cell-free cDNA, or any combination of the above.

[0137]

[0144] The polynucleotides may include cell-free DNA, circulating tumor DNA, genomic DNA, and DNA from formalin-fixed paraffin-embedded (FFPE) samples. In some cases, the DNA extracted from FFPE samples may be damaged, and such damaged DNA can be repaired by available FFPE DNA repair kits. The samples may include any suitable DNA and / or cDNA samples, such as urine, stool, blood, saliva, tissue, biopsy, body fluids, or tumor cells.

[0138]

[0145] The multiple polynucleotides may be single-stranded or double-stranded.

[0139]

[0146] A polynucleotide sample can be from any suitable source, for example, a sample can be obtained from a patient, an animal, a plant, or the environment, such as naturally occurring or man-made air, water, soil, airborne pathogen collection systems, subsurface sediments, groundwater, or a sewage treatment plant.

[0140]

[0147] The polynucleotides from the sample may include one or more different polynucleotides, such as, for example, DNA, RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), messenger RNA (mRNA), a fragment of any of the foregoing, or a combination of any of the foregoing. The sample may include DNA. The sample may include genomic DNA. The sample may include mitochondrial DNA, chloroplast DNA, plasmid DNA, bacterial artificial chromosomes, yeast artificial chromosomes, oligonucleotide tags, or a combination of any of the foregoing.

[0141]

[0148] A polynucleotide can be single-stranded, double-stranded, or a combination thereof. A polynucleotide can be a single-stranded polynucleotide, which may or may not be in the presence of a double-stranded polynucleotide.

[0142]

[0149] The starting amount of polynucleotide in a sample can be less than 50 ng, such as, for example, 45 ng, 40 ng, 35 ng, 30 ng, 25 ng, 20 ng, 15 ng, 10 ng, 5 ng, 4 ng, 3 ng, 2 ng, 1 ng, 0.5 ng, 0.1 ng or less. The starting amount of polynucleotide in a sample can be more than 0.1 ng, such as, for example, 0.5 ng, 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45 ng, 50 ng or more. The amount of starting polynucleotide can be, for example, 0.1 ng to 100 ng, 1 ng to 75 ng, 5 ng to 50 ng, or 10 ng to 20 ng.

[0143]

[0150] The polynucleotides in the sample may be single-stranded, either as obtained or after processing (e.g., denaturation). Further examples of suitable polynucleotides are described herein, including with respect to any of the various aspects of the present disclosure. The polynucleotides can be subjected to subsequent steps (e.g., circularization and amplification) without an extraction step and / or without a purification step. For example, a fluid sample can be subjected to a process to remove cells without an extraction step to produce a purified liquid sample and a cell sample, and then polynucleotides can be isolated from the purified fluid sample. Various procedures are available for isolating polynucleotides, such as by precipitation or non-specific binding to a substrate followed by washing of the substrate to release the bound polynucleotides. When polynucleotides are isolated from a sample without a cell extraction step, the polynucleotides will generally be extracellular or "cell-free" polynucleotides that may correspond to dead or damaged cells. The identity of such cells can be used to characterize the original cell or cell population from which it originated, such as in a microbial community.

[0144]

[0151] The sample may be from a subject. The subject may be any suitable organism, including, for example, a plant, an animal, a fungus, a protist, a monera, a virus, a mitochondrion, and a chloroplast. The specimen polynucleotide may be isolated from a subject, such as a cell sample, a tissue sample, a body fluid sample, or an organ sample, or a cell culture derived from any of these, including a cell-containing fluid sample, such as a cultured cell line, a biopsy, a blood sample, a buccal swab, or saliva. The subject may be an animal, such as a mammal, such as a cow, a pig, a mouse, a rat, a chicken, a cat, a dog, or a human. The sample may include tumor cells, such as in a sample of tumor tissue from a subject.

[0145]

[0152] The sample may not contain intact cells, may be subjected to a process to remove cells, or polynucleotides may be isolated without a cell extraction step, such as to isolate cell-free polynucleotides, such as cell-free DNA.

[0146]

[0153] Other examples of sample sources include those from blood, urine, feces, nasal passages, lungs, intestines, other bodily fluids or excretions, derivatives thereof, or combinations thereof.

[0147]

[0154] A sample from a single individual can be divided into multiple separate samples, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate samples, which are independently subjected to the method of the present disclosure, such as analysis in duplicate, triplicate, quadruplicate or more.When a sample is from a subject, the reference sequence can also be derived from the subject, such as a consensus sequence from the sample under analysis or a sequence of polynucleotide from another sample or tissue of the same subject.For example, a blood sample can be analyzed for ctDNA mutations, and cellular DNA from another sample from the subject, such as a buccal or skin sample, can be analyzed to determine the reference sequence.

[0148]

[0155] The polynucleotides may be extracted from the sample by any suitable method, with or without extraction from the cells in the sample.

[0149]

[0156] The plurality of polynucleotides can include cell-free polynucleotides, such as cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). Cell-free DNA circulates in both healthy and diseased individuals. cfDNA (ctDNA) from tumors is not limited to any particular cancer type and appears to be a common finding across a variety of malignancies. Free circulating DNA concentration in plasma can be reduced in control subjects compared to patients with or suspected of having a pathology. In some instances, free circulating DNA concentration in plasma can be, for example, 14ng / mL to 18ng / mL in control subjects and 18ng / mL to 318ng / mL in patients with neoplasms.

[0150]

[0157] Apoptotic and necrotic cell death may contribute to cell-free circulating DNA in body fluids. For example, a significant increase in circulating DNA levels can be observed in the plasma of prostate cancer patients and other prostate diseases such as benign prostatic hyperplasia and prostatitis. In addition, circulating tumor DNA NA can be present in fluids derived from organs where primary tumors arise. In some examples, breast cancer detection can be achieved in ductal lavage fluid; colorectal cancer detection in stool; lung cancer detection in sputum and prostate cancer detection in urine or ejaculate. Cell-free DNA can be obtained from various sources. An exemplary source can be a subject's blood sample. However, cfDNA or other fragmented DNA can be derived from various other sources, including, for example, urine and stool samples, and can be the source of cfDNA, including ctDNA.

[0151]

[0158] In some embodiments, the target molecule may be indicative of a condition or disease. In some cases, the disease may be a tumor or cancer. Non-limiting examples of antigens that may be bound by the binding units of the subject systems include, but are not limited to, 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A-kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenoceptor β3 (ADRB3), AGS-22M6, alpha folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis (Bacillus anthracis, B cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), Brother of the Regulator of Imprinted Sites (BORIS), C242 Antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC binding factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD 40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile, Claudin amplifying factor A, CLCA2, colony-stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CXC chemokine receptor type 4, cyclin B1, cytochrome P4501B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, Escherichia coli (E. coli) Shiga toxin type 1, Escherichia coli (E. coli Shiga toxin type 2, ecto-ADP-ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like domain multiplex 7 (EGFL7), elongation factor 2 mutated (ELF2M), endotoxin, ephrin A2, ephrin B2, ephrin type A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), episialin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), F protein of respiratory syncytial virus, FAP, Ig Fc fragment of A receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II beta chain, fibroblast activation protein alpha (FAP), fibronectin extra domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor alpha, folate receptor beta, Fos-related antigen 1, Frizzled receptor, fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor alpha chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 mutant (mut hsp70-2), hemagglutinin, hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexasaccharide moiety of globoH glycoceramide (GloboH), HGF, HHGFR, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor,Interferon-gamma-inducible protein, interleukin-11 receptor alpha (IL-11Rα), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1 cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis Y antigen Hara, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex, locus K9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1) , melanoma cancer testis antigen-2 (MAD-CT-2), melanoma inhibitor of apoptosis (ML-IAP), melanoma associated antigen 1 (MAGE-A1), mesothelin, mucin 1, cell surface associated (MUC1), MUC-2, mucinCanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyltransferase V (NA17), NCA-90 (granulocyte antigen), nerve growth factor (NGF), neuronal apoptosis-regulating proteinase 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitors (e.g. For example, NOGO-A, NOGO-B, and NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), carcinoembryonic antigen (h5T4), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl), Oryctolagus cuniculus, OX-, 40, oxLDL, p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), phosphate-sodium cotransporter, phosphatidylserine, placenta-specific 1 (PLAC1 ), platelet-derived growth factor receptor alpha (PDGF-Rα), platelet-derived growth factor receptor beta (PDGFR-β), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteasome (prosome, macropain) subunit, beta, type 9 (LMP2) , Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rh factor, sarcoma translocation breakpoint, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigens 1, 2, and 3 recognized by T cells (SART1, SART2, and SART3), stage-specific fetal antigen-4 (SSEA-4), Staphylococcus aureus, ST EAP1, survivin, syndecan 1 (SDC1)+A314, SOX10, survivin, survivin-2B, synovial sarcoma, X breakpoint 2 (SSX2), T cell receptor, TCR gamma alternative reading frame protein (TARP), telomerase, TEM1, tenascin-C, TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3), thyroid-stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen (Tn Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-α, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAA16.88, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), tumor protein p53 (p53), tumor-specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosinase, tyrosinase-related protein 1 (TYRP1 or glycoprotein 75), tyrosinase-related protein 2 (TYRP2), uroplakin 2 (UPK2), vascular endothelial growth factors (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), von Willebrand syndrome (VHS) These include varicella-wrinkle factor (VWF), Wilms tumor protein (WT1), X antigen family, member 1A (XAGE1), β-amyloid and κ light chain.

[0152]

[0159] III. Nanopore

[0160] The sequencing system can include a reaction chamber containing one or more nanopore devices. The nanopore device can be an individually addressable nanopore device. The individually addressable nanopores can be individually readable. The individually addressable nanopores can be individually writeable. The individually addressable nanopores can be individually readable and individually writeable. The system can include one or more computer processors that facilitate sample preparation and various operations of the present disclosure, such as polynucleotide sequencing. The processor can be coupled to the nanopore device.

[0153]

[0161] The nanopore device may include multiple individually addressable sensing electrodes. Each sensing electrode may include a membrane adjacent to the electrode and one or more nanopores in the membrane. The nanopore may be in a membrane, such as a lipid bilayer, located adjacent to an electrode that is part of an integrated circuit or coupled thereto, or in sensing proximity thereto. The nanopore may be associated with a separate electrode and sensing integrated circuit or multiple electrodes and sensing integrated circuits. The nanopore may include a solid-state nanopore.

[0154]

[0162] The devices and systems used in the methods provided by the present disclosure can accurately detect individual nucleotide incorporation events, such as when a nucleotide is incorporated into a growing strand complementary to a template. Nucleotides can be incorporated into a growing polynucleotide strand by enzymes such as DNA polymerase, RNA polymerase, or ligase. Enzymes such as polymerases can result in polynucleotide strands.

[0155]

[0163] The nucleotides added may be complementary to the corresponding template polynucleotide strand hybridized to the growing strand. The nucleotides may include tags or tag species coupled to any position of the nucleotide, including but not limited to the phosphate, such as the gamma phosphate, the sugar, or the nitrogenous base moiety of the nucleotide. Optionally, the tag is detected during incorporation of the nucleotide tag while the tag is associated with the polymerase. The tag may remain detected until the tag moves through the nanopore after nucleotide incorporation, followed by cleavage and / or release of the tag. The nucleotide incorporation event releases the tag from the nucleotide and passes through the nanopore, where it may be detected. The tag may be released by the polymerase or may be cleaved / released in any suitable manner, including, without limitation, cleavage by an enzyme located in the vicinity of the polymerase. In this way, a unique tag is released from each type of nucleotide (i.e., adenine, cytosine, guanine, thymine, or uracil), allowing the incorporated base (i.e., A, C, G, T, or U) to be identified. In non-release nucleotide incorporation events, a tag coupled to the incorporated nucleotide is detected using the nanopore. In some cases, the tag translocates through or in close proximity to the nanopore, where it can be detected using the nanopore.

[0156]

[0164] The disclosed methods and systems may allow for detection of polynucleotide incorporation events, such as with a resolution of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000, 5000, 10000, 50000, or 100000 polynucleotide bases in a given time. For example, a nanopore device may be used to detect individual polynucleotide incorporation events, where each event is associated with an individual nucleic acid base. In other examples, a nanopore device may be used to detect events associated with multiple bases. For example, the signal sensed by the nanopore device may be a combination of signals from at least 2, 3, 4, or 5 bases.

[0157]

[0165] In certain sequencing methods, the tag does not pass through the nanopore. The tag can be detected by the nanopore and exit the nanopore without passing through it, such as exiting in the opposite direction from the way the tag entered the nanopore. The sequencing device can be configured to actively expel the tag from the nanopore.

[0158]

[0166] In certain sequencing methods, a nucleotide incorporation event does not release a tag. A nucleotide incorporation event may present a tag to a nanopore without releasing the tag. The tag may be detected by the nanopore without being released. The tag may be attached to the nucleotide by a linker of sufficient length to present the tag to the nanopore for detection.

[0159]

[0167] Nucleotide incorporation events can be detected by the nanopore in real time as they occur. An enzyme, such as a DNA polymerase, attached to or adjacent to the nanopore can facilitate the flow of polynucleotides through or adjacent to the nanopore. A nucleotide incorporation event, i.e., incorporation of multiple nucleotides, can release or present one or more tags, which can be detected by the nanopore. Detection can occur as the tag flows through or adjacent to the nanopore, as the tag resides in the nanopore, and / or as the tag is presented to the nanopore. In some cases, detection of the tag associated with incorporation of one or more nucleotides can be aided by an enzyme attached to or adjacent to the nanopore.

[0160]

[0168] The tag can be an atom, a molecule, a group of atoms or a group of molecules. The tag can provide an optical, electrochemical, magnetic or electrostatic, such as inductive or capacitive, signature that can be detected using the nanopore.

[0161]

[0169] The nanopore may be formed in or otherwise embedded in a membrane disposed adjacent to a sensing electrode of a sensing circuit, such as an integrated circuit. The integrated circuit may be an application specific integrated circuit (ASIC). The integrated circuit may be a field effect transistor or a complementary metal oxide semiconductor (CMOS). The sensing circuit may be located on a chip or other device that has the nanopore, or may be located off the chip or device, such as in an off-chip configuration.

[0162]

[0170] As the nucleic acid or tag flows through or adjacent to the nanopore, the sensing circuitry detects an electrical signal associated with the nucleic acid or tag. The nucleic acid may be a subunit of a larger chain. The tag may be a by-product of a nucleotide incorporation event or other interaction between the tagged nucleic acid and the nanopore or a species adjacent to the nanopore, such as an enzyme that cleaves the tag from the nucleic acid. The tag may remain attached to the nucleotide. The detected signal may be collected, stored in a memory location, and later used to construct the sequence of the nucleic acid. The collected signal may be processed to account for any anomalies in the detected signal, such as errors.

[0163]

[0171] The nanopore can be used to indirectly sequence the polynucleotide, optionally with electrical detection. Indirect sequencing can be any method in which the nucleotide incorporated into the growing chain does not pass through the nanopore. The polynucleotide can pass within any suitable distance from the nanopore and / or in close proximity thereto, optionally within a distance such that the tag released from the nucleotide incorporation event is detected at the nanopore.

[0164]

[0172] The nanopore can detect by-products of nucleotide incorporation events. A nucleotide incorporation event refers to the incorporation of a nucleotide into a growing polynucleotide chain. The by-products can be correlated with the incorporation of a given type of nucleotide. Nucleotide incorporation events can be catalyzed by enzymes such as DNA polymerases, which can use base pairing interactions with the template molecule to select among the available nucleotides for incorporation at each position.

[0165]

[0173] A nucleic acid sample may be sequenced using tagged nucleotides or nucleotide analogs. In some examples, a method of sequencing a nucleic acid molecule includes (a) incorporating (e.g., polymerizing) tagged nucleotides, whereby a tag associated with the individual nucleotide is released upon incorporation, and (b) detecting the released tag with a nanopore. In some examples, the method further includes directing the tag attached to or released from the individual nucleotide through the nanopore. The released or attached tag may be detected by any suitable technique. Alternatively, a free or attached tag can be induced to pass through a nanopore without the use of an enzyme. For example, a tag can be induced by a voltage difference across a nanopore, as described herein.

[0166]

[0174] The tag may be detected using a nanopore device having at least one nanopore in the membrane. The tag may be associated with an individual tagged nucleotide upon incorporation of the individual tagged nucleotide. The nanopore device may detect the tag associated with the individual tagged nucleotide upon incorporation. The tagged nucleotide, whether incorporated or not into the growing nucleic acid strand, may be detected, determined or identified by the nanopore device, optionally using electrodes and / or nanopores of the nanopore device, over a given period of time. The time during which the nanopore device detects the tag may be short, and optionally substantially short, compared to the time that the tag and / or nucleotide coupled to the tag is retained by an enzyme, such as an enzyme (e.g., a polymerase) that facilitates incorporation of the nucleotide into the nucleic acid strand. The tag may be detected by the electrodes multiple times over the time that the incorporated tagged nucleotide is associated with the enzyme. For example, the tag can be detected by the electrode at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 10,000, 100,000 or 1,000,000 times over the time that the incorporated tagged nucleotide is associated with the enzyme.

[0167]

[0175] Sequencing can be accomplished using preloaded tags. Preloading the tag can include inducing at least a portion of the tag to pass through at least a portion of the nanopore while the tag can be attached to a nucleotide and the nucleotide is in a state of incorporation into a nucleic acid strand (e.g., a growing nucleic acid strand), while incorporation into the nucleic acid strand is occurring, or while not yet incorporation into the nucleic acid strand but incorporation into the nucleic acid strand can occur. Preloading the tag can include inducing at least a portion of the tag to pass through at least a portion of the nanopore before or while the nucleotide is incorporation into the nucleic acid strand. Preloading the tag can include inducing at least a portion of the tag to pass through at least a portion of the nanopore after the nucleotide is incorporation into the nucleic acid strand.

[0168]

[0176] Tags associated with individual nucleotides can be detected by the nanopore without being released from the nucleotide upon incorporation. Tags can be detected during synthesis of a nucleic acid strand complementary to a target strand without being released from the incorporated nucleotide. Tags can be attached to nucleotides using a linker such that the tag is presented to the nanopore (e.g., the tag hangs down or otherwise extends through at least a portion of the nanopore). The length of the linker can be long enough to allow the tag to extend to or through at least a portion of the nanopore. In some cases, the tag is presented to the nanopore (i.e., travels into it) by a voltage difference. Other methods of presenting the tag into the pore can also be suitable (e.g., using enzymes, magnets, electric fields, pressure differences). In some cases, no active force is applied to the tag (i.e., the tag diffuses into the nanopore).

[0169]

[0177] The chip for sequencing nucleic acid samples may include a plurality of individually addressable nanopores. The individually addressable nanopores in the plurality may include at least one nanopore formed in a membrane disposed adjacent to an integrated circuit. Each of the individually addressable nanopores may have the ability to detect a tag associated with an individual nucleotide. The nucleotide may be incorporated (e.g., polymerized), and upon incorporation, the tag may not be released from the nucleotide.

[0170]

[0178] The tag may be presented to the nanopore following a nucleotide incorporation event and released from the nucleotide. The released tag may pass through the nanopore. In some cases, the tag does not pass through the nanopore. The tag released following a nucleotide incorporation event may flow through the nanopore, but is distinguished from tags not released following a nucleotide incorporation event, at least in part, by their residence time in the nanopore. In some cases, a tag that resides in the nanopore for at least 100 milliseconds (ms) is released following a nucleotide incorporation event, and a tag that resides in the nanopore for less than 100 ms is not released following a nucleotide incorporation event. The tag may be captured in and / or guided through the nanopore by a second enzyme or protein (e.g., a nucleic acid binding protein). The second enzyme may cleave the tag following (e.g., during or after) the incorporation of the nucleotide. A linker between the tag and the nucleotide may be cleaved.

[0171]

[0179] The tag coupled to the incorporated nucleotide is distinguished from the tag associated with the non-incorporated nucleotide in the growing complementary strand by the nanopore based on the residence time of the tag in the nanopore or the signal detected from the non-incorporated nucleotide. The non-incorporated nucleotide can generate a detectable signal (e.g., voltage difference, current) for a time of 1 nanosecond (ns) to 100 ms or 1 ns to 50 ms, while the incorporated nucleotide can generate a signal with a lifetime of 50 ms to 500 ms or 100 ms to 200 ms. The non-incorporated nucleotide can generate a detectable signal for a time of 1 ns to 10 ms or 1 ns to 1 ms. The non-incorporated tag is detectable by the nanopore for a longer time (on average) than the incorporated tag is detectable by the nanopore.

[0172]

[0180] An incorporated nucleic acid may be detected and / or is detectable by a nanopore for a shorter time than an unincorporated nucleotide. Alternatively, an incorporated nucleic acid may be detected and / or is detectable by a nanopore for a longer time than an unincorporated nucleotide. These time differences and / or ratios can be used to determine whether a nucleotide detected by a nanopore as described herein has been incorporated.

[0173]

[0181] The detection time can be based on the free flow of nucleotides through the nanopore. Unincorporated nucleotides can reside at or near the nanopore for a time of 1 nanosecond (ns) to 100 ms or 1 ns to 50 ms, while incorporated nucleotides can reside at or near the nanopore for a time of 50 ms to 500 ms or 100 ms to 200 ms. These times can vary based on the process conditions. However, incorporated nucleotides can have a longer residence time than unincorporated nucleotides.

[0174]

[0182] A tag or tag species may include a detectable atom or molecule or multiple detectable atoms or molecules. A tag may include one or more adenines, guanines, cytosines, thymines, uracils, or derivatives thereof linked to any position of a nucleic acid molecule, including a phosphate group, a sugar, or a nitrogenous base. A tag may include one or more adenines, guanines, cytosines, thymines, uracils, or derivatives thereof covalently linked to a phosphate group of a nucleic acid base.

[0175]

[0183] The tag can be at least 0.1 nanometer (nm), 1 nm, 2 nm, 3 nm, 4, nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm or 1000 nm in length.

[0176]

[0184] The tag may comprise a tail of repeating subunits such as a plurality of adenine, guanine, cytosine, thymine, uracil or derivatives thereof. For example, the tag may comprise a tail portion having at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 or 100,000 adenine, guanine, cytosine, thymine, uracil or derivatives thereof. The subunits may be linked at their terminal ends linked to each other and to the phosphate groups of the nucleic acid. Other examples of tag moieties include any polymeric material such as polyethylene glycol (PEG), polysulfonates, amino acids or any fully or partially positively, negatively or uncharged polymer.

[0177]

[0185] IV. Polymerase

[0186] A DNA polymerase can bind to the 3' end of the nicked strand of the polynucleotide at the nicking site. DNA sequencing can be accomplished by amplifying and transcribing the polynucleotide in proximity to the nanopore and the tagged nucleotide using an enzyme such as a DNA polymerase. The sequencing method can involve incorporating or polymerizing the tagged nucleotide using a polymerase such as a DNA polymerase or a transcriptase. The polymerase can be mutated to enable it to accept the tagged nucleotide. The polymerase can also be mutated to increase the time at which the tag is detected by the nanopore.

[0178]

[0187] The sequencing enzyme can be any suitable enzyme that creates a polynucleotide chain by, for example, phosphate linkage of nucleotides. The DNA polymerase can be, for example, 9°Nm™ polymerase or a variant thereof, E. coli DNA polymerase I, bacteriophage T4 DNA polymerase, Sequenase, Ta q DNA polymerase, 9°Nm™ polymerase (exo-) A485L / Y40 The DNA polymerase may be 9V, Φ29 DNA polymerase, Bst DNA polymerase, or a variant, mutant, or homologue of any of the foregoing. The homologue may have any suitable percentage of homology, such as, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0179]

[0188] In some cases, a polymerase may be attached to or located in close proximity to the nanopore for nanopore sequencing. A suitable method for attaching a polymerase to a nanopore includes bridging the enzyme to or in close proximity to the nanopore, such as by forming an intramolecular disulfide bond. The nanopore and enzyme may also be fusions, such as encoded by a single polypeptide chain. A method for making a fusion protein may include fusing the coding sequence of the enzyme in frame and adjacent to the coding sequence of the nanopore and expressing the fusion sequence from a single promoter. The polymerase may be attached or coupled to the nanopore using molecular staples or protein fingers. The polymerase may be attached to the nanopore via an intermediate molecule, such as, for example, biotin conjugated to both the enzyme and the nanopore, with a streptavidin tetramer linked to both biotins. This intermediate molecule may be referred to as a linker.

[0180]

[0189] Sequencing enzymes can also be attached to the nanopore with antibodies. Polymerases can be attached to the nanopore using proteins that form covalent bonds between each other. Phosphatase enzymes or enzymes that cleave tags from nucleotides can also be attached to the nanopore.

[0181]

[0190] The polymerase can be mutated to facilitate and / or improve the efficiency of incorporation of tagged nucleotides into a growing polynucleotide by the mutant polymerase compared to the non-mutated polymerase. The polymerase can be mutated to improve entry of nucleotide analogs, such as tagged nucleotides, into the active site region of the polymerase and / or to cooperate with nucleotide analogs at the active site.

[0182]

[0191] Other mutations, such as amino acid substitutions, insertions, deletions and / or exogenous features to a polymerase may result in enhanced metal ion coordination, decreased exonuclease activity, decreased reaction rate in one or more steps of the polymerase kinetics cycle, decreased branching rate, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased read length, increased salt tolerance compared to the non-mutated polymerase.

[0183]

[0192] A suitable polymerase may have a kinetic profile suitable for detection of the tag by the nanopore, which generally refers to the overall rate of nucleotide incorporation and / or the rate of any step of nucleotide incorporation, such as nucleotide addition, enzymatic isomerization to or from a closed ring state, cofactor binding or release, product release, incorporation or translocation of a polynucleotide into a growing polynucleotide, etc.

[0184]

[0193] The polymerase can be adapted to allow for detection of sequencing events. The kinetic profile of the polymerase can be such that the tag is loaded into (and / or detected by) the nanopore over an average of 0.1 milliseconds (ms), 1 ms, 5 ms, 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 80 ms, 100 ms, 120 ms, 140 ms, 160 ms, 180 ms, 200 ms, 220 ms, 240 ms, 260 ms, 280 ms, 300 ms, 400 ms, 500 ms, 600 ms, 800 ms, or 1000 ms. For example, the kinetic profile of the polymerase can be such that the tag is loaded into and / or detected by the nanopore over an average of at least 5 ms, at least 10 ms, at least 20 ms, at least 30 ms, at least 40 ms, at least 50 ms, at least 60 ms, at least 80 ms, at least 100 ms, at least 120 ms, at least 140 ms, at least 160 ms, at least 180 ms, at least 200 ms, at least 220 ms, at least 240 ms, at least 260 ms, at least 280 ms, at least 300 ms, at least 400 ms, at least 500 ms, at least 600 ms, at least 800 ms, or at least 1000 ms. The tag can be detected by the nanopore over an average of 80 ms to 260 ms, 100 ms to 200 ms, or 100 ms to 150 ms.

[0185]

[0194] The nanopore / polymerase complex can be configured to allow for detection of one or more events associated with the amplification and transcription of the circular polynucleotide. The one or more events can be kinetically observable and / or non-kinetically observable, such as a nucleotide translocating through the nanopore without contacting the polymerase.

[0186]

[0195] In some cases, the polymerase reaction exhibits two kinetic steps, one proceeding from an intermediate in which the nucleotide or polyphosphate product is bound to the polymerase enzyme, and the other proceeding from an intermediate in which the nucleotide and polyphosphate product are not bound to the polymerase enzyme. These two kinetic steps may include enzyme isomerization, nucleotide incorporation, and product release. In some cases, these two kinetic steps are template translocation and nucleotide binding.

[0187]

[0196] Suitable polymerases may exhibit strong or enhanced strand displacement.

[0188]

[0197] V. Identification of sequence variants

[0198] The method provided by the present disclosure can be used to identify sequence variants in polynucleotide samples.The sequence difference between sequencing reads and reference sequences is called a genuine sequence variant if the sequence difference appears in at least two different polynucleotides, for example two different circular polynucleotides, and can be distinguished as a result of having different junctions.Since the position and type of sequence variants resulting from amplification or sequencing errors are unlikely to be exactly repeated in two different polynucleotides that contain the same target sequence, including this validation parameter can reduce the background of false sequence variants, while at the same time increasing the sensitivity and accuracy of detecting real sequence mutations in samples. A sequence variant may occur at a frequency of less than 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, 0.1%, 0.075%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.001% or less, sufficiently above background to allow accurate identification. A sequence variant may occur at a frequency of less than 0.1%. A sequence variant frequency may be sufficiently above background when such frequency is, for example, less than 0.05, 0.01, 0.001 or 0.0001, and is statistically significantly above background error rate. The frequency of a sequence variant may be sufficiently above background when the frequency is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 25x, 50x, 100x or more above the background error rate. The background error rate for accurately determining a sequence at a given position may be less than 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001% or 0.0005%.

[0189]

[0199] Identifying sequence variants may include optimally aligning one or more sequencing reads with a reference sequence to identify the difference between the two and to identify junctions.Alignment may involve placing one sequence along another, repeatedly introducing gaps along each sequence, scoring how well the two sequences match, and repeating for different positions along the reference.The best-scoring match is considered to be the alignment, which represents an inference about the degree of relationship between sequences.

[0190]

[0200] The reference sequence to which the sequencing reads are compared is a reference genome, such as the genome of a member of the same species as the subject. The reference genome can be complete or incomplete. The reference genome can consist of only the region containing the target polynucleotide, such as from the reference genome or from a consensus generated from the sequencing reads under analysis. The reference sequence can include or consist of sequences of polynucleotides of one or more organisms, such as sequences from one or more bacteria, archaea, viruses, protists, fungi, or other organisms. The reference sequence can consist of only a portion of the reference genome, such as a region corresponding to one or more target sequences under analysis. For example, for pathogen detection, the reference genome can be the entire genome of the pathogen or a portion thereof useful for identification, such as a pathogen of a particular strain or serotype. The sequencing reads can be aligned with multiple different reference sequences, such as for screening for multiple different organisms or strains.

[0191]

[0201] VI. Therapeutic applications

[0202] The methods, systems and compositions provided herein may relate to one or more therapeutic applications, such as in characterizing patient samples and optionally diagnosing a condition in a subject. Therapeutic applications include providing information based on the results of the methods provided by the present disclosure regarding the selection of therapies to which a patient may best respond and / or the treatment of a subject in need of therapeutic intervention. may be included.

[0192]

[0203] For example, the method provided by the present disclosure can be used to diagnose the presence or absence of tumors, tumor progression and / or metastasis, such as when the polynucleotide under analysis comprises or consists of cfDNA, ctDNA or fragmented tumor DNA. A subject can be monitored for the effectiveness of tumor treatment, for example by monitoring ctDNA over time, where a decrease in ctDNA can be used as an indicator of treatment effectiveness, and an increase in ctDNA can inform the selection of different treatments and / or different dosages. Other uses include the assessment of organ rejection in transplant recipients, such as when an increase in the amount of circulating DNA corresponding to the transplant donor genome is used as an early indicator of transplant rejection, and genotyping / isotyping of pathogen infections, such as viral or bacterial infections. Detection of sequence variants in circulating fetal DNA can be used to diagnose fetal pathologies.

[0193]

[0204] Methods provided by the present disclosure may include diagnosing a subject based on the sequencing results, such as diagnosing the subject with a disease associated with the detected causative genetic variant, or reporting the likelihood that the patient has or will develop such a disease.

[0194]

[0205] Causative genetic variants may include sequence variants associated with a particular type or stage of cancer or sequence variants of cancers with particular characteristics, such as metastatic potential, drug resistance and / or drug responsiveness. The methods provided by the present disclosure can be used to inform treatment decisions, guidance and monitoring of cancer therapy. For example, treatment efficacy can be monitored by comparing patient ctDNA samples before, during and after treatment, including targeted therapies such as monoclonal drugs, chemotherapy drugs, radiation therapy protocols and combinations of any of the above, among others. For example, ctDNA can be monitored to see if certain mutations increase or decrease after treatment, or if new mutations appear, which may allow physicians to modify treatment in a much shorter time than is provided by monitoring methods that track the patient's symptoms. The methods may include diagnosing a subject based on the results of polynucleotide sequencing, such as diagnosing the subject with a particular stage or type of cancer associated with the detected sequence variant, or reporting the likelihood that the patient has or will develop such cancer.

[0195]

[0206] For example, for a therapy that is specifically targeted to a patient based on molecular markers, the patient can be tested to find out whether certain mutations are present in the tumor, and those mutations can be used to predict response or resistance to the therapy and guide the decision to use the therapy.Detection and monitoring of ctDNA during the course of treatment can be useful to guide treatment selection.

[0196]

[0207] Sequence variants associated with one or more cancers can be used for diagnosis, prognosis or treatment decisions.For example, suitable target sequences with oncological significance include alterations in TP53, ALK, KRAS, PIK3CA, BRAF, EGFR and KIT genes.The target sequences that can be specifically amplified and / or specifically analyzed for sequence variants can be all or part of cancer-related genes.

[0197]

[0208] The methods provided by the present disclosure can be useful for discovering novel rare mutations associated with one or more cancer types, stages, or cancer characteristics. For example, in a population of individuals sharing a characteristic under analysis, such as a particular disease, cancer type, and / or cancer stage, the methods provided by the present disclosure can be used to identify sequence variants that reflect mutations in specific genes or gene parts. Individuals sharing the characteristic are more likely to have the same mutation than individuals who do not have the characteristic. Identified sequence variants that occur at a statistically significantly higher frequency in a population can be assigned a degree of association with the trait, and the sequence variants or types of sequence variants so identified can then be used to diagnose or treat individuals found to have them.

[0198]

[0209] Further therapeutic applications include use in non-invasive fetal diagnosis. Fetal DNA may be present in the blood of a pregnant woman. Using the methods provided by the present disclosure, sequence variants of circulating fetal DNA may be identified and thus used to diagnose one or more genetic disorders in the fetus, such as genetic disorders associated with one or more causative genetic variants. Examples of causative genetic variants include trisomy, cystic fibrosis, sickle cell anemia, and Tay-Sachs disease. The mother may provide a control sample and a blood sample used for comparison. The control sample may be any suitable tissue, which can then be sequenced to provide a reference sequence. The sequence of the cfDNA corresponding to the fetal genomic DNA may then be identified as a sequence variant compared to the maternal reference. The father may also provide a reference sample to aid in the identification of fetal sequences and sequence variants.

[0199]

[0210] Another therapeutic application may include detection of exogenous polynucleotides, including those from pathogens such as bacteria, viruses, fungi and microorganisms, the information of which may inform treatment.

[0200]

[0211] VII. Computer Systems

[0212] The present disclosure provides a computer system programmed to implement one or more methods of the present disclosure. The computer system of the present disclosure may be used to control various operations of the sensor, such as detecting one or more signals indicative of the impedance or impedance change of the sensor when at least a portion of the target molecule is bound by the binding moiety of the sensor.

[0201]

[0213] 6 shows a computer system 601 programmed or otherwise configured to communicate with and control various aspects of the procedures of the present disclosure. The computer system 601 can communicate with, for example, one or more circuits coupled to or including sensors and one or more devices (e.g., machines) used to prepare, process, or hold one or more reaction mixtures for sensing. The computer system 601 can also communicate with one or more controllers or processors of the present disclosure. The computer system 601 can be a user's electronic device or a computer system located remotely relative to the electronic device. The electronic device can be a mobile electronic device.

[0202]

[0214] The computer system 601 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 605, which may be a single-core or multi-core processor or multiple processors for parallel processing. The computer system 601 also includes memory or storage locations 610 (e.g., random access memory, read-only memory, flash memory), electronic storage 615 (e.g., hard disk), communication interface 620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 625, such as cache, other memory, data storage, and / or electronic display adapters. The memory 610, storage 615, interface 620, and peripheral devices 625 are in communication with the CPU 605 through a communication bus (solid lines), such as a motherboard. The storage 615 may be a data storage device (or data repository) for storing data. The computer system 601 may be operatively coupled to a computer network ("network") 630 using the communication interface 620. Network 630 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. Network 630 may be a data network and / or a data network. Network 630 may include one or more computer servers, which may enable distributed computing, such as cloud computing. Network 630 may optionally implement a peer-to-peer network with computer system 601, allowing devices coupled to computer system 601 to behave as clients or servers.

[0203]

[0215] CPU 605 may execute a series of machine-readable instructions, which may be embodied as a program or software. The instructions may be stored in a memory location, such as memory 610. The instructions may be directed to CPU 605, which may then program or otherwise configure CPU 605 to implement the methods of the present disclosure. Examples of operations performed by CPU 605 may include fetch, decode, execute, and write.

[0204]

[0216] The CPU 605 may be part of a circuit, such as an integrated circuit. The circuit may include one or more other components of the system 601. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0205]

[0217] The storage device 615 can store files such as drivers, libraries, and saved programs. The storage device 615 can store user data, such as user settings and user programs. The computer system 601 may optionally include one or more additional data storage devices that are external to the computer system 601, such as located on a remote server in communication with the computer system 601 through an intranet or the Internet.

[0206]

[0218] Computer system 601 can communicate with one or more remote computer systems through network 630. For example, computer system 601 can communicate with a remote computer system of a user. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a phone, a smart phone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access computer system 601 through network 630.

[0207]

[0219] Methods as described herein may be implemented using machine (e.g., a computer processor) executable code stored in electronic storage locations of computer system 601, such as, for example, memory 610 or electronic storage 615. Machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by processor 605. In some cases, the code may be retrieved from storage 615 and stored in memory 610 for easy access by processor 605. In some circumstances, electronic storage 615 may be excluded and machine executable instructions are stored in memory 610.

[0208]

[0220] The code may be precompiled and configured for use with a machine having a processor adapted to execute the code, or it may be compiled at run time. The code may be provided in a programming language, which may be selected to allow execution of the code in a precompiled or compiled manner.

[0209]

[0221] The systems and methods provided herein, such as computer system 601 Aspects may be embodied in programming. Various aspects of the technology are typically presented as a "product" or "article" in the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. A machine executable code may be considered an "article of manufacture". The machine executable code may be stored in electronic storage devices such as memory (e.g., read only memory, random access memory, flash memory) or hard disks. A "storage" type medium may include any tangible memory of a computer, processor, etc., or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which may provide permanent, non-transitory storage for software programming. All or part of the software may sometimes be communicated through the Internet or various other telecommunications networks. Such communication may, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used through physical interfaces between local devices, through wired and optical landline networks, and via various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media that carry the software. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0210]

[0222] Thus, a machine-readable medium, such as a computer-executable code, may take many forms, including but not limited to a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices in any one or more computers, such as may be used to implement the databases, etc., shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer readable media thus include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or a DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium having a perforated pattern, a RAM, a ROM, a PROM and an EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link which transports such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0211]

[0223] The computer system 601 may include or be in communication with an electronic display 635 that includes a user interface (UI) 640 to provide, for example, (i) the progress of the reaction mixture, (ii) the progress of the sequencing, and (iii) sequencing information obtained from the sequencing. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces.

[0212]

[0224] The methods and systems of the present disclosure may be implemented using one or more algorithms. The algorithms may be implemented using software when executed by the central processing unit 605. The algorithms may be implemented, for example, using a target nucleotide, polynucleotide, peptide, polypeptide, or nucleic acid sequence. Sequence readouts for polypeptides, proteins, etc. may be determined.

[0213]

[0225] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it is to be understood that none of the aspects of the present invention are limited to the specific depictions, configurations or relative proportions shown herein, which depend upon a variety of conditions and variables. It must be understood that various alternatives to the embodiments of the present invention described herein may be utilized in the practice of the present invention. It is therefore contemplated that the present invention shall embrace any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of the claims and their equivalents are encompassed within the scope of the present invention.

Claims

1. 1. A system for analyzing or identifying a target molecule, comprising: (i) a sensing electrode; (ii) a binding unit coupled to the sensing electrode and configured to bind to at least a portion of the target molecule; and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of a surface of the sensing electrode. Equipped with the sensor is configured to detect one or more signals indicative of an impedance or an impedance change of the sensor when the at least the portion of the target molecule is bound by the binding unit, the one or more signals being usable to analyze or identify the target molecule. system.

2. 2. The system of claim 1, wherein the one or more signals are indicative of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, or (iii) the electrical inductance of the sensor or a change therein.

3. The system of claim 1 , wherein the one or more signals is a current or a voltage.

4. The system of claim 3 , wherein the one or more signals are not a tunneling current.

5. The system of claim 1 , wherein the average cross-sectional dimension of the sensing electrode is no more than 20 times the average size of the target molecules.

6. The system of claim 5 , wherein the average cross-sectional dimension of the sensing electrodes is no more than twice the average size of the target molecules.

7. The system of claim 1 , wherein the average cross-sectional dimension of the sensing electrode is smaller than the average size of the target molecules.

8. The system of claim 1 , wherein the binding unit is coupled to the sensing electrode via a conductive material.

9. The system of claim 1 , wherein the dielectric material is a self-assembled monolayer.

10. The system of claim 1 , wherein the target molecule comprises a tag, the tag configured to induce a change in the one or more signals.

11. 10. The system of claim 1, wherein the sensor further comprises a reference electrode in electrical communication with the sensing electrode, and the one or more signals are indicative of the impedance or impedance change between the sensing electrode and the reference electrode.

12. 12. The system of claim 11, wherein the sensing electrode and the reference electrode are configured to provide a first electric field along a first direction, the system further comprising an additional electric field generator configured to apply a second electric field along a second direction different from the first direction.

13. The system of claim 12 , wherein the second direction is substantially perpendicular to the first direction.

14. The system of claim 1 , wherein the sensor is further configured to determine a residence time of the at least the portion of the target molecule on the binding unit.

15. The system of claim 1 , wherein the binding unit comprises one or more members selected from the group consisting of a small molecule, an enzyme, an antibody, a functional fragment thereof, and a functional variant thereof.

16. The system of claim 1 , wherein the target molecule comprises one or more members selected from the group consisting of small molecules, nucleotides, polynucleotides, amino acids, peptides, polypeptides, and variants thereof.

17. 1. A method for analyzing or identifying a target molecule, comprising: (a) providing a sensor comprising: (i) a sensing electrode; (ii) a binding unit coupled to the sensing electrode and configured to bind to at least a portion of the target molecule; and (iii) a dielectric material coupled to the sensing electrode and covering at least a portion of a surface of the sensing electrode; (b) detecting one or more signals indicative of the impedance or impedance change of the sensor when the at least the portion of the target molecule is bound by the binding unit; and (c) analyzing or identifying the target molecule using the one or more signals. The method includes:

18. 20. The method of claim 17, wherein the one or more signals are indicative of (i) the electrical resistance of the sensor or a change therein, (ii) the electrical capacitance of the sensor or a change therein, or (ii) the electrical inductance of the sensor or a change therein.

19. The method of claim 17 , wherein the one or more signals is a current or a voltage.

20. 20. The method of claim 19, wherein the one or more signals are not a tunneling current.

21. The method of claim 17 , wherein the average cross-sectional dimension of the sensing electrode is no more than 20 times the average size of the target molecules.

22. The method of claim 21 , wherein the average cross-sectional dimension of the sensing electrode is no more than twice the average size of the target molecules.

23. The method of claim 17 , wherein the average cross-sectional dimension of the sensing electrode is smaller than the average size of the target molecules.

24. The method of claim 17 , wherein the binding unit is coupled to the sensing electrode via a conductive material.

25. The method of claim 17 , wherein the dielectric material is a self-assembled monolayer.

26. 20. The method of claim 17, wherein the target molecule comprises a tag, the tag configured to induce a change in the one or more signals.

27. 20. The method of claim 17, wherein the sensor further comprises a reference electrode in electrical communication with the sensing electrode, and the one or more signals are indicative of the impedance or impedance change between the sensing electrode and the reference electrode.

28. 30. The method of claim 27, wherein the sensing electrode and the reference electrode provide a first electric field along a first direction, the method further comprising applying a second electric field along a second direction different from the first direction using an additional electric field generator.

29. 30. The method of claim 28, wherein the second direction is substantially perpendicular to the first direction.

30. 20. The method of claim 17, further comprising determining a residence time of the at least the portion of the target molecule on the binding unit.

31. 20. The method of claim 17, wherein the binding unit comprises one or more members selected from the group consisting of a small molecule, an enzyme, an antibody, a functional fragment thereof, and a functional variant thereof.

32. 20. The method of claim 17, wherein the target molecule comprises one or more members selected from the group consisting of small molecules, nucleotides, polynucleotides, amino acids, peptides, polypeptides, and variants thereof.

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